exploratory research and hypothesis in medicine - NET

EXPLORATORY RESEARCH AND HYPOTHESIS IN MEDICINE

CONTENTS 2017 2(4):77–151

Editorial

Editorial: Novel and Contemporary Perspectives in Medical NutritionMark Lucock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Young Scholars Section

Xenohormesis: Applying Evolutionary Principles to Contemporary Health IssuesShelley Suter, Mark Lucock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79

Review Articles

Effect of Folate Supplementation on Inflammatory Markers in Individuals Susceptible to Depres-sion: A Systematic ReviewHelen Barnett, Nathan M. D’Cunha, Ekavi N. Georgousopoulou, Jane Kellett, Duane D. Mellor, Andrew J. McKune, Nenad Naumovski . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86

Dietary Treatment for Crohn’s Disease—Old Therapy, New InsightsRakesh Vora, John W.L. Puntis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Nutrigenetics—Personalized Nutrition in the Genetic AgeEmma L. Beckett, Patrice R. Jones, Martin Veysey, Mark Lucock . . . . . . . . . . . . . . . . . . . . 109

Maternal Undernutrition and Type 2 Diabetes in Australian Aboriginal and Torres Strait Islander People: History and Future DirectionDean V. Sculley, Mark Lucock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Nutrition as Medicine to Improve Outcomes in Adolescents Sustaining a Sports-related ConcussionKrista Casazza, Erin Swanson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Original Articles

Vitamin D-related Nutrigenetics and Cognitive Decline in an Elderly PopulationCharlotte Martin, Zoe Yates, Martin Veysey, Katrina King, Suzanne Niblett, Mark Lucock . . . . . . . . . . 131

Cytotoxic Effect of Bitter Melon (Momordica charantia L.) Ethanol Extract and Its Fractions on Pancreatic Cancer Cells in vitroRebecca A. Richmond, Quan V. Vuong, Christopher J. Scarlett . . . . . . . . . . . . . . . . . . . . . . 139

Reviewer Acknowledgement

2017 Reviewer Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Exploratory Research and Hypothesis in Medicine 2017 vol. 2 | 77–78

Copyright: © 2017 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Editorial

Editorial: Novel and Contemporary Perspectives in Medical Nutrition

Mark Lucock*

School of Environmental & Life Sciences, University of Newcastle, PO Box 127, Brush Rd, Ourimbah, NSW 2258, Australia

I was originally asked to put together a special issue for Ex-ploratory Research and Hypothesis in Medicine (ERHM) on the topic of “Medical Nutrition”. I scratched my head, and considered how far human nutritional sciences have evolved over the past two decades, and suggested to the Journal’s editorial staff that an alter-native theme—“Novel and Contemporary Perspectives in Medical Nutrition”—might fit more comfortably with where the state of the art now resides.

This revised title appealed because it had a more contemporane-ous perspective, encompassing a potential breadth of ideas from the role of maternal diet in early human development within the burgeoning field of “Developmental Origins of Adult Disease” through to the concept of “Functional Foods” and related bioac-tives in maintenance of human health. These and other concepts, such as “Personalized Nutrition”, “Nutrigenetics/Nutrigenomics” and “Epigenetics”, are cognate subdiciplines of human nutrition that have grown to become hugely significant in the modern era of “Molecular Nutrition”. The goal was to have an issue that best re-flects how nutrition now serves the prevailing paradigm(s) of what is meant by optimal human health.

The medical application of human nutrition has been a long and interesting journey. Hippocrates was born 2,500 years ago (460 BC) on Kos, and famously stated, “Let food be thy medicine and medi-cine be thy food”. It seems appropriate to me that, as the “Father of Medicine”, Hippocrates’ rationalization/professionalization of the approach to treating disease advocated lifestyle approaches, such as exercise and diet, to treat pathology. Indeed, a lesser cited aphorism attributed to Hippocrates was “Walking is man’s best medicine”. I’m sure time has reshaped these much-quoted tenets that are as relevant today as they were two and a half millennia ago—and I’m happy to be a firm believer in the message they convey.

Although Hippocrates sagely advice was lost over the years, understanding the role of diet and health really ramped up in the early 1900s. Sir Frederick Gowland-Hopkins was awarded the No-bel Prize in Physiology of Medicine in 1929 along with the Dutch Physician Christiaan Eijkman.1 They received this preeminent award for the discovery of vitamins. However, this achievement should really also include the Polish biochemist Casimir Funk, who was another important architect in developing our under-standing of micronutrients in the maintenance of human health.2

The first half of the twentieth century was devoted to curing deficiency syndromes—it was all about under-nutrition. In 1912, Gowland-Hopkins conducted seminal work, demonstrating that a

highly refined diet comprised of pure proteins, carbohydrates, fats, minerals and water fail to support growth in weanling rats. This led him to discover that tiny quantities of an as yet unidentified dietary substance (from milk) was essential for animal growth and survival. These unidentified, speculative substances were, in 1913, given the name “vitamins” (from vital amines).

If the first half of the last century was about vitamin discovery and treating deficiency diseases like pellagra and beriberi, the sec-ond half of the twentieth century was all about addressing problems of over-consumption and the associated disease burden (increased obesity, type II diabetes, cardiovascular disease and cancers). Pub-lic health guidelines in the USA identified the issues and developed sound public health guidelines. In 1988, C Everett Koop, USA’s Surgeon General, published a report that spawned further reports and recommendations.3 Basically, the nation had to face up to diet-disease associations linked to a diet high in saturated fatty acids, total fat, cholesterol, salt and sugars, but low in unsaturated fatty acids, complex carbohydrates and fiber. A diet that is basically high in animal products and which is largely energy rich-nutrient poor with too little plant-based materials.

However, over the last 20 years, with an increasingly affluent and aging society, concepts such as functional foods and use of supplements/nutraceuticals have come into favor to support health and the compression of morbidity. Academia and industry have developed synergies to develop this concept, and since the 1990s, scientists have been actively examining zoochemical and phyto-chemical bioactives as potential new agents to promote health, prevent disease and improve longevity.

One of the most interesting ideas in the field of phytochemical bioactives is that of xenohormesis: phytochemicals are produced by plants under stress, and when consumed, are able to activate longevity pathways in other organisms, including humans.

This special issue of ERHM examines many of these contem-porary issues. Given the seemingly intractable problems society faces today, and even more so tomorrow in dealing with an ageing population structure (and the corollary of increased Alzheimer’s disease, diabetes, cardiovascular disease, etc.), perhaps diet should be reexamined and framed once again as “Food is Medicine”.

As an Associate Editor at ERHM, I’d like to thank all contribu-tors to this issue, it has turned out to be a well-rounded selection of topics that deal with leading edge issues of huge relevance to society. Our contributors have dealt with many of the topics I have alluded to above: from xenohormesis to leading-edge research on the role of phytochemicals in pancreatic cancer cell models; from nutrition in sports injury to vitamins in depression. The articles also span the lifecycle from pediatric applications of enteral nutri-tion in Crohn’s disease to vitamin genetics and cognitive decline in the elderly.

What goes ‘round, comes ‘round—time to return to food as medicine.

*Correspondence to: Mark Lucock, School of Environmental & Life Sciences, Uni-versity of Newcastle, PO Box 127, Brush Rd, Ourimbah, NSW 2258, Australia. Tel: +61 2 4348 4109, Fax:+61 2 4348 4145, E-mail: [email protected] to cite this article: Lucock M. Editorial: Novel and Contemporary Perspec-tives in Medical Nutrition. Exploratory Research and Hypothesis in Medicine 2017;2(4):77–78. doi: 10.14218/ERHM.2017.EDITORIAL.

http://creativecommons.org/licenses/by-nc/4.0/

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Lucock M.: EditorialExplor Res Hypothesis Med

References

[1] Thomas A. Who was Fredrick Gowland-Hopkins? The Biologist 2011; 58:33–37.

[2] Griminger P. Casimir Funk – A Biographical Sketch (1884-1967). J Nutr 1972;102:1105–1113.

[3] SurgeonGeneral’sNutrition&HealthReport.Availablefrom:https://www. c-span.org/video/?3713-1/surgeon-generals-nutrition-health-report.

Mark Lucock PhD, CBiol, FRSB, FRCPath

https://www.c-span.org/video/?3713-1/surgeon-generals-nutrition-health-report

https://www.c-span.org/video/?3713-1/surgeon-generals-nutrition-health-report



Young Scholars Section

Introduction

Hormesis is the biological process where a low dose exposure to a toxin or environmental condition, which would otherwise be dam-aging at a higher dose, induces an adaptive response that is actually beneficial.1 It is a concept embedded in evolutionary theory, and essentially it supports the idea that what does not kill us makes us stronger. The xenohormesis hypothesis, first coined by Howitz and Sinclair, explains how stressed plants and autotrophs produce compounds that offer survival benefits to animals that consume them.2,3 It specifically proposes that the majority of health benefits from plant consumption come not only from their known antioxi-dant and micronutrient properties, but also from an evolutionary adaptation of enzyme and receptor pathways. According to the hy-pothesis, mammals and fungi have the ability to utilize and react to information plants provide about the environment, offering a distinct selective advantage.3

The molecular mechanisms underlying the hypothesis are not

yet fully understood; however, the philosophical perspective pro-vides insight on stress responses and their biochemical purpose. Stress is a universal state experienced by all living organisms in response to their environment. Plants are particularly vulnerable because they are unable to remove themselves from danger and have highly developed coping mechanisms to ensure survival. Plants experiencing mild stress in the form of severe tempera-ture, dehydration, nutrient deprivation, sun exposure, toxins and predators produce a variety of protective compounds or second-ary metabolites known as phytochemicals. These allow plants to overcome continuous and temporary threats to their survival; such phytochemicals act as UV filters, antibiotics, insecticides and fun-gicides, while also defending against herbivores, competitive plant species and pollutants.4 When consumed, these bioactive plant molecules have the ability to induce and up-regulate specific bio-logical pathways associated with endurance, longevity and disease resistance in animals. Unsurprisingly, survival, reproductive abil-ity and natural selection favors those that activate longevity and cellular defensive pathways, and so facilitate the natural cycle of plant stress and conferred resistance in animals.

The success of an evolutionary process partially relies on the concept that environmental exposure represents a relevant and sig-nificant threat to survival. Considering the increasing occurrence of contemporary health conditions relating to affluence and exces-sive consumption, it is possible to draw a link between dietary habits that do not reflect physiological needs (and agricultural practices that do not reflect a balanced environment) and the inter-ruption of survival processes. The xenohormesis hypothesis can be used not only as a way of identifying mechanisms that aid in our understanding of disease etiology but also as an exciting modern concept embracing nutritional medicine, targeting treatment and

Xenohormesis: Applying Evolutionary Principles to Contemporary Health Issues

Shelley Suter and Mark Lucock*

School of Environmental & Life Sciences, University of Newcastle, Ourimbah, NSW 2258, Australia

Abstract

The ability of plants to exert health benefits beyond antioxidant and micronutrient capacity introduces a gap in scientific understanding. The xenohormesis hypothesis aims to fill this gap, proposing that an evolutionary adap-tation of enzyme and receptor pathways allow us to react to information that plants provide about the environ-ment, offering a distinct survival advantage. The concept suggests that phytochemicals produced by plants under stress are able to activate longevity pathways in other organisms when consumed. The same pathways activated by calorie restriction, the highly conserved sirtuin enzymes and cellular homeostasis mechanisms provide an ex-citing perspective for treating chronic conditions related to excessive consumption. Harnessing the biological ac-tivity associated with the xenohormesis paradigm could provide a simple and achievable therapeutic alternative, although it needs to be considered within the confounding framework. The objective of this paper is to provide an update on the role of xenohormesis within nutritional medicine and to discuss the impact of modern food supply and consumption practices on evolutionary processes.

Keywords: Xenohormesis; Polyphenols; Phytochemicals; Nutritional medicine; Resveratrol; Stress response; Sirtuin.Abbreviations: CVD, cardiovascular disease; ER, endoplasmic reticulum; NIDDM, non-insulin dependent diabetes mellitus; PERM, proteasome, endoplasmic reticulum and mitochondria; ROS, reactive oxygen species; UPR, unfolded protein response; UPRMT, mitochondrial unfolded protein response.Received: July 21, 2017; Revised: October 26, 2017; Accepted: November 06, 2017*Correspondence to: Mark Lucock, School of Environmental & Life Sciences, Uni-versity of Newcastle, PO Box 127, Brush Rd, Ourimbah, NSW 2258, Australia. Tel: +61 2 4348 4109, Fax: +61 2 4348 4145, E-mail: [email protected] to cite this article: Suter S, Lucock M. Xenohormesis: Applying Evolutionary Principles to Contemporary Health Issues. Exploratory Research and Hypothesis in Medicine 2017;2(4):79–85. doi: 10.14218/ERHM.2017.00023.


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prevention. This paper aims to review the current understanding regarding xenohormesis and associated biological pathways as well as the realistic application of related compounds in response to contemporary health problems.

Evolution of the human diet and contemporary health issues

Throughout the human lineage, consumption of plant and ani-mal food products has changed in response to environmental and lifestyle factors. The xenohormesis hypothesis suggests that non-nutrient plant molecules assist in human stress resistance and survival during harsh conditions; however, it is unlikely that this activity alone dictated survival. Energy dense animal food sources and their associated stress signals played a central role in evolu-tionary development and natural selection.5 Humans have a long and seemingly successful history of meat consumption, with the use of animal food products dating back at least 5 million years.6 In modern society however, excessive meat consumption, in com-bination with other high-energy foods and a sedentary lifestyle, is associated with a growing list of chronic conditions.6 A recent population-based cohort study showed an increased risk of mortal-ity from nine different causes directly linked with red meat and processed meat consumption,7 while excessive sugar consumption plays a key role in metabolic disease by altered lipid and carbohy-drate metabolism, positive energy balance and weight gain.8 These results are important to consider in the context of xenohormesis because they identify problems arising from nutritional practices that do not reflect physiological needs in our contemporary envi-ronment.

It is difficult to predict the extent to which dietary behaviors affect health; however, the etiology of most conditions is in some way related to an individual’s past or present nutritional status. Non-insulin dependent diabetes mellitus (NIDDM) and cardiovas-cular disease (CVD) are often referred to as diseases of affluence, where prevalence rises with economic development. While afflu-ence is no longer considered the major factor it once was, these diet-related chronic diseases impose a significant healthcare bur-den. In 2010, dietary risk factors such as low fruit intake, in com-bination with excess energy and physical inactivity were estimated to account for 10% of global disability and years of life lost.9 Therefore, it is possible to associate contemporary health issues with inappropriate biological stress and environmental disconnec-tion. Treating diet-related disease with dietary intervention is not a novel concept; however, considering the complex evolution of the human diet and the risks associated with contemporary food choices, a deeper understanding of xenohormesis could provide a specific direction for nutritional intervention.

Calorie restriction

Calorie excess is a primary risk factor in a variety of modern health problems; therefore, it is not surprising that calorie restriction is associated with increased lifespan and improved health. First iden-tified in rats over 75 years ago,10 the relationship between fast-ing and longevity has been observed in a variety of organisms, including yeast, flies, rodents and monkeys.11,12 While the exact mechanism remains relatively unknown, various relevant meta-bolic pathways have been identified.13 Calorie restriction, but not starvation, initiates mild stress in the deprived organism and acti-vates pathways related to increased metabolic efficacy and protec-tion from cellular damage.13 These pathways are the result of a highly conserved evolutionary response, where improved health

from fasting ensures survival in times of restriction and thus the ability to reproduce when suitable conditions return. In a time where many chronic conditions are associated with obesity, the concept that calorie restriction could improve population health status seems obvious yet remarkably difficult to put into practice. A key point here is that plant compounds are known to activate the same longevity pathways associated with calorie restriction when consumed.14

Xenohormetic pathways

Many non-nutritional dietary components activate stress responses and homeostasis mechanisms in animals. Polyphenols are a group of phytochemicals closely associated with plant stress and sec-ondary resistance in animals. Bioactive polyphenols are known to have antioxidant and anti-inflammatory properties, and have been directly linked to reduced mortality rates in humans.15,16 One of the most promising and well-researched xenohormetic polyphe-nols is resveratrol, a stilbene commonly known for its presence in red wine.

Resveratrol activates the same pathways as calorie restriction, with early research showing the compound was able to activate sirtuin (SIRT2) enzymes in the yeast strain Saccharomyces cer-evisiae, resulting in improved DNA stability and a dramatic 70% increase in lifespan.17 This observation essentially formed the foundations of the xenohormesis hypothesis, sparking interest in phytochemically activated enzyme/receptor pathways and their origin. The mammalian sirtuin homologs, a group of 7 NAD+ de-pendent histone deacetylases (SIRT 1-7), act on a variety of physi-ological processes including metabolism, apoptosis, DNA repair and DNA transcription.18 Due to the synthesis of resveratrol in re-sponse to stress, grapes grown in undesirably cool environments, at high elevation or in alkaline soil produce the best wine in re-lation to taste and health.19 It is because of resveratrol and other polyphenolic compounds that mild to moderate wine drinking has been linked to cancer protection and reduced cardiovascular disease, as well to slowing of neurodegenerative conditions.20–23 While antioxidant activity is partially responsible for resveratrol’s protective action, it is also thought to be the result of a highly adap-tive stress response and various signaling pathways activated by SIRT1 enzymes in mammals.

Other biological pathways involved in stress response and sur-vival mechanisms should be considered alongside or within the xenohormesis paradigm. The proteasome, endoplasmic reticulum and mitochondria (PERM) hypothesis aims to explain how xenobi-otic compounds, including trace metals and phytochemicals, exert beneficial effects via homeostatic mechanisms.24 The hypothesis explains stress response on a cellular level, where proteasomes, the endoplasmic reticulum (ER), mitochondria and peroxisomes, collectively form a functional structure labeled the proterome. The proterome works to regulate cell apoptosis or autophagy under oxidative stress by mechanisms of altered calcium homeostasis, mitochondrial polarization and chaotic oscillation. It is thought that reactive oxygen species (ROS) produced by exposure to phy-tochemicals and xenobiotic compounds act as signaling molecules that trigger ER stress and subsequent proterome formation. While extended or excessive exposure to ROS leads to protein, lipid and nucleic acid degradation, low amounts exert therapeutic like ef-fects by regulation of cell signaling cascades.25 The outcome is cell conservation or death, and the resulting pathway ultimately supports survival of the remaining living cells.

ER stress and mitochondrial stress occur in response to genetic and environmental factors. Cells under physiological stress pro-

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duce unfolded proteins, the ER’s primary role is to ensure only folded proteins exit the cell. The unfolded protein response (UPR) occurs as a homeostatic mechanism of the ER and its purpose is to monitor protein-folding capacity and ER abundance to ensure quality and control of protein transcription.26,27 Similarly, the mi-tochondrial stress response, coined the mitochondrial unfolded protein response (UPRMT), is a quality control system comprised of signaling pathways to the nucleus and ER. Damaged proteins or a disrupted membrane potential in response to ROS accumulation activate the UPRMT in pursuit of mitochondrial homeostasis.28

Mitochondria and ER communication is essential for appropri-ate apoptosis and autophagy; dysfunction is directly linked to the etiology of many chronic diseases, including the development of NIDDM and CVD.29,30 For example, in response to excessive con-sumption and obesity, there is an increased demand on pancreatic beta cells for insulin production, causing cellular stress and pro-tein mutation.31 ER hormesis can trigger and up-regulate the UPR, meaning mild stress provides protection in certain disease models and is considered a plausible therapeutic target.32 It is also pro-posed that sirtuin activation is linked to the UPR, where up-regula-tion of sirtuins and subsequent deacetylation of the XPB1 protein controls UPR signaling and further prevents cellular dysfunction.33

The PERM hypothesis, ER and mitochondrial stress responses, can be considered alongside the xenohormesis hypothesis and

sirtuin activation to understand cellular stress resistance and its implication on human health. Figure 13,18,24,31,32 summarizes the occurrence of sirtuin activation, cellular homeostasis mechanisms and their relevant biological pathways, and identifies areas suscep-tible to therapeutic intervention.

Xenohormesis and the modern diet

Xenohormetic awareness raises important questions about the food supply chain; the way we eat, source and respond to our food is continuously changing in response to a growing population and climate change. It is well known that modern agricultural practices aim for large yields and uniformed produce in order to optimize fi-nancial profit. Crops are provided with ideal conditions for growth in the form of environmental or chemical protection, removing any form of stress that could inhibit or alter the final product. Many studies have identified composition differences between organic and conventional plants and while results are often conflicting with regard to nutritional value, there is a general consensus that con-ventional inorganic practices produce larger yields and fewer stress compounds.34–36 Previous reviews on xenohormesis have raised concern regarding the increasing popularity of mono-cropping and the subsequent loss of nutritional benefit.37 While growth and har-

Fig. 1. Mechanism of xenohormesis from plant stress to SIRT1 activation and cellular homeostasis. Mild stress and the consumption of plant stress com-pounds activate SIRT1 enzymes and subsequent pathways associated with increased stress resistance and survival.3 Biological targets of sirtuin enzymes include the liver, brain, heart, pancreas, adipose tissue and skeletal muscle, conferring a diverse range of health benefits.18 In pursuit of cellular homeostasis and survival after mild physiological stress, the ER UPR is up-regulated.31,32 The ROS in response to mild ER stress serves as a signal to the proposed PERM mechanism, further ensuring homeostasis and organism survival via cell conservation or cell death.24 Therapeutic intervention could be achieved by agri-cultural, dietary or supplementation intervention. Abbreviations: AMPK, adenosine monophosphate-activated protein kinase; ATF6, activating transcription factor 6; ER, endoplasmic reticulum; FoxO, forkhead box class O; IRE1, inositol-requiring enzyme 1; NAD, nicotinamide adenine dinucleotide; NF-κB, nuclear factor-κB; PERK, protein kinase-like ER kinase; PERM: proteasome, endoplasmic reticulum and mitochondria; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator-1α; ROS: reactive oxygen species; UPR: unfolded protein responses; XBP1, X-box binding protein 1.



vesting techniques that remove plant stress essentially eradicate the traditional application of xenohormesis, it could be argued that this is simply an evolutionary reflection of a time where the envi-ronment does not represent an immediate threat. Despite this, and in response to increasing concern, a growing consumer demand for higher quality fruit and vegetables has promoted the exploration of breeding programs focused on improving the nutritional qualities of fresh produce.

Applying mild stress in the form of high light, heat shock and chilling shock can increase the concentration of phenolic com-pounds in lettuce without inhibiting overall growth or yield,38 while the cultivation period and phytochemical concentration in broccoli can be increased by low radiation exposure under con-trolled temperature.39 The concept provides an opportunity for population level nutritional intervention; however, it is not without practicality issues. Most polyphenols and bioactive compounds are bitter or astringent; therefore, increasing nutritional value is either limited or achieved only by sacrificing sensory quality.40 While na-ture’s regulation of polyphenol intake presents limitations for the deliberate application of xenohormesis, this remains an exciting area of food and nutritional science and provides scientists with the ability to generate plants that could address diet-related chronic disease and possibly global scale health issues.41,42

Xenohormesis and nutritional medicine

Xenohormesis, via nutrition, is associated with survival, uninten-tional disease prevention and general wellbeing. Phytochemicals have a secure place in the nutraceutical market; however, their suitability for prevention of chronic disease remains largely un-defined. Plant compounds that activate longevity pathways and cellular homeostasis mechanisms have successfully demonstrated medicinal activity for NIDDM, CVD, hypertension and other conditions associated with aging and diet.43,44 However, despite

obvious therapeutic potential and commonly supported non-toxic-ity,45,46 many factors have prevented definitive recommendations regarding medicinal use at this time. It is important to note that plants interact with many biological pathways and demonstrate diverse therapeutic activity, which means their physiological ef-fect can be inconsistent and altered by many variables. Table 147–58 outlines a selection of clinical evidence supporting the therapeutic use of certain phytochemical compounds.

It is thought that dietary polyphenols can provide relief to sub-jects with NIDDM by obvious anti-inflammatory and antioxidant capacity, but also by offering protection to pancreatic beta cells against glucose toxicity.59 Resveratrol has been widely studied for its ability to interact with insulin-regulated blood glucose path-ways. Use of the polyphenol was shown to extend the lifespan and exert a wide range of health benefits on overweight mice subjected to a high-calorie diet60; however, the same meaningful results are yet to be achieved in subjects of normal weight.61 Another animal study has confirmed the context-dependent activity of resveratrol, with variables such as sex, diet and metabolic condition directly influencing the results.62 Human trials have also shown that ben-eficial activity is dictated by dosage, length of exposure and the pa-tient’s health status. Twenty-six weeks of resveratrol intake, in oth-erwise healthy overweight subjects, was able to improve memory and brain function in addition to improved glucose metabolism,63 which is supported by another study reporting beneficial effects on blood glucose levels in overweight participants.64 In contrast how-ever, 8 weeks of red wine polyphenol supplementation in obese volunteers did not improve insulin sensitivity, and when trialed on healthy non-obese patients, resveratrol was shown to have little to no effect.65,66 Based on current knowledge, resveratrol activity might be more beneficial when administered as a smaller dose over a long period of time. Furthermore, its therapeutic affect appears to favor those with already compromised health, which is significant when considering prevention of NIDDM in overweight subjects.

Many plant compounds have demonstrated beneficial cardio-

Table 1. Phytochemical compounds associated with hormetic pathways and a selection of clinical evidence supporting their therapeutic potential

Compound Classification Food Source Therapeutic Potential

Sulphoraphane Organosulphur Isothiocyanate

Cruciferous vegetables (broccoli, brussels sprouts, cabbage, kale)

Improved insulin resistance in NIDDM patients47

Defence against oxidative stress and Cardiovascular disease48

Catechins Flavanol Green tea, dark chocolate, red wine, apples

Reduce body fat and low-density lipoprotein in healthy men49

Modulation of oxidative stress in subjects with heart failure and type 2 diabetes50

Curcumin Diarylheptanoid Turmeric Prevention of NIDDM in a pre-diabetic population and improved function of beta cells51

Cholesterol improvement in subjects with metabolic syndrome52

Resveratrol Stilbene Grapes, peanuts, blueberries, cocoa, dark chocolate

Exercise mimetic activity, SIRT1 activation and improved energy expenditure in patients with NIDDM53

Reduced lipoprotein particle production in patients with hypertriglyceridemia54

Lignan Phytoestrogen Flaxseeds, whole grains, sesame seeds

High lignan intake can decrease oxidized low-density lipoprotein in healthy men and women55

Antihypertensive activity associated with changes in diastolic blood pressure in patients with cardiovascular disease56

Quercetin Flavonol Cherries, berries, tomatoes, apples, peppers, red wine, citrus fruits

Improved glycemic and insulin response in NIDDM patients57

Combined green tea, resveratrol and quercetin supplementation reduced diastolic pressure and improved blood pressure in hypertensive subjects58

Abbreviation: NIDDM, non-insulin dependent diabetes mellitus.



vascular effects, including antioxidant, antithrombotic and anti-inflammatory properties.67 Phytochemicals play a multi-faceted role in the treatment and prevention of CVD; alteration of endothe-lial cell function, blood lipid profile and blood pressure are areas susceptible to phytochemical therapy. Human trials have shown that polyphenols can significantly reduce fasting and postprandial plasma triglyceride concentrations in obese metabolically compro-mised subjects.68 Furthermore, participants at high CVD risk, who consumed high amounts of stilbene polyphenols and lignin from a Mediterranean diet, demonstrated a reduced risk of overall mortal-ity after 5 years of dietary intervention.69 Interestingly, grape-seed polyphenol supplementation in hypertensive adults did not signifi-cantly influence blood pressure measurements and combined poly-phenol and vitamin C supplementation over 6 weeks negatively increased blood pressure variation, suggesting combined therapy could be detrimental.70 While the benefits of polyphenol intake on CVD risk is evident, it remains unclear whether a typical intake of polyphenol-rich foods offers cardio-protection.71,72

Limitations

The xenohormesis hypothesis represents a concept with evolution-ary biology at the heart of the paradigm; however, purposeful ap-plication of the concept presents limitations. Current research sug-gests resveratrol treatment is only beneficial in certain population groups, with many studies having focused on overweight, meta-bolically-challenged or elderly subjects. While it is ethically and practically difficult to establish a causal connection between plant compounds and the extension of human longevity, the relevance of extrapolation from animal and in vitro studies remains unknown. From a philosophical perspective, exploitation of evolutionary processes could be counterproductive, and limitations regarding suitable use could be a reflection of this. Previous reviews have highlighted the inconsistency of phytochemical bioavailability in humans and how medicinal qualities are difficult to reproduce due to composition variation from one plant to the next.37 Additionally, despite the fact that resveratrol and other polyphenols are found in many foods, in reality they are not very abundant in a normal diet. While the recommended daily dosage is varied and supplements range from 2 mg up to 500 mg, the average resveratrol and resver-atrol-derivative intake in certain wine-drinking population groups is just 100 µg/day and 933 µg/day respectively.73 Studies concern-ing appropriate dosage, where smaller amounts appear to be more beneficial,74,75 further reiterate the idea that polyphenol activity is part of a wider, context-dependent, biological occurrence.

Perspective

Xenohormesis and its medicinal scope is a broad concept, diffi-cult to comprehend on a large scale. In order to gain a wider un-derstanding and to overcome the limitations discussed previously, research should be focused on smaller, independent areas. Further identification of hormetic compounds and the food sources that provide them is an essential part of this process. Agricultural fac-tors restricting the traditional application of xenohormesis should be identified along with advantageous environmental stresses and consumer tolerance levels that allow for maximum therapeu-tic benefit and sensory satisfaction. Future research should focus on the human bioavailability of related compounds in addition to other metabolic factors that effect a therapeutic benefit, including phytochemical interaction with other bioactive compounds. The

relationship between PERM cellular homeostasis and sirtuin ac-tivation would also aid in the understanding of aging and longev-ity factors. Long-term human clinical trials including non-obese, healthy subjects would replicate xenohormesis in a controlled environment and provide valuable insight into the potential of its medicinal applications, both preventative and curative. Consoli-dating current research will establish the need for agricultural and food supply chain practices that ensure evolutionary processes are preserved or encouraged.

Conclusions

The xenohormesis hypothesis of plant stress and secondary resist-ance by sirtuin activation and cellular homeostasis mechanisms not only provides a rational explanation for the diverse therapeutic activity of phytochemicals, but also offers an avenue for realistic health intervention. In a time where excessive and inappropriate food consumption has led to an increase in chronic health con-ditions and reduced lifespan, understanding and applying evolu-tionary principles to nutritional medicine is a novel, yet promis-ing, concept. Other contemporary factors, including the way we produce and source food, pose a significant threat to evolutionary and/or adaptive processes, and should be considered in relation to contemporary health concerns. Research regarding xenohormetic compounds has produced conflicting results regarding dosage and metabolic activity. Despite confusion, the nutritional and medici-nal potential of plant polyphenols represents an area of research likely to produce alternative therapeutic models in the future.

Conflict of interest

The authors have no conflict of interests related to this publication.

Author contributions

Idea researched and developed (SS, ML), article crafted in final form (SS, ML).

References

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Review Article

Introduction

The identification of folate as a key nutrient in the prevention of hu-man disease, and particularly neural tube defects (NTD) in utero,1 has led to public health interventions based on mandatory folic acid (FA) fortification. These measures have been deemed necessary to reduce the incidence of NTD; and, while FA supplementation has been found to be successful in reducing NTD,1,2 there remains controversy surrounding the safety of mandatory fortification.3–5 Mandatory fortification exposes the whole population to a syn-thetic form of folate, rather than just the target population, while consumption of foods containing natural forms of folate is decreas-ing.6–8

Over 80 countries have implemented a mandatory FA fortifi-cation policy. The intake of folate in countries with mandatory fortification is often higher than the recommended ranges, calling into question whether mandatory fortification is warranted in other countries.9 In countries with mandatory fortification, FA is avail-

Effect of Folate Supplementation on Inflammatory Markers in Individuals Susceptible to Depression: A Systematic Review

Helen Barnett1, Nathan M. D’Cunha1,2, Ekavi N. Georgousopoulou1,2,3, Jane Kellett1,2, Duane D. Mellor1,2,4, Andrew J. McKune1,2,5 and Nenad Naumovski1,2,6*

1Faculty Health, University of Canberra, Kirinari Street, Bruce, Canberra, ACT, 2617, Australia; 2Collaborative Research in Bioactives and Biomarkers (CRIBB) Group, Kirinari Street, Bruce, Canberra, ACT, 2617, Australia; 3Department of Nutrition-Dietetics, School of

Health and Education, Harokopio University, Athens, 17671, Greece; 4School of Life Sciences, Coventry University, Coventry, CV1 2DS, UK; 5University of Canberra Research Institute for Sport and Exercise (UCRISE), University of Canberra, Bruce, Canberra, ACT, 2617, Australia; 6University of Canberra Health Research Institute (UCHRI), University of Canberra, Bruce, Canberra, ACT, 2617, Australia

Abstract

Folate has been proposed to be an efficacious treatment strategy for depression. The mandatory fortification of flour with synthetic folic acid (FA) in over 80 countries has yielded improvements in folate intake; however, depression is still a considerable public health concern. While there are established benefits of FA fortification in reducing risk of neural tube defects, the implications regarding depression are unclear, especially in individu-als with certain genetic polymorphisms. Therefore, a systematic review was conducted to examine the effects of folate to treat depression. Following PRISMA guidelines, a systematic review was conducted of electronic data-bases (PUBMED, Scopus, CINAHL, and Cochrane Library) to identify human clinical trials examining the effects of folate (including FA) supplementation in the management or prevention of depression, the impact on inflam-matory markers and if genetic polymorphisms were considered. Ten trials met the inclusion criteria. Seven trials examined effects of either adjunctive FA or L-methylfolate (L-MTHF) supplementation with antidepressants in the management of depression and three examined effects of FA supplementation alone for prevention of depres-sion. No benefit of FA was found compared to placebo (all, p > 0.05). The single L-MTHF trial that explored the interplay of genetic polymorphisms and methylation status found benefit in the Hamilton depression rating scale from adjunctive treatment with 15 mg/day of L-MTHF compared with placebo (−6.8 ± 7.2 vs. −3.7 ± 6.5; p = 0.017) and improvement with L-MTHF for most genetic markers. Currently, there is no evidence to support FA supple-mentation for the management or prevention of depression. More research is required to determine the efficacy of L-MTHF and folinic acid in certain clinical populations.

Keywords: Folic acid; Homocysteine; Depression; Epigenomics.Abbreviations: 4-HNE, 4-hydroxy-2-nonenal; 5-HIAA, 5-hydroxyindoleacetic acid; 5-MTHF, 5-methyltetrahydrofolate; BDI, Beck depression inventory; BMI, body mass index; CI, confidence interval; CVD, cardiovascular disease; DHF, dihydro-folate; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders IV; FA, folic acid; Hcy, homocysteine; HDRS, Hamilton depression rating scale; hs-CRP, high-sensitivity C-reactive protein; IL, interleukin; L-MTHF, L-methylfolate; MINI, mini international neuropsychiatric interview; MTHFR, methylenetetrahydrofolate reduc-tase; NS, not specified; NTD, neural tube defects; OR, odds ratio; RCF, red blood cell folate; SAH, S-adenosylhomocysteine; SAMe, S-adenosylmethionine; THF, tetrahy-drofolate; TNF-α, tumor necrosis factor-alpha.Received: July 31, 2017; Revised: October 31, 2017; Accepted: November 14, 2017*Correspondence to: Nenad Naumovski, Room 1C130; Faculty of Health; Locked Bag 1; University of Canberra, Canberra, ACT, 2601, Australia. Tel: +61 2 62068719, Fax: +61 2 62015999, E-mail: [email protected] to cite this article: Barnett H, D’Cunha NM, Georgousopoulou EN, Kel-lett J, Mellor DD, McKune AJ, Naumovski N. Effect of Folate Supplementation on Inflammatory Markers in Individuals Susceptible to Depression: A System-atic Review. Exploratory Research and Hypothesis in Medicine 2017;2(4):86–100. doi: 10.14218/ERHM.2017.00025.



Barnett H. et al: Folate supplementation and depression Explor Res Hypothesis Med

able as either a supplement (400 µg to 4 mg/day recommended in pregnancy, depending on medical history) or through the fortifica-tion of flour and cereal products.10 Fortification levels vary world-wide, with a FA level of 140 µg/100 g implemented in the United States in 19985 and 200–300 µg/100 g in Australia as of 2009.11 Although increased folate reduces plasma homocysteine (Hcy),6 excessive FA consumption from fortified foods may impair intra-cellular folate metabolism and play a role in epigenetic changes linked to increased inflammation and non-communicable diseases including depression.12–16

Types of folate

Folate was first discovered in 1931 by Wills, as a substance in yeast extract, and it was found to be effective in the treatment of pregnancy-related anemia.17 Currently, there are multiple struc-tural forms of the vitamin that have been identified (‘synthetic’ and ‘natural’ forms), as well as a result of metabolic processes, which makes understanding the role of folate in human health and disease incredibly complex.17 Due to humans not being able to synthe-size folate, it must be obtained from dietary sources, such as leafy green vegetables, with the main forms in food being 5-methyltet-rahydrofolate (5-MTHF) and formyltetrahydrofolate.18

The main structural differences between natural and synthetic forms of folate are the oxidation state, the number of conjugated glutamic acid moieties and the type of one-carbon substituents at the N5 and N10 positions.17 Dietary folate is predominantly 5-MTHF, which is the biologically active form of the vitamin but may also exist as formyltetrahydrofolate or in the oxidized form 5-methyl-5,6-dihydrofolate. The latter is rapidly degraded; however, the secretion of ascorbic acid into the gastric lumen appears to be a critical mechanism to reduce it back to the more stable 5-MTHF; therefore, increasing the bioavailability of food folates.17 Accordingly, 5-MTHF is demethylated to form tetrahy-drofolate (THF) by methionine synthase, so that it can be used for the synthesis of nucleotides.

In contrast, synthetic FA exists as the fully oxidized pteroyl-monoglutamic acid,19 which is chemically more stable and cheaper to produce than most other forms of folate.4 Like 5-MTHF, FA is also metabolized to THF; however, it is via a much ‘slower’ two-step process catalyzed by the enzyme dihydrofolate reductase and producing the intermediate dihydrofolate (DHF).20 Consequently, human ability to absorb and process FA to biologically active forms of folate is saturated at daily intakes of between 266–400 µg.21,22 Folinic acid is another form of folate supplement available and is commonly used after methotrexate treatment for various types of cancers.17

Roles of folate in health

Folate, as 5-MTHF, is required for the synthesis of the DNA nucle-otide thymidine from the RNA nucleotide uracil and is, thus, vital for the stability of DNA. Therefore, folate deficiency is associated with misincorporation of uracil into DNA and DNA strand break-ages.23 As DNA is incorporated into all cells, folate is especially important for rapidly dividing cells, such as red blood cells, and is consequently associated with anemia.24 Similarly, folate plays a conflicting role in cancer development, depending on the stage of cancer. Adequate folate levels prevent the expression of oncogenes and promote DNA stability before cancer initiation; however, after cancer cells have become established, folate can stimulate cell di-vision and growth.25–28

Folate also provides a methyl group for the conversion of Hcy back to methionine, which leads on to the production of S-aden-osylmethionine (SAMe). The reduction of Hcy also appears to be a vital aspect of this cycle, as elevated Hcy has been associated with disorders ranging from cardiovascular disease (CVD)29,30 to mental health disorders.31–35 The importance to CVD has primar-ily been investigated due to the role of FA in lowering Hcy, and also due to its ability to enhance nitric oxide production, thereby reducing the endothelial dysfunction associated with CVD.36,37 Similarly, folate plays a role in the recycling of tetrahydrobiop-terin, a cofactor involved in the synthesis of the neurotransmitters dopamine, serotonin and noradrenaline.32,34

Folate and mental health

Folate was first observed to be associated with perturbations in mental health when Victor Herbert consumed a diet deficient in folate in 1962.38 In 1978, Boetz et al.39 observed that both under- and over-supplementation of FA resulted in low brain serotonin levels in rats, indicating that folate was involved in the synthesis of neurotransmitters. Epidemiological studies have suggested that low folate status is associated with depres-sion,40,41 severity of depression40,42,43 and response to antide-pressant treatment,41,43,44 though the exact mechanisms of action are still not understood.

To date, there is focus on: the relevance of folate in recycling tetrahydrobiopterin, a cofactor in the synthesis of the neurotrans-mitters;32,34 the function of folate as a methyl donor and the role it plays in the production of SAMe;32,34 the importance of SAMe in the methylation of neurotransmitters;32,45 and, the accumulation of both Hcy and its precursor metabolite S-adenosylhomocysteine (SAH), which has been shown to be neurotoxic due to its inhibi-tion of monoamine metabolism.46–48 Similarly, genome-wide as-sociation studies have shown a link between the methylenetetrahy-drofolate reductase (MTHFR) C677T mutation and certain mental health disorders, such as depression and schizophrenia.49–51

Many trials have been conducted to understand the role that folate plays in human neurochemistry.31,39,52–55 The function of folate as a methyl donor has led to a focus on the inflammatory marker Hcy, and whether lowering of this amino acid has a ben-eficial effect on depressive symptoms. While folate is vital for neurotransmitter production and mental health, it has become in-creasingly questioned whether FA is the best form of folate,4,20,56,57 and even if it may result in detrimental effects in some individuals which could be due to one or more of the many potential genetic polymorphisms within the folate pathway.58–61 Importantly, there is a need to consider whether folate biochemistry works similarly in different tissues. As the brain is a post-mitotic tissue, it does not synthesize nucleotides, and therefore the role of folate in the central nervous system is different. However, the ability to meas-ure biomarkers accurately in the brain is challenging due to the invasive nature of such testing.

Therefore, the aim of this literature review is to examine hu-man clinical trials to determine what type of folate supplemen-tation have been used, what inflammatory markers have been measured, whether genetic polymorphisms have been explored and their effects, and measures of depressive symptoms. As there are concerns being expressed over the safety of FA sup-plementation and fortification, it appears timely to examine the evidence surrounding FA supplementation in the treatment of depression. In addition, this review will attempt to examine whether there are other mechanisms involved which have not yet been explored.


Barnett H. et al: Folate supplementation and depressionExplor Res Hypothesis Med

Methods

A systematic review of published literature was performed to iden-tify the evidence for folate supplementation to treat depression. A flow chart describing the study selection is presented in Figure 1.

Search strategy

Four electronic databases (PUBMED, Scopus, CINAHL and the Cochrane Library) were searched independently by two authors following the PRISMA 2009 guidelines,62 to identify human clini-cal trials examining the effects of folate (including FA) supple-mentation on the inflammatory markers Hcy, interleukins (ILs), tumor necrosis factor-alpha (TNF-α) and high-sensitivity C-reac-tive protein (hs-CRP) in the treatment or prevention of depression. The database search used keyword terms related to the aim: “fo-lic acid”, “folate”, “Folinic Acid”, “methylfolate”, “5-MTHF” in combination with “homocysteine”, “interleukin*”, “tumour necro-sis factor alpha”, “high sensitive C-reactive protein” AND “mental health” OR “depression”. Two independent authors conducted the searches (H.B. and N.D.), and a manual search of the reference lists of review articles was also performed.

Inclusion criteria

The search included articles that measured depression as a pri-mary or secondary outcome measure. We included studies if they measured Hcy or one and more inflammatory markers and if ge-netic polymorphisms were considered in the analysis. Only peer-reviewed journal articles published in English were included. To assess the recent evidence regarding folate and depression, only randomized, placebo-controlled, human clinical trials conducted between the years of 1991 and 2017 were included. The year 1991 was selected due to the establishment of the role of FA in the pre-vention of NTD.1

Exclusion criteria

Observational studies were excluded due to considerable het-erogeneity, difficulty in identifying the type of folate consumed, confounding due to socioeconomic factors and a reliance on self-reported data. During the search, two trials satisfied the inclusion criteria;63,64 however, they were excluded as the hyperhomocyst-einemia that was being studied was secondary to, or a result of, the disease process, medication and/or treatment for the specific

Fig. 1. PRISMA flow diagram.



disease or condition. Consequently, the results of these trials were not generalizable to the wider population.

Assessment of risk of bias

The risk of bias was independently assessed by two reviewers (H.B. and N.D.) (Table 138,65–73) using the criteria suggested in the Cochrane guidelines.74 The country and source of funding were also considered to illustrate if any bias may have been introduced into the study. Differences in opinion between reviewers were re-solved through discussion until consensus was reached. When con-sensus could not be reached, co-authors (N.N. and E.G.) provided input until a final decision was agreed upon.

Results

Description of studies

In total, 338 studies were identified from the initial electronic database search. Of these, 61 titles were extracted for further analysis of their abstracts, with only 10 satisfying the inclusion criteria (Fig. 1).62 Six trials examined the effect of either FA or L-methylfolate (L-MTHF) as an adjunct to antidepressants in the treatment of depression,65–70 three examined the effect of FA sup-plementation on the prevention of depression as a standalone treat-ment,38,71,72 and one trial included participants who were all on antidepressant medication throughout the trial period (although the antidepressant was not part of the intervention).73 Nine of the trials measured Hcy,38,65–68,70–73 with one measuring hs-CRP and SAMe/SAH ratio.69 No trials measured TNF-α, IL or supplementation with folinic acid. Five trials considered the interaction between folate supplementation and genetic polymorphisms. Two studies analyzed single nucleotide polymorphisms in detail and presented their results,65,69 two made reference to the relevance of genetic polymorphisms in the folate pathway (mainly MTHFR),65,70 and one study excluded participants who were homozygous for the C677T polymorphism on the basis that it impairs folate status.38 A range of outcomes pertaining to depression was found in the included studies to assess depressive symptoms, such as the mini international neuropsychiatric interview (MINI) and the Beck de-pression inventory (BDI). The outcomes measures used in the in-cluded studies are briefly described in Supplementary Information 1.

For this review, the articles were categorized into either: 1. FA/methylfolate as an adjunct in the treatment of depression (descrip-tion of studies provided in Table 265–70,73); 2. FA as a standalone supplement in the prevention or treatment of depression (descrip-tion of studies provided in Table 338,71,72).

Folate (FA and/or L-MTHF) as an adjunct to antidepressants in the treatment of depression

Five included studies65–68,70 examined the effect of FA in enhanc-ing the efficacy of antidepressants in the treatment of depression, while one study examined the use of L-MTHF as an adjunct to antidepressant treatment.69 The study by Loria-Kohen et al.73 did not include the use of antidepressants in the study design or inter-vention. However, it was noted that all study participants were on either an antidepressant, anxiolytic or mood stabilizer. A summary of each of the studies is included in Table 1.

The duration of trials ranged from 6 weeks to 2 years, with sam-ple sizes ranging from 27 to 900 participants. In the FA trials, al-most two-thirds of the 1706 participants were female (n = 1060). In four of the studies,65,68–70 participants were recruited following di-agnosis by the Diagnostic and Statistical Manual of Mental Disor-ders IV (DSM-IV) of Major Depression.75 Two studies examined adults with either depressive symptoms,67 or moderate to severe depression.66 Inclusion into the remaining study was based on a diagnosis of Restrictive Anorexia Nervosa or Eating Disorder Not Otherwise Specified.73 In this study, a food frequency and “3-day food and drink record” also assessed low folate intake. All studies included measurements of either Hcy or hs-CRP.65–70,73 Levels of FA supplementation ranged from 0.4–10 mg/day for FA and 15 mg/day for the L-MTHF trial.

Three studies observed a positive effect of adjunctive FA sup-plementation alongside antidepressants for the treatment of de-pression,68,70,73 while two studies observed no significant differ-ence between placebo and intervention (all, p > 0.05).66,67 The trial by Almeida et al.65 only observed a difference over 52 weeks of treatment, but not over 12 weeks.

The study by Coppen et al.68 found an overall positive effect of 500 µg/day FA supplementation over 10 weeks alongside fluox-etine in the reduction of plasma Hcy concentration (placebo: 9.52 ± 3.22 µmol/L; FA: 8.01 ± 2.23 µmol/L, p < 0.02) and depressive scores (Hamilton depression rating scale (HDRS): 26.8 ± 5.0 de-creasing to 8.1 ± 5.4, p < 0.05). However, when the results were analyzed by sex, the beneficial effect of FA in the reduction of Hcy only extended to females (placebo: 8.56 ± 2.34 µmol/L to 9.36 ± 4.25 µmol/L, p < 0.025; FA: 9.46 ± 3.69 µmol/L to 7.51 ± 1.63 µmol/L) and not males (placebo: 9.92 ± 3.11 µmol/L to 10.21 ± 3.88 µmol/L, p > 0.05; FA: 9.65 ± 2.05 µmol/L to 9.01 ± 2.90 µmol/L, p > 0.05). The same sex relationship was also observed with respect to HDRS in females (placebo: 26.7 ± 4.4 to 11.4 ± 6.9; FA: 27.0 ± 4.8 to 6.8 ± 4.1, p < 0.05) compared with males (pla-cebo: 26.4 ± 5.1 to 9.7 ± 7.9; FA: 26.6 ± 5.3 to 10.9 ± 6.8, p > 0.05). This was the also the only trial that represented its results by sex.

Also providing equivocal results was the trial by Almeida et al.,65 which indicated that FA did not increase the efficacy of an-tidepressants at 12 weeks. For the criteria for major depression, the placebo group (n = 73) had a 78.1% improvement rate, while the FA group (n = 73) improved by 79.4% (between-group p = 0.84). However, after 52 weeks, the FA group (n = 62) responded positively compared with the placebo group (n = 66). In this trial, intervention participants received 2 mg/day FA, 0.5 mg vitamin B12 and 25 mg vitamin B6 alongside 20–40 mg citalopram for 52 weeks. The primary outcome was remission of major depression (as defined by the DSM-IV-TR) and measured by the MINI. The Hcy, red blood cell folate (RCF) and serum vitamin B12 were collected at baseline, and after 12, 26 and 52 weeks. FA supplementation result-ed in an increased RCF (+608.4 nmol/L, 95% confidence interval (CI): 487.8 nmol/L to 729.1 nmol/L; p-value not specified (NS)), and a reduction in Hcy relative to baseline (11.2 µmol/L to 9.1 µmol/L; p = NS). Positive effects of FA supplementation after 52 weeks included reduced rate of relapse amongst those who were no longer depressed by week 12, and greater odds of remission com-pared with placebo for those participants with a baseline Hcy >10.4 µmol/L (odds ratio (OR): 3.47, 95% CI: 1.22–9.84), compared to those with Hcy ≤10.4 µmol/L (OR: 1.09, 95% CI: 0.32–3.75).

The two remaining trials70,73 determined a positive effect of FA, using 10 mg/day of FA supplementation with mostly female partici-pants (>85%). Interestingly, this dose is double the already high dose recommended for women with a high risk of NTD.76 In the study by Loria-Kohen et al.,73 24 patients with an eating disorder, low dietary folate intake and depressive symptomatology (as assessed



Tabl

e 1.

Sum

mar

y of

the

risks

of b

ias f

or in

clud

ed st

udie

s

Bias

Ca

tego

ry

Rand

om

sequ

ence

ge

nera

tion

(sel

ectio

n bi

as)

Allo

catio

n co

ncea

lmen

t (s

elec

tion

bias

)

Blin

ding

of

parti

cipa

nts

and

pers

onne

l (p

erfo

rman

ce b

ias)

Blin

ding

of o

utco

me

asse

ssm

ent

(det

ectio

n bi

as)

(mor

talit

y)

Inco

mpl

ete

data

ad

dres

sed

(att

rition

bia

s)

Sele

ctive

re

porti

ng

(rep

ortin

g bi

as)

Oth

er

bias

Coun

try

Sour

ce o

f fu

ndin

g

Stud

ies o

f fol

ic a

cid

supp

lem

enta

tion

as a

djun

ct to

anti

depr

essa

nts i

n th

e tr

eatm

ent o

f dep

ress

ion

Alm

eida

et

al.65

Low

Low

Low

Low

Low

Low

Low

Aust

ralia

Gove

rnm

enta

l

Beds

on

et a

l.66Lo

wLo

wLo

wLo

wLo

wLo

wLo

wU

nite

d Ki

ngdo

mGo

vern

men

tal

Papa

kost

as

et a

l.69Lo

wLo

wLo

wLo

wLo

wLo

wLo

wU

nite

d St

ates

Phar

mac

euti-

cal i

ndus

try

Resle

r et

al.70

Low

Low

Low

High

Low

Unc

lear

Low

Vene

zuel

aGo

vern

men

tal

Copp

en

et a

l.68Lo

wLo

wLo

wU

ncle

arU

ncle

arU

ncle

arLo

wU

nite

d Ki

ngdo

mPh

arm

aceu

ti-ca

l ind

ustr

y

Loria

-Koh

en

et a

l.73U

ncle

arLo

wLo

wLo

wLo

wLo

wHi

ghSp

ain

Phar

mac

euti-

cal i

ndus

try

Chris

tens

en

et a

l.67Lo

wLo

wLo

wLo

wLo

wLo

wLo

wAu

stra

liaGo

vern

men

tal

Stud

ies o

f fol

ic a

cid

supp

lem

enta

tion

as a

stan

dalo

ne tr

eatm

ent f

or d

epre

ssio

n

De K

onin

g et

al.71

Low

Low

Low

Low

Low

Low

High

Net

herla

nds

Gove

rnm

enta

l/Da

iry in

dust

ry

Oke

reke

et

al.72

Low

Low

Low

Low

Low

Low

High

Uni

ted

Stat

esGo

vern

men

tal/

Insti

tutio

nal

Will

iam

s et

al.38

Low

Low

Low

Low

Low

Low

Low

Nor

ther

n Ire

land

Gove

rnm

enta

l



Tabl

e 2.

Stu

dies

of f

olic

aci

d su

pple

men

tatio

n as

adj

unct

to a

ntide

pres

sant

s in

the

trea

tmen

t of d

epre

ssio

n

Auth

orFo

late

ty

peCo

un-

try

Desi

gnDu

ratio

nSu

bjec

tsIn

terv

entio

nO

utco

me

mea

sure

sRe

sults

Alm

eida

et

al.65

FA 2

mg,

vi

tam

in

B 12 0

.5

mg,

vi

tam

in

B 6 25

mg

Aus-

tral

iaDo

uble

-blin

d,

para

llel p

lace

bo-

cont

rolle

d RC

T

1 y

n =

153

(F =

86,

M

= 67

); Ag

e =

61.7

±

8.2

(pla

cebo

), 63

.4

± 7.

4 (t

reat

men

t)

Plac

ebo:

cita

lopr

am

plus

pla

cebo

.Tr

eatm

ent:

cita

lo-

pram

plu

s 2 m

g FA

, 0.

5 m

g vi

tam

in B

12,

25 m

g vi

tam

in B

6

MIN

I, M

ADRS

.Ho

moc

yst-

eine

, RCF

, and

se

rum

B12

.

No

signi

fican

t diff

eren

ce b

etw

een

grou

ps

at 1

2 w

eeks

, but

anti

depr

essa

nt re

spon

se

was

enh

ance

d an

d su

stai

ned

by a

ddi-

tion

of B

-vita

min

s ove

r 52

wee

ks (O

R:

2.49

, 95%

CI:

1.12

–5.5

1). N

o gr

oup

diffe

r-en

ces i

n M

ADRS

scor

es (p

> 0

.05)

; how

-ev

er, B

-vita

min

supp

lem

enta

tion

redu

ced

risk

of su

bseq

uent

rela

pse

in th

ose

who

ha

d ac

hiev

ed re

miss

ion

of sy

mpt

oms a

t 12

wee

ks (O

R: 0

.33,

95%

CI:

0.12

–0.9

4).

Beds

on

et a

l.66FA

5 m

gU

nite

d Ki

ng-

dom

Doub

le-b

lind,

m

ulti-

cent

er R

CT12

wee

ksn

= 47

5 (F

= 3

04, M

=

171)

; Age

= 4

5 ±

12 y

(pla

cebo

), 45

±

14 y

(tre

atm

ent)

.

Plac

ebo:

any

an

tidep

ress

ant

at a

dequ

ate

dose

&

pla

cebo

.Tr

eatm

ent:

antid

e-pr

essa

nt &

FA 5

mg

BDI-I

I, CG

I, M

ADRS

, UKU

Si

de e

ffect

s sc

ale,

MIN

I, SF

-12,

EQ

-5D.

Seru

m fo

late

, se

rum

B12

, Hcy

Mor

isky

Que

stion

naire

Cl

ient

Ser

vice

Re

ceip

t Q

uesti

onna

ire

No

evid

ence

that

FA w

as e

ffec-

tive

in a

ugm

entin

g an

tidep

ress

ants

in

the

trea

tmen

t of d

epre

ssio

n.

Papa

-ko

stas

et

al.69

L-M

THF

15 m

gU

nite

d St

ates

Doub

le-b

lind,

pl

aceb

o-co

n-tr

olle

d, se

quen

-tia

l par

alle

l RCT

60 d

ays

(pha

se 1

: 30

day

s;

phas

e 2:

30

day

s)

n =

75 (F

= 5

3, M

=

22);

Age

= 45

.4

± 11

.6 y

(pla

cebo

), Ag

e =

49.6

± 1

6.6

y (L

-MTH

F) A

ge

= 50

.86

± 10

.6 y

(p

lace

bo/L

-MTH

F)

3 ar

ms:

1. L

-MTH

F fo

r 60

days

; 2.

plac

ebo

for 3

0 da

ys

follo

wed

by

L-M

THF

for 3

0 da

ys; 3

. pla

-ce

bo fo

r 60

days

.Al

l par

ticip

ants

w

ere

also

on

a st

able

SSR

I dos

e.

HDRS

-28,

HD

RS-7

, CP

FQ, C

GI-S

Hs-C

RP,

4-HN

E,

SAM

e/SA

H

Pool

ed m

ean

chan

ge si

gnifi

cant

ly g

reat

er

with

adj

uncti

ve L

-MTH

F 15

mg/

d co

m-

pare

d w

ith p

lace

bo (p

= 0

.017

).

Resle

r et

al.70

FA 1

0 m

g/da

yVe

n-ez

uela

Doub

le-b

lind,

pla

-ce

bo-c

ontr

olle

d pa

ralle

l RCT

6 w

eeks

n =

27 (F

= 2

3, M

=

4); A

ge =

34.

13

± 2.

05 y

(pla

cebo

), 35

.04

± 2.

63 y

(t

reat

men

t):

3 Ar

ms:

1. 2

0 m

g flu

oxeti

ne +

10

mg/

day

FA; 2

. 20

mg

fluox

etine

+

plac

ebo;

3. c

ontr

ol

HDRS

-17

Plas

ma

fola

te,

seru

m v

itam

in

B 12, H

cy, l

ym-

phoc

yte

con-

cent

ratio

ns o

f 5-

HT, 5

-HIA

A an

d 5-

HT/5

-HI

AA ra

tio

Sero

toni

n sig

nific

antly

redu

ced

in ly

mph

o-cy

tes a

fter fl

uoxe

tine

eith

er w

ith fo

late

(p

= 0.

03) o

r pla

cebo

(p =

0.0

1) d

ue to

blo

ck-

ade

actio

n of

anti

depr

essa

nt. 5

-HIA

A (5

-HT

met

abol

ite) w

as lo

wer

in p

atien

ts re

ceiv

ing

fola

te (p

= 0

.04)

. FA

resu

lted

in h

ighe

r pla

sma

fola

te a

nd lo

wer

hom

ocys

tein

e (p

< 0

.05)

.



Auth

orFo

late

ty

peCo

un-

try

Desi

gnDu

ratio

nSu

bjec

tsIn

terv

entio

nO

utco

me

mea

sure

sRe

sults

Copp

en

et a

l.68FA

0.5

m

g/da

yDo

uble

-blin

d,

plac

ebo-

con-

trol

led

RCT

10 w

eeks

n =

127

(F =

82,

M

= 45

) Age

: 44.

3 ±

14.6

y, (p

lace

bo),

41.9

± 1

2.0

y (t

reat

men

t)

Plac

ebo:

fluo

xetin

e 20

mg

+ pl

aceb

o or

Tre

atm

ent:

fluox

etine

20

mg

+ FA

0.5

mg/

day

HDRS

-17

Seru

m v

itam

in

B 12, H

cy,

plas

ma

fola

te

Bene

fit o

f FA

was

con

fined

to w

omen

on

ly. In

the

GA g

roup

, 93.

9% o

f F sh

owed

go

od re

spon

se (>

50%

redu

ction

in

HDRS

-17

scor

e) c

ompa

red

with

61.

1%

of F

rece

ivin

g pl

aceb

o (p

< 0

.005

).

Loria

-Ko

hen

et a

l.73

FA 1

0 m

g/da

ySp

ain

Doub

le-b

lind,

pa

ralle

l, pl

aceb

o-co

ntro

lled,

RCT

6 m

onth

sn

= 24

(F =

23,

M

= 1)

; Age

: 26.

7 ±

10.0

y (p

lace

bo),

22.3

± 7

.6 y

Plac

ebo

or tr

eat-

men

t con

sistin

g of

2 ×

5 m

g FA

ta

blet

s per

day

.

BDI,

Stro

op

test

, TM

T.Se

rum

&

RCF,

seru

m

vita

min

B12

, Pl

asm

a Hc

y

Trea

tmen

t gro

up si

gnifi

cant

ly in

crea

sed

seru

m a

nd R

CF a

nd d

ecre

ased

Hcy

. Tim

e sp

ent o

n pa

rt B

of T

MT

was

low

er, a

nd th

ere

was

an

incr

ease

d nu

mbe

r of w

ords

read

in

all p

arts

of t

he S

troo

p te

st. B

DI sc

ores

wer

e al

so si

gnifi

cant

ly lo

wer

(all,

p <

0.0

5). B

MI

for t

he tr

eatm

ent g

roup

incr

ease

d fr

om

18.9

± 3

.2 v

s. 2

0.1

± 2.

6 kg

/m2 ;

p <

0.05

.N

o sig

nific

ant c

hang

es in

any

of

the

test

s ass

esse

d fo

r the

pla

cebo

gr

oup

and

BMI d

id n

ot c

hang

e.

Chris

-te

nsen

et

al.67

FA 4

00

mcg

+ v

i-ta

min

B12

10

0 m

cg

Aus-

tral

iaDo

uble

-blin

d,

para

llel,

plac

ebo

cont

rolle

d RC

T w

ith 4

gro

ups a

s a

func

tion

of 2

fa

ctor

s: FA

+ B

12

or P

lace

bo; a

nd

self-

repo

rted

an

tidep

ress

ant

use

(Yes

/No)

2 y

n =

900

(F =

542

, M

= 35

8); A

ge: 6

5.95

±

4.22

y (p

lace

bo

+ an

tidep

ress

ant)

, 65

.97

± 4.

18 y

(pla

-ce

bo),

65.8

2 ±

4.05

(t

reat

men

t + a

nti-

depr

essa

nt),

65.9

5 ±

(tre

atm

ent o

nly)

Plac

ebo:

pla

cebo

ta

blet

s (n

= 10

9 re

-po

rted

anti

depr

es-

sant

use

) OR

Trea

t-m

ent:

initi

ally

1 x

ta

blet

con

tain

ing

400

µg FA

and

100

µg

B12

. Cha

nged

to

2 x

tabl

ets c

onta

in-

ing

200

µg FA

and

50

µg

vita

min

B12

PHQ

-9,

PRIM

E-M

D,

K-10

.Se

rum

vita

min

B 12

, Hcy

, RCF

Onl

y sig

nific

ant e

ffect

was

that

of t

reat

-m

ent a

nd a

ntide

pres

sant

gro

up a

t 24

mon

ths o

n K-

10 sc

ores

(p =

0.0

414)

. For

al

l oth

er m

easu

res,

dep

ress

ion

scor

es

wer

e no

n-sig

nific

ant (

p >

0.05

).

Abbr

evia

tions

: 4-H

NE,

4-h

ydro

xy-2

-non

enal

; 5-H

IAA,

5-h

ydro

zyin

dole

aceti

c ac

id; 5

-HT,

5-h

ydro

xytr

ypta

min

e; B

DI, B

eck’

s Dep

ress

ion

Inve

ntor

y; B

MI,

body

mas

s ind

ex; C

GI, C

linic

al G

loba

l Im

pres

sions

Sca

le; C

PFQ

, Cog

nitiv

e an

d Ph

ysic

al F

uncti

onin

g Q

uesti

onna

ire; E

Q-5

D, E

uroQ

ol F

ive

Dim

ensio

ns Q

uesti

onna

ire; F

A, fo

lic a

cid;

F, fe

mal

e; H

cy, h

omoc

yste

ine;

HDR

S, H

amilt

on D

epre

ssio

n Ra

ting

Scal

e; h

s-CR

P, h

igh-

sens

itivi

ty C

-rea

ctive

pro

tein

; K-1

0, K

es-

sler P

sych

olog

ical

Dist

ress

Sca

le; L

-MTH

F, L-

met

hylfo

late

; M, m

ale;

MAD

RS: M

ontg

omer

y-As

berg

Dep

ress

ion

Scal

e; M

INI,

Min

i Int

erna

tiona

l Neu

rops

ychi

atric

Inte

rvie

w; O

R, o

dds

ratio

; PHQ

-9, P

atien

t Hea

lth Q

uesti

onna

ire-9

; PR

IME-

MD,

Prim

ary

Care

Eva

luati

on o

f Men

tal D

isord

ers;

RCF

, red

blo

od c

ell f

olat

e; R

CT, r

ando

mize

d co

ntro

lled

tria

l; SA

Me/

SAH,

S-a

deno

sylm

ethi

onin

e to

S-a

deno

sylh

omoc

yste

ine

ratio

; SF-

12, S

hort

For

m H

ealth

Sur

vey;

SSR

I, se

lecti

ve se

rato

nin

reup

take

inhi

bito

r; TM

T, T

rail

mak

ing

test

; UKU

, Udv

alg

for K

linisk

e U

nder

soge

lser S

cale

; y, y

ears

.

Tabl

e 2.

Stu

dies

of f

olic

aci

d su

pple

men

tatio

n as

adj

unct

to a

ntide

pres

sant

s in

the

trea

tmen

t of d

epre

ssio

n - (

conti

nued

)



Tabl

e 3.

Stu

dies

of f

olic

aci

d su

pple

men

tatio

n as

a st

anda

lone

trea

tmen

t for

dep

ress

ion

Auth

orFo

late

ty

peCo

un-

try

Desi

gnDu

ra-

tion

Subj

ects

Inte

rven

tion

Out

com

e m

easu

res

Resu

lts

De K

onin

g et

al.71

FA 4

00 µ

g +

vita

min

B 12

500

µg

Net

h-er

-la

nds

Pros

pecti

ve,

para

llel,

plac

ebo

cont

rolle

d,

doub

le-

blin

d RC

T

2 y

n =

2919

(F

= 14

60, M

=

1459

) Age

74.

1 ±

6.5

y (6

9–78

) (b

oth

grou

ps).

Plac

ebo

cont

aini

ng

15 m

cg v

itam

in

D 3 OR

Trea

t-m

ent:

FA 4

00 µ

g,

vita

min

B12

500

µg,

vi

tam

in D

3 15

µg.

GDS-

15, S

F-12

&

EQ-5

D, H

R-Q

oL.

Hcy,

seru

m B

12,

seru

m h

olot

rans

co-

bala

min

, ser

um

met

hylm

alon

ic

acid

, ser

um fo

late

No

effec

t of F

A su

pple

men

tatio

n on

dep

res-

sive

sym

ptom

s in

eith

er g

roup

(OR:

1.1

3, 9

5%

CI: 0

.83–

1.53

; p =

0.4

5). I

n su

bsam

ple

that

had

de

pres

sion

at b

asel

ine,

FA su

pple

men

tatio

n di

d no

t hav

e a

signi

fican

t effe

ct o

n de

pres

sive

sym

ptom

s (p

= 0.

55).

No

asso

ciati

on fo

und

betw

een

a re

ducti

on in

Hcy

and

dep

ress

ive

sym

ptom

s. E

Q-5

D de

clin

ed le

ss in

the

trea

tmen

t gr

oup

sugg

estin

g ro

le o

f B-v

itam

ins i

n lo

wer

ing

hom

ocys

tein

e m

ay sl

ight

ly in

crea

se H

R-Q

oL.

Oke

reke

et

al.72

FA 2

.5 m

g,

vita

min

B 6 5

0 m

g,

vita

min

B 12

1 m

g

Uni

ted

Stat

esDo

uble

-bl

ind,

pl

aceb

o-co

ntro

lled

para

llel R

CT

7.3

yn

= 43

31 (a

ll,

F); A

ge: 6

3.6

yPl

aceb

o O

R Tr

eat-

men

t: FA

(2.5

m

g/da

y), v

itam

in

B 6 (50

mg/

day)

an

d vi

tam

in B

12

(1 m

g/da

y)

Self-

repo

rted

phy

si-ci

an/c

linic

ian

diag

-no

sed

depr

essio

n,

or se

lf-re

port

ed d

e-pr

essiv

e sy

mpt

oms

base

d on

the

Men

-ta

l Hea

lth In

dex.

Subg

roup

ana

lysis

of

pla

sma

fola

te

and

hom

ocys

tein

e.

No

diffe

renc

e be

twee

n gr

oups

in d

epre

s-sio

n ov

er 7

y (R

R: 1

.02,

95%

CI:

0.86

–1.2

1; p

=

0.81

) des

pite

sign

ifica

nt re

ducti

on in

Hcy

.Si

mila

rly, n

o di

ffere

nces

in e

ffect

of F

A/B-

vita

min

s on

dep

ress

ion

risk

acco

rdin

g to

age

, or a

cros

s su

b-gr

oups

(i.e

. pre

viou

s tre

atm

ents

gro

ups i

n W

ACS)

, nor

did

it re

duce

the

risk

of la

te-li

fe d

e-pr

essio

n am

ong

parti

cipa

nts ≥

65 y

rs (a

ll, p

> 0

.05)

.

Will

iam

s et

al.38

FA 1

00

µg O

R FA

20

0 µg

Nor

th-

ern

Irela

nd

Doub

le-

blin

d Pl

aceb

o co

ntro

lled

RCT

12

wee

ksn

= 23

(all,

M);

Age:

32

(21–

39)

Plac

ebo

OR

Trea

t-m

ent:

FA 1

00

µg fo

r 6 w

eeks

fo

llow

ed b

y FA

200

µg

for 6

wee

ks

PAN

ASW

hole

blo

od

5-HT

, ser

um &

RC

F, pl

asm

a Hc

y

Seru

m a

nd R

CF in

crea

sed

and

Hcy

decr

ease

d in

supp

lem

ente

d gr

oup;

how

ever

, the

re w

ere

no d

iffer

ence

s in

who

le b

lood

5-H

T le

vels

or su

bjec

tive

moo

d be

twee

n th

e gr

oups

.

Abbr

evia

tions

: 5-H

T, 5

-hyd

roxy

tryp

tam

ine;

CI,

confi

denc

e in

terv

al; E

Q-5

D, E

uroQ

ol F

ive

Dim

ensio

ns Q

uesti

onna

ire; F

, fem

ale;

FA,

folic

aci

d; G

DS, G

eria

tric

Dep

ress

ion

Scal

e; H

cy, h

omoc

yste

ine;

HR-

QoL

, Hea

lth-R

elat

ed Q

ualit

y of

Life

; M, m

ale;

OR,

odd

s rati

o; R

CF, r

ed b

lood

cel

l fol

ate;

RCT

, ran

dom

ized

cont

rolle

d tr

ial;

RR, r

elati

ve ri

sk; S

F-12

, Sho

rt F

orm

Hea

lth S

urve

y; W

ACS,

Wom

en’s

Antio

xida

nt C

ardi

ovas

cula

r Stu

dy; y

: yea

rs.



by the BDI) were randomized to either placebo or an intervention group supplementing with 10 mg/day of FA for 6 months. Although antidepressant medication was not part of the intervention, 57.1% of the intervention group were taking antidepressants, 42.9% were tak-ing anxiolytics, and 7% used mood stabilizers. In the placebo group, 70% were taking antidepressants, and 30% were taking anxiolytics. The main outcome measures were depressive symptomatology us-ing the BDI and cognitive status using the Stroop and trail making tests. Data on serum folate, RCF, Hcy and serum vitamin B12 were also collected. Results indicated that RCF increased in the interven-tion group (634.3 ± 300.0 ng/mL to 1521.7 ± 167.0 ng/mL) com-pared with the placebo group (844.4 ± 285.4 ng/mL to 945.0 ± 347.0 ng/mL; p < 0.0001). However, plasma Hcy concentrations in both groups decreased (placebo: 10.0 ± 2.05 µmol/L to 8.0 ± 1.8 µmol/L; FA: 9.4 ± 2.4 µmol/L to 7.5 ± 1.7 µmol/L). However, interestingly, only the reduction of Hcy in the supplemented group was reported to be significant (p < 0.01). Depression scores (as measured by the BDI) decreased significantly in the FA (placebo: 17.3 ± 12.1 to 13.4 ± 11.8; FA: 22.9 ± 8.1 to 15.2 ± 9.9; p < 0.05).

The study by Resler et al.70 explored the effect of 6 weeks of 10 mg/day FA supplementation in combination with fluoxetine on plasma Hcy and serotonin levels in lymphocytes. Primary outcome measures were a reduction in depressive symptoms measured by the HDRS-17, plasma folate, Hcy, serum vitamin B12, serotonin and 5-hydroxyindoleacetic acid (5-HIAA). Results indicated that plasma folate increased significantly in the supplementation group (9.22 ± 1.97 nM to 47.81 ± 6.66 nM) compared with the placebo group (9.10 ± 1.66 nM to 11.61 ± 3.53 nM; p = 0.0005), while plasma Hcy decreased significantly in the supplementation group from baseline (9.49 ± 0.7 pM to 7.35 ± 0.61 pM; p = 0.02) (placebo group values were not reported). Mean HDRS scores were reduced from 22.50 ± 0.98 to 7.43 ± 1.65 in the FA group compared with 21.85 ± 0.94 to 11.43 ± 1.31 in the placebo group (p = 0.04). As expected, the serotonin concentration was reduced in lymphocytes due to the administration of fluoxetine and did not differ between the FA group (p = 0.03) or placebo group (p = 0.01). The main difference, however, was that in the FA group, 5-HIAA was signifi-cantly decreased (p = 0.04).

The trials by Bedson et al.66 and Christensen et al.67 reported no benefit from the adjunctive use of 5 mg/day of FA for 12 weeks, and 0.4 mg/day of FA with 0.1 mg/day of vitamin B12 for 24 months, respectively. In the study by Bedson et al.,66 475 participants were included initially (females, 304; males, 171) with the outcome measure, assessed by the BDI-II at 25 weeks, showing no evidence that FA was more effective than the placebo (OR: 1.09; 95% CI: 0.75–1.59; p = 0.65). All other outcome measures showed no signif-icant difference between FA and placebo (p > 0.05), except for the SF-12 mental component for the placebo group (p = 0.017). Simi-larly, the trial by Christensen et al.67 (n = 900), in which 209 report-ed antidepressant use during follow-up, reported no clear evidence that FA enhanced the efficacy of antidepressants. Primary outcome measures were depressive symptoms measured by the PHQ-9 and PRIME-MD, and serum B12, RCF and Hcy. Results showed that there was no significant interaction between antidepressant use on depression as measured by PHQ-9 and K-10 (p = 0.868). However, there was a significant interaction effect between antidepressant use, FA and time, but only at 24 months (F4 799.5 = 2.50, p = 0.041). The K-10 scores were lower in the FA with vitamin B12 group at 24 months than the placebo group (t789 = −2.24, p = 0.025; 95% CI: −3.68 to −0.24). The FA supplementation increased RCF levels in the supplemented group from 573 ± 266 nmol/L to 1019 ± 410 nmol/L at 12 months and to 951 ± 423 nmol/L at 24 months (F2 729.0 = 75.9, p < 0.0001). This was in comparison to the placebo group that only had a slight increase from 557 ± 277 nmol/L to 616 ± 360

nmol/L at 12 months and 568 ± 326 nmol/L at 24 months. The Hcy increased significantly (p < 0.0001) in the placebo group (9.8 ± 2.8 µmol/L to 11.6 ± 2.7 µmol/L at 12 months and 12.0 ± 2.8 µmol/L at 24 months) compared with the supplementation group (9.6 ± 2.6 µmol/L to 9.8 ± 2.4 µmol/L at 12 months to 10.4 ± 4.5 µmol/L at 24 months). These results showed that, within the FA group, Hcy levels increased significantly more in those taking antidepressants compared with those not taking antidepressants (p = 0.021).

One study69 determined the effect of adjunctive L-MTHF sup-plementation in the treatment of major depression amongst patients who had previously failed to adequately respond to selective sera-tonin reuptake inhibitors. In this study, participants were stratified according to baseline body mass index (BMI), levels of plasma hs-CRP, 4-hydroxy-2-nonenal (4-HNE), SAMe/SAH ratio, and various genetic polymorphisms. Overall results indicated that ad-junctive treatment with 15 mg/day of L-MTHF resulted in a greater mean change on the HDRS than placebo (−6.8 ± 7.2 vs. −3.7 ± 6.5; p = 0.017). When the results were further analyzed by subgroups, there were significant changes (all, p < 0.05). Firstly, patients with a baseline plasma SAMe/SAH ratio below the study median value, hs-CRP or 4-HNE blood levels above the study median value or a BMI ≥30 kg/m2, had a greater mean change on the HDRS-28 in the L-MTHF group compared with placebo (p ≤ 0.05). This significant effect of L-MTHF on HDRS scores was also observed for most genetic markers including COMT GG (p < 0.001) COMT CC (p < 0.001), MTR AG/GG (p = 0.001) and RFC1 AA (p = 0.003) and for most combinations of both biological and genetic markers and different genetic markers. These included the following: MTHFR 677 CT/TT and MTR 2756 AG/GG (p < 0.001); BMI ≥30 kg/m2 and MTR 2756 AG/GG (p < 0.001); DNAMT3B AG/AA and MTR 2756 AG/GG (p < 0.001), MTHFR 677 CT/TT with BMI ≥30 kg/m2 (p < 0.001). This highlights that folate metabolism is influenced by individual metabolic and genetic factors, which in turn could identify people both at risk of major depressive disorder and those who may not respond adequately to antidepressant treatment.

FA supplementation as standalone therapy in prevention of de-pression

Three trials38,71,72 examined the effect of FA supplementation as a standalone treatment in the prevention of depression, and all three studies found no difference in depressive symptoms between the groups. A summary of each of the studies is included in Table 2. In the trial by De Koning et al.,71 2919 participants (mean age, 74.1 ± 6.5) were randomized to receive either a placebo containing 15 µg of vitamin D3, or the intervention tablet containing 40 µg of FA, 500 µg vitamin B12 and 15 µg of vitamin D3 per day for 2 years. While the primary outcome measure was assessment of the impact of this supplement regime on bone fracture risk, a second-ary outcome measure was depressive symptoms as measured by the HDRS-17. Participants were included in this study if they had elevated Hcy concentrations (range: 12–50 µmol/L), with the mean baseline concentrations being 14.3 µmol/L in the intervention group and 14.5 µmol/L in the placebo group. The aim of the study was to determine whether the FA and vitamin B12 supplementation would decrease Hcy levels and, in turn, have an impact on depressive symptoms. The Hcy did indeed decrease significantly more in the supplemented group compared to placebo over the 2-year interven-tion (placebo: −0.2 ± 4.1 µmol/L; intervention: −4.4 ± 3.3 µmol/L; p < 0.001). However, the rate of depressive symptoms between the groups did not differ (OR: 1.13, 95% CI: 0.83–1.53; p = 0.45).

In a similarly large trial (n = 4331), Okereke et al.72 studied the effect of supplementing 2.5 mg FA, 50 mg vitamin B6 and 1 mg



vitamin B12 for 7.3 years in older women (mean age, 63.6 ± 8.7 years). As with the De Koning study,71 depression was a secondary outcome (CVD was the primary outcome), and it was determined using the Mental Health Inventory. Despite a reduction in Hcy lev-els in the supplementation group, there was no significant effect on depressive symptoms in comparison to placebo (relative risk: 1.02; 95% CI: 0.86–1.21; p = 0.81). The findings did not change when analyzed according to age (i.e. rates of depression varied for <65 years compared to >65 years) and there was no impact of B-vitamins on depression risk according to age (all, p > 0.05).

The only trial to examine the effect of FA supplementation on mood in non-depressed, otherwise healthy individuals, was by Williams et al.38 in 2005. This small trial randomized 28 males (mean age, 32 years) to receive either placebo or intervention of 100 µg FA for 6 weeks followed by 200 µg FA for 6 weeks. Sub-jective mood was measured and inferred using the Positive and Negative Affect Score, and biochemical markers included RCF, serum folate, Hcy and whole blood serotonin. Although this was a folate-replete sample, the participants had Hcy levels within the normal range. Following supplementation, even the relative low dose (100–200 µg/day) significantly increased serum folate levels at 100 µg/day (p = 0.043) and 200 µg/day (p = 0.024). Hcy also de-creased at both 100 µg/day (p = 0.032) and 200 µg/day (p = 0.015) supplementation levels. However, neither subjective mood (all, p > 0.05) nor whole blood serotonin (p = 0.816) differed between the two groups post-intervention.

We did not attempt to meta-analyze the data due to the wide va-riety of depression outcomes measured, the differences in the dos-es given for each intervention, the inclusion of vitamins in some of the trials, and the considerable heterogeneity observed between the cohorts studied.

Discussion

The majority of the ten studies included were human clinical trials that explored the relationship between Hcy, folate and depression scores at baseline, and whether FA supplementation improved de-pressive symptoms. Only the trial of L-MTHF explored the inter-play of genetic polymorphisms and underlying inflammatory bio-markers. The overall findings suggest that there is little evidence to support the use of FA as an adjunct to antidepressants in the treat-ment of depression, or as a standalone therapy in the prevention of depression. There may be some benefit in the use of L-MTHF. However, as there was only one trial of L-MTHF included in this review, with a relatively small number of participants (n = 75),69 further research is needed to guide recommendations for this form of folate. Similarly, there was significant heterogeneity amongst the trials within this review, making it difficult to elucidate a clear effect or benefit of FA on either Hcy or depression.

Although it was observed in four trials65,68,70,73 that FA supple-mentation lowered Hcy, the effect on depressive symptoms was somewhat equivocal. It should be noted that these trials were of a relatively small size (range, 24–153), recruited predominantly females (65%), and apart from the trial by Almeida et al.,65 were relatively short in duration. Interestingly, the trial by Coppen et al.68 is the earliest published trial in this review, and it is the only trial where data was analyzed based on differences in sex. While the authors found an overall positive effect for FA on both Hcy and depression, when analyzed by sex, they found this effect only extended to females on both measures. The authors noted that the males had a smaller increase in plasma folate compared with the females (males, 4.58 ± 1.68 ng/mL to 8.70 ± 3.50 ng/mL, p

< 0.001; females, 4.04 ± 1.64 ng/mL to 12.70 ± 1.65 ng/mL, p < 0.001) and this may have been insufficient to illicit a decrease in plasma Hcy. Further, they argued that the failure to decrease Hcy in males may explain why they did not improve their depressive symptoms as measured by the HDRS.

The study by Almeida et al.65 did not analyze data by sex, despite being of a much longer duration (52 weeks) and using a higher dose of FA (2 mg). Moreover, the trial also contained a larger proportion of males (44%) and was conducted in an older population (treatment, mean age: 61.7 ± 8.2 years); placebo, mean age: 63.4 ± 7.4 years). This is an important consideration as Hcy is known to increase with age.77,78 In contrast, the trials by Resler et al.70 and Loria-Kohen et al.73 contained mostly female participants (>85%), with an average age of 24.2 years and 35.04 years respec-tively, and supplemented with 10 mg/day of FA. Although Resler et al.70 reported a lowering of Hcy in the supplemented group, this was not associated with a change in clinical symptoms. Rather, in this trial, a reduction in 5-HIAA was noted in patients receiving FA and it was postulated that the main effect of FA was due to modi-fication of the serotonergic system in lymphocytes. Loria-Kohen et al.73 also reported a lowering of Hcy; however, the results show that Hcy decreased comparably in both intervention and placebo (intervention: 9.4 ± 2.4 µmol/L to 7.5 ± 1.7 µmol/L; placebo: 10.0 ± 2.05 µmol/L to 8.0 ± 1.8 µmol/L). The authors also reported this result as a significant reduction for the intervention group (p < 0.01) but not in the placebo group. In addition, the baseline RCF (intervention: 634.3 ± 300.0 ng/mL vs. placebo: 844.4 ± 285.4 ng/mL) and vitamin B12 levels (intervention: 562.6 ± 209.5 pg/mL vs. placebo: 782.0 ± 387.0 pg/mL) were higher pre-intervention in the placebo group compared to the supplemented group. Based on these results, it is difficult to attribute the reduction in depressive symptoms to the lowering of Hcy, while the discrepancy in base-line biochemical status may be confounding the results.

Of the five remaining trials, there was no evidence that FA en-hanced the effect of antidepressants or prevented depression as a standalone treatment.38,66,67,71,72 Four were large trials (range, 475–4331) and three had a duration of 2 years or more,66,67,71,72 while dosage ranged from 0.1–5 mg/day. Overall, these trials found there was no correlation between a reduction in Hcy and de-pressive symptoms. Interestingly, in contrast to the study by Cop-pen et al.,68 the trial by Okereke et al.72 found that a reduction in Hcy did not reduce depressive symptoms in women in comparison with placebo. This was a large trial with only female participants (n = 4331), used a relatively high FA dosage (2.5 mg/day), and was conducted for 7.3 years; therefore, the findings suggested that the conclusion reached by Coppen et al.68 may have been confounded by other factors. Similarly, in two other relatively long term trials (2 years) with large cohorts, Christensen et al.67 and De Koning et al.71 found no relationship between a reduction in Hcy and depres-sion scores. Subsequent analysis of the participants’ DNA revealed altered methylation patterns in the FA and vitamin B12 group ver-sus the placebo group,14 further supporting the impact of long-term FA exposure on the epigenome and thus gene expression, not just during vital developmental periods but into older age as well.

Only one included trial examined the use of L-MTHF (15 mg/day) and the relevance of biomarkers other than Hcy (hs-CRP, SAMe/SAH ratio, 4-HNE),69 and the effect of genetic markers on treatment response in comparison to placebo. As L-MTHF is the form of folate predominantly found in food,17 it could be argued that it makes more sense to examine how this naturally occur-ring form interacts with human biochemistry, neurochemistry and genetics. In this trial, L-MTHF was significantly more effective overall than placebo in reducing depression as measured by the HDRS-28. The results became more interesting when they were



analyzed according to subgroup, which showed that L-MTHF was more effective in patients with elevated hs-CRP or 4-HNE, a BMI >30 kg/m2, or a plasma SAMe/SAH ratio below the study mean. Similarly, patients with the MTR 2756 AG/GG or MTRR 66 AG/GG genotypes, compared with the homozygous dominant al-leles, had a significantly greater HDRS-28 response on L-MTHF compared with placebo. However, this was not observed for the MTHFR 677CT/TT or MTHFR 1298 AC/CC genotypes. This ob-servation is interesting given that research has been conducted to identify the potential role of MTHFR in altered folate metabo-lism.50,51,60,79–81 Similarly, the measurement of SAMe/SAH is in-teresting as this is an indicator of methylation status, with a low SAMe/SAH ratio indicating impaired methylation. Although the trial was only 60 days in duration, and had only 75 participants, this study served to highlight the fundamental importance of ge-netics in folate metabolism.

No relevant randomized controlled trials were located for this review examining the efficacy of folinic acid in the treatment or prevention of depression. Future randomized controlled trials investigating folinic acid and depressive symptoms would be of interest to evaluate in this population. Folinic acid is a natural me-tabolite of THF; therefore, its pathway through the folate cycle is regulated by homeostatic processes.17 This contrasts with L-MTHF supplementation, which may lead to risk of B12 deficiency if con-sumed at supraphysiological doses.17,82 Similarly, this review did not examine trials in individuals with autism, schizophrenia or bi-polar depression due to the more complex neurochemical process-es and different pathophysiology of these disorders. However, it is acknowledged that the role of folate and FA in these disorders has caused similar controversy and warrants further research that may provide insight into the treatment of depressive symptoms.83–87

Future research directions

Historically, most of the research has focused on exploring whether lowering Hcy with FA supplementation reduces depression symp-toms or risk of depression. However, findings of this review suggest that the link between Hcy and depression is tenuous. The trial by Papakostas et al.69 disbanded examination of Hcy in favor of ex-ploring the interplay of genetic polymorphisms, inflammatory bio-markers (i.e. hs-CRP and 4-HNE) and methylation status (SAMe/SAH ratio).88 To our knowledge, this is the only human randomized controlled trial using L-MTHF in individuals with depression.

Further research into the use of L-MTHF as an alternative to FA is clearly needed, as L-MTHF is both the naturally occurring and the biologically active form of folate.17 While Papakostas et al.69 demonstrated a differential effect of 5-MTHF based on numerous genetic and biological markers, it would be pertinent to explore if these same markers would be indicative of an impaired or similarly differential response with FA treatment. This trial also highlights that further research is needed into the genetic polymorphisms that impact upon folate metabolism, and how in turn they may impact upon depression risk or symptoms.

Research into the differential metabolism of FA compared to 5-MTHF in relation to its impact on depression is also needed. DHF is the intermediate metabolite in FA metabolism and acts to inhibit key enzymes in the folate cycle, while slowing down the biotransformation of FA to its active forms.20 The possible con-sequences may be that non-metabolized FA in the plasma is left to compete with biologically active forms for transport into the central nervous system, and that intracellular folate metabolism is impaired by the FA intermediates.4,21 Moreover, the MTHFR gene

has been extensively studied due to the observation that carriers of the C677T variant have reduced enzyme function, resulting in both increased levels of Hcy and an increased risk of depression.89,90 Therefore, it is hypothesized (Fig. 2) that supplemental FA is in-hibiting MTHFR by DHF, thus impairing folate metabolism fur-ther in people with the MTHFR C677T mutation and resulting in a functional deficiency of folate.

Perhaps an important point to consider is that the fundamen-tal role of folate is the donation and de novo synthesis of methyl groups. While both 5-MTHF and FA are capable of de novo syn-thesis, only 5-MTHF contributes new methyl groups to the human system. Consequently, research that adequately compares the bio-logical functioning of the natural versus synthetic forms of folate, and the impact on depression symptoms and risk of developing depression, would be extremely valuable.

Furthermore, exploring how existing FA supplementation and fortification may be affecting genetic expression, and in turn whether FA is increasing the risk, incidence and prevalence of de-pression is needed. There is increasing evidence that folate plays a fundamental role in the epigenetic modification of the human genome, with methylation in particular serving to either silence or promote the transcription of genes. While impaired folate metabo-lism caused by FA supplementation can result in hypomethyla-tion, it is unknown what impact un-metabolized FA has on human health and disease. If it can be demonstrated that FA is altering methylation patterns in genetically-susceptible individuals, then the implications for depression and many modern noncommunica-ble diseases could be quite profound.

Finally, increases in latitude are associated with an increased frequency of the MTHFR-C677T and MTHFR-A1298C polymor-phisms, suggesting a link between latitude and depression in the context of folate intake is worthy of further investigation.91

Conclusions

The biochemistry of folate and FA is both complex and fascinating. Folate plays an integral role at our most fundamental level, that of controlling and modifying genetic expression. The findings of this review suggest that there is limited evidence to suggest ben-efits to folate supplementation in the treatment or management of depression. However, further research investigating the benefits of L-MTHF and folinic acid supplementation in individuals with depression is warranted. In addition, methylation status and the in-teraction of various genetic polymorphisms associated with folate metabolism may influence the efficacy of folate supplementation.

Depressive symptoms can be debilitating and many of the phar-macological agents employed to treat them can have significant side effects. The challenge of the human condition is that we desire to find an answer in a capsule rather than consuming a dietary pat-tern based on minimally processed foods such as green leafy veg-etables. Future research should assess the potential for these sup-plements to cause epigenetic changes to our DNA in individuals with certain genetic polymorphisms that may be heritable across future generations. Therefore, it is worth questioning whether the current supplementation of FA is appropriate, and whether other forms of folate, such as L-MTHF or folinic acid, may prove to be safer alternatives.


The authors have no conflict of interest related to this publication.




Conducted initial searches and formulated the first draft of the man-uscript (HB, NMD), critically evaluated the papers, search proce-dures and assisted with the risk of bias assessment (ENG), critically reviewed the paper and provided the input into the study design and assisted with analysis and interpretation of studies (JK, DM, AM); designed the study, monitored the study design, all protocols and supervised the students involved in this paper (NN, HB, NMD); All authors included contributed significantly to the final manuscript.

Supporting information

Supplementary material for this article is available at https://doi.org/10.14218/ERHM.2017.00025.

Supplementary information 1. Measures of depressive symptoms.

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Fig. 2. Relationship between folate, methylation and neurotransmitters involved with depression. It is hypothesized that consumption of FA may result in decreased neurotransmitter production due to decreased availability of biologically active folate 5-MTHF and a resulting decrease in SAMe. Moreover, the addition of FA into a finely balanced yet complicated cycle in susceptible individuals (e.g., those with MTHFR, MTR, MTRR and COMT polymorphisms) may serve to greatly slow the cycle via the inhibition of MTHFR by DHF. As the population continues to consume less folate from food sources such as leafy green vegetables, then it is postulated that the rates and severity of depression will increase as 5-MTHF decreases. Abbreviations: DHF, dihydrofolate; FA, folic acid; 5-MTHF, 5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; SAMe, S-adenosylmethionine.

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[71] de Koning EJ, van der Zwaluw NL, van Wijngaarden JP, Sohl E, Brou-wer-Brolsma EM, van Marwijk HW, et al. Effects of two-year vitamin B12 and folic acid supplementation on depressive symptoms and quality of life in older adults with elevated homocysteine concentra-tions: additional results from the B-PROOF study, an RCT. Nutrients 2016;8(11):E748. doi:10.3390/nu8110748.

[72] Okereke OI, Cook NR, Albert CM, Van Denburgh M, Buring JE, Man-son JE. Effect of long-term supplementation with folic acid and B vitamins on risk of depression in older women. Br J Psychiatry 2015;206(4):324–331. doi:10.1192/bjp.bp.114.148361.

[73] Loria-Kohen V, Gomez-Candela C, Palma-Milla S, Amador-Sastre B, Hernanz A, Bermejo LM. A pilot study of folic acid supplementation for improving homocysteine levels, cognitive and depressive status in eating disorders. Nutr Hosp 2013;28(3):807–815. doi:10.3305/nh.2013.28.3.6335.

[74] Shuster JJ. Cochrane handbook for systematic reviews of interven-tions Version 5.1.0 [updated March 2011]. Higgins JPT, Green S, Edi-tors. Research Synthesis Methods. 2011;2(2):126–130.

[75] American Psychiatric Association. Diagnostic and statistical manual of mental disorders DSM-IV-TR. Washington, DC: American Psychi-atric Association, 2000. Available from: http://dsm.psychiatryonline.org/dsmPreviousEditions.

[76] World Health Organization. Periconceptional folic acid supplementa-tion to prevent neural tube defects 2017 [updated 9 January 2017]. Available from: http://www.who.int/elena/titles/folate_periconcep-tional/en/.

[77] Wald DS, Wald NJ, Morris JK, Law M. Folic acid, homocysteine, and cardiovascular disease: judging causality in the face of inconclu-sive trial evidence. BMJ 2006;333(7578):1114–1117. doi:10.1136/bmj.39000.486701.68.

[78] van Wijngaarden JP, Doets EL, Szczecinska A, Souverein OW, Duffy ME, Dullemeijer C, et al. Vitamin B12, folate, homocyst-eine, and bone health in adults and elderly people: a system-atic review with meta-analyses. J Nutr Metab 2013;2013:486186. doi:10.1155/2013/486186.

[79] Narayanan S, McConnell J, Little J, Sharp L, Piyathilake CJ, Powers H, et al. Associations between two common variants C677T and A1298C in the methylenetetrahydrofolate reductase gene and measures of folate metabolism and DNA stability (strand breaks, misincorporated uracil, and DNA methylation status) in human lymphocytes in vivo. Cancer Epidemiol Biomarkers Prev 2004;13(9):1436–1443.

[80] Jiang W, Xu J, Lu XJ, Sun Y. Association between MTHFR C677T poly-morphism and depression: a meta-analysis in the Chinese popula-tion. Psychol Health Med 2016;21(6):675–685. doi:10.1080/13548506.2015.1120327.

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[82] Smulders YM, Smith DE, Kok RM, Teerlink T, Swinkels DW, Stehou-wer CD, et al. Cellular folate vitamer distribution during and after correction of vitamin B12 deficiency: a case for the methylfolate trap. Br J Haematol 2006;132(5):623–629. doi:10.1111/j.1365-2141.2005.05913.x.

[83] Wang M, Li K, Zhao D, Li L. The association between maternal use of folic acid supplements during pregnancy and risk of autism spec-trum disorders in children: a meta-analysis. Mol Autism 2017;8:51. doi:10.1186/s13229-017-0170-8.

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[84] Roffman JL, Petruzzi LJ, Tanner AS, Brown HE, Eryilmaz H, Ho NF, et al. Biochemical, physiological and clinical effects of l-methylfolate in schizophrenia: a randomized controlled trial. Mol Psychiatry 2017. doi:10.1038/mp.2017.41.

[85] Wang D, Zhai JX, Liu DW. Serum folate levels in schizophrenia: A meta-analysis. Psychiatry Res 2016;235:83–89. doi:10.1016/j.psy-chres.2015.11.045.

[86] Ghanizadeh A, Singh AB, Berk M, Torabi-Nami M. Homocysteine as a potential biomarker in bipolar disorders: a critical review and sugges-tions for improved studies. Expert Opin Ther Targets 2015;19(7):927–939. doi:10.1517/14728222.2015.1019866.

[87] Mitchell ES, Conus N, Kaput J. B vitamin polymorphisms and behavior: evidence of associations with neurodevelopment, depression, schiz-ophrenia, bipolar disorder and cognitive decline. Neurosci Biobehav Rev 2014;47:307–320. doi:10.1016/j.neubiorev.2014.08.006.

[88] Melnyk S, Pogribna M, Pogribny IP, Yi P, James SJ. Measurement of

plasma and intracellular S-adenosylmethionine and S-adenosylho-mocysteine utilizing coulometric electrochemical detection: altera-tions with plasma homocysteine and pyridoxal 5′-phosphate concen-trations. Clin Chem 2000;46(2):265–272.

[89] Reif A, Pfuhlmann B, Lesch KP. Homocysteinemia as well as meth-ylenetetrahydrofolate reductase polymorphism are associated with affective psychoses. Prog Neuropsychopharmacol Biol Psychiatry 2005;29(7):1162–1168. doi:10.1016/j.pnpbp.2005.06.027.

[90] Liang S, Zhou Y, Wang H, Qian Y, Ma D, Tian W, et al. The effect of mul-tiple single nucleotide polymorphisms in the folic acid pathway genes on homocysteine metabolism. Biomed Res Int 2014;2014:560183. doi:10.1155/2014/560183.

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Review Article

Introduction

Exclusive enteral nutrition (EEN) is a nutritional therapy used for treatment of Crohn’s disease (CD), particularly in children. In practical terms, it requires stopping all usual foods and substitut-ing an exclusive liquid low-residue feed, using either a polymeric or an elemental formula as the sole source of nutrients for 6 to 8 weeks. Historically, use of EEN aimed at improving nutritional status of patients with inflammatory bowel disease (IBD), espe-cially in those who were not amenable to surgical treatment. The observed reduction in symptoms with EEN was thought to be sec-

ondary to improvements in nutritional status. In the 1970’s, a group of surgeons first reported efficacy of EEN in CD, when they treated 13 patients with elemental diet and showed a significant decrease in the inflammatory markers in a majority, along with nutritional improvement1; at that time, steroids continued to be the mainstay of treatment. In 1982, Navarro et al.2 was able to show that EEN in pediatric CD induced remission and decreased steroid dependency. In addition, prolonged EEN (from two to seven months) was able to reduce or resolve stenotic bowel disease.2 O’Morain published the first controlled trial in 21 adults in 1987,3 wherein an elemen-tal diet given for four weeks was compared with oral steroids and showed that the efficacy of EEN in inducing remission was similar to that of treatment with corticosteroids.

Rigaud et al.4 (1991) conducted a prospective randomized clini-cal trial (RCT) with elemental or polymeric diet for four to six weeks in 30 steroid-unresponsive CD patients and showed signifi-cant improvement in mucosal lesions seen at colonoscopy, inflam-matory markers and nutritional state on follow-up; however, the majority of patients suffered relapse within a year. In a double-blind RCT, Royall et al.5 compared amino acid with peptide-based formula in adults with CD over 3 weeks and showed that the rate of clinical remission in the two groups was similar. In 1997, Zoli et al.6 published a RCT showing that, in adult CD, EEN was as effective as steroids in initiating clinical remission. These authors hypothesized that the mechanism of action was probably second-ary to the effects of EEN in normalizing intestinal permeability.6 A multicenter RCT, wherein 33 children with CD were rand-

Dietary Treatment for Crohn’s Disease—Old Therapy, New Insights

Rakesh Vora* and John W.L. Puntis

Department of Paediatric Gastroenterology and Nutrition, Leeds Children’s Hospital, UK

Abstract

Crohn’s disease in childhood accounts for about 25% of the overall prevalence of this condition, and compared with adult-onset disease has unique characteristics in being more likely to involve the colon, being more aggres-sive in behavior, and requiring early escalation of therapy. Exclusive enteral nutrition has proven to be an effec-tive therapy, especially in children. There are various hypotheses regarding mode of action; however, the precise mechanisms are yet to be established. The aim of this paper is to provide an up-to-date review of the efficacy and mechanism of action of exclusive enteral nutrition in Crohn’s disease. A PubMed search was performed using the terms ‘mechanism of action’, ‘exclusive enteral nutrition’, ’partial enteral nutrition’, ‘nutritional therapy’, ‘chil-dren’, ‘paediatric’, ‘Crohn’s disease’. Relevant articles were selected from this search. In addition, the reference lists of these papers were scrutinized for further relevant publications. There is significant evidence for efficacy of exclusive enteral nutrition and some evidence for a number of mechanisms, including alteration of the gut mi-crobiome, a direct anti-inflammatory effect at the mucosal level, and through alteration in the fat content within the diet. Exclusive enteral nutrition provides benefits beyond disease remission, especially through promoting growth; further studies are required to elucidate exactly how it works and the longer-term outcomes. This is par-ticularly important given the lack of negative effects compared with the significant side-effect profile of biological therapies. Improving resources to minimize the psycho-social impact of exclusive enteral nutrition may open the way for wider use in adult patients through the development of solid diet alternatives to liquid feeds.

Keywords: Exclusive enteral nutrition; Crohn’s disease; Nutritional therapy; Pediat-ric; Adult; Inflammatory bowel disease.Abbreviations: CD, Crohn’s disease; CI, confidence interval; CRP, C-reactive pro-tein; ECCO, European Crohn’s and Colitis Organization; EEN, exclusive enteral nu-trition; FODMAP, fermentable oligo-, di- and monosaccharides and polyols; IBD, inflammatory bowel disease; IFX, infliximab; IP, intestinal permeability; OR, odds ratio; PCDAI, pediatric Crohn’s disease activity index; PEN, partial enteral nutrition; RCT, randomized clinical trial; SCD, specific carbohydrate diet; SFD, solid food diet; TNFα, tumor necrosis factor alpha.Received: July 31, 2017; Revised: October 31, 2017; Accepted: November 24, 2017*Correspondence to: Rakesh Vora, Paediatric Offices, off A Floor corridor, Old Main Site, The General Infirmary at Leeds, Great George Street, Leeds LS1 3X, UK. Tel: 0113 392 3828, E-mail: [email protected] to cite this article: Vora R, Puntis JWL. Dietary Treatment for Crohn’s Dis-ease—Old Therapy, New Insights. Exploratory Research and Hypothesis in Medicine 2017;2(4):101–108. doi: 10.14218/ERHM.2017.00026.



Vora R. et al: Dietary treatment in Crohn’s diseaseExplor Res Hypothesis Med

omized to receive either elemental or polymeric formula, showed no significant differences in remission rates of the two groups.7 A Cochrane meta-analysis in 2007, which included six studies with 192 patients receiving EEN and 160 patients receiving steroids, showed that steroids were superior to EEN in inducing remission (with an odds ratio (OR) of 0.33 and 95% confidence interval (CI) of 0.21 to 0.53).8

EEN in current management of CD

There is clearly some variation in clinical practice as far as EEN management is concerned. An international survey in childhood CD found that 23 different liquid feed formulas were being used among the units that responded.9 There were also variations in the length of therapy, with some centers prescribing EEN for four to six weeks and others for six to eight weeks. Not only did the liquid feeds differ but some centers used polymeric and other elemen-tal formulas also, and some preferred nasogastric to oral feeding. There are no specific controlled trials comparing the efficacy of EEN that have been based on types of formula feed or duration of treatment. In some centers, semi-elemental diets are used10; al-though, these have been largely superseded by polymeric feeds, which are more palatable and more likely to be taken by mouth. The period of EEN is followed by a gradual introduction and es-calation of a low-residue diet (i.e. low-fiber diet, limiting foods like raw fruits and vegetables), usually over a few days; however, the literature regarding reintroduction of foods is very limited, and speed of return to usual diet varies from a few days to several weeks. The optimal method of food reintroduction has yet to be established.7 There is some evidence to suggest a beneficial effect in CD from combining partial EEN together with usual diet and immunosuppressive medications.11

Efficacy of EEN in CD

Induction of remission at new diagnosis

There are no RCTs comparing EEN with placebo in pediatric CD. EEN, when compared to steroids in multiple clinical trials subject-ed to meta-analysis, has shown an overall induction of remission rate of around 75%. Pediatric trials and two meta-analyses also demonstrate similar efficacy of both EEN and corticosteroids in in-ducing remission.12–15 Schwab et al.14 performed a meta-analysis of 15 studies which included 571 patients receiving EEN and con-cluded that there was no difference in the remission rates between EEN and corticosteroid-treated groups. In addition, the meta-analysis showed that the efficacy of elemental and polymeric diets was similar.16 A further pediatric meta-analysis, which included five pediatric trials involving 147 children, showed that EEN and corticosteroids had similar efficacy. In children, better compliance was found with elemental and semi-elemental diets compared to adults, who had a high drop-out rate of 40%.17

Dziechciarz et al.13 analyzed four RCTs involving 144 pediatric patients and showed the remission rates in the EEN and corticos-teroid groups were similar (relative risk of 0.97, 95%CI: 0.7–1.4). In contrast, the 2007 Cochrane meta-analysis by Zachos et al.,8 which included both pediatric and adult trials comparing EEN to corticosteroids with 192 patients receiving EEN and 160 receiv-ing corticosteroids, demonstrated a pooled OR of 0.33 (CI 0.21–0.53), favoring corticosteroid therapy. The same meta-analysis also looked at 10 trials involving 334 patients, and showed no dif-

ference in the efficacy of elemental versus non-elemental formula (OR of 1.10; 95% CI: 0.69–1.75). A North American Pediatric Gastroenterology, Hepatology and Nutrition working group on IBD has examined this meta-analysis and raised questions over-interpretation. They found that many of the pediatric trials that had been excluded for methodological reasons showed that EEN had similar or higher efficacy than the steroids. The difference in the results from trials in adult patients showing lower rates of remission compared to pediatric studies are probably due to lower compliance rates with EEN and possibly less experience among adult gastroenterology teams with supporting the use of this treat-ment.10

Of additional interest is the study by Sigall-Boneh et al.16 show-ing that partial enteral nutrition (PEN) can also lead to high re-mission rates in both children and adults with CD. Forty-seven patients (34 children, 13 young adults) with mild to moderate CD were treated with a 6-week structured CD exclusion diet, wherein they had access to certain solid foods and restricted exposure to other foods, and derived 50% of their caloric intake from a poly-meric formula (i.e. the PEN). Response was seen in 78% and re-mission in 70% of these patients, as measured by decrease in the Harvey-Bradshaw index and pediatric Crohn’s disease activity in-dex (PCDAI), in addition to normalization of C-reactive protein (CRP). There was no significant difference in remission rates for the groups of pediatric and young adult patients.18

Based on the above evidence, the current European Crohn’s and Colitis Organisation (ECCO) and European Society of Paediatric Gastroenterology, Hepatology and Nutrition consensus guideline from 2014 recommends the use of EEN as first-line therapy to in-duce remission in children with active luminal CD, and against using PEN.19 For adult CD, the ECCO evidence-based consensus from 2017 recommends using systemic corticosteroids as first-line therapy.20 The European Society of Parenteral and Enteral Nutri-tion currently also considers that EEN is not proven to be effective in inducing remission in adults with CD. The more limited data from adult CD patients on inducing remission is different from pediatric experience, probably due to lesser experience and exper-tise in EEN use, and lower compliance rates–problems that will require significant resources, such as more dietetic support, to ad-dress.21

Re-induction of remission in patients with relapsed disease or disease flare-ups

The relapse rates in patients treated with EEN at initial diagno-sis and going into remission are between 60–70% within the first year.15,22 Seidman et al.21 and Day et al.22 performed studies in-cluding children with relapsing CD; the results showed that effi-cacy of EEN in inducing remission during a relapse was 50%, with five out of 10 and seven out of 12 patients, respectively, respond-ing. Both studies noted that even in children who were non-re-sponders to EEN the disease activity decreased and nutritional sta-tus improved. A randomized trial involving 32 adult patients with active CD showed similar remission rates for the patients receiving EEN and those receiving corticosteroids. However, after follow-up for one year, there was a higher relapse rate in patients who received EEN, as compared to the steroid-treated group.23 Grogan et al.15 performed a double-blind RCT of EEN in 34 children with CD, with the treatment given over a six week period; patients with large bowel disease alone were excluded from the study. The au-thors compared the efficacy of polymeric to elemental enteral feed, with a 2-year follow up period. There were no significant differ-ences in the clinical and biochemical remission rates between the


Vora R. et al: Dietary treatment in Crohn’s disease Explor Res Hypothesis Med

two groups. Two-thirds of children in both groups relapsed within a year; the majority of children (82%) who relapsed used EEN as treatment for relapse. There are no studies showing EEN efficacy during flare-ups in patients who did not receive EEN during initial presentation or who are EEN-naïve. Other issues for investigation are whether EEN-exposed patients naturally perform worse on EEN therapy after a second or a third flare-up and whether this is secondary to poor compliance or mucosal changes with chronic disease.

Maintenance of remission

The role of EEN as maintenance therapy is unclear. There have been multiple studies examining the use of PEN as a possible way of prolonging remission. Verma et al.24 concluded from their study of 30 adult CD patients that PEN is safe, well-tolerated and effec-tive in quiescent CD, and showed significant reduction in relapse rates based on disease activity scores. A prospective study in 2010 highlighted that concomitant PEN during infliximab (IFX) mainte-nance therapy does not significantly increase the maintenance rates of clinical remission.25 A multicenter trial in Japan recruiting 102 patients showed that PEN combined with IFX maintenance use was associated with significantly reduced relapse rates, as com-pared to IFX alone.26

Esaki et al.27 performed a single-center retrospective study in adult CD patients who had entered remission after parenteral nutrition. One group received more than 1200 kcal per day from supplementary nutrition (the ‘enteral nutrition’ group), and another group received less than 1200 kcal per day from supplementary enteral nutrition (the ‘non-enteral nutrition’ group). The authors concluded that clinical remission can be prolonged by supplemen-tary nutrition (with relapse rates higher in the ‘non-enteral nutri-tion’ group), and that the risk of relapse in the ‘enteral nutrition’ group was significantly increased if they had penetrating CD or had undergone previous surgery.

A recent systematic review of 12 studies including 1,169 pa-tients (95 of them children) with inactive CD concluded that PEN was more effective than a regular diet and that PEN used in com-bination with standard immunosuppression produced results either better than or as effective as the comparator group without the PEN.28 Sigall Boneh et al.29 reported on 21 children and adults with CD who had lost response to biologics and were treated with PEN using a polymeric formula after two weeks of EEN. The study subjects were allowed an oral diet based on fruit, vegetables, meats and complex and simple carbohydrates (termed the Crohn’s Disease Exclusion Diet) (Table 1), which was hypothesized to modify the microbiome or intestinal permeability. Clinical remis-sion, as measured by the Harvey-Bradshaw index, occurred in 62% of the children, along with decreased inflammation, as shown by biochemical evidence (i.e. reduction in CRP and increase in

mean serum albumin concentration). The authors concluded that nutritional treatment, which combines an exclusion diet based on a range of solid foods together with PEN, might be an effective salvage therapy in some patients.

These studies collectively suggest that the quantity of enteral formula feed used is important. The higher the calories provided by the enteral formula, the higher were the remission rates29; how-ever, large RCTs are necessary to define the role of manipulation of enteral nutrition for the maintenance of remission in CD. In contrast, Johnson et al.,30 who looked at 50 children with active CD based on PCDAI >20, showed that long-term PEN does not suppress bowel inflammation and is unlikely to prevent disease re-lapse. The authors randomly recruited patients to receive total ca-loric requirements from either 50% PEN from elemental formula with unrestricted regular diet or 100% of calories from EEN with an elemental formula. At follow-up, clinical disease scores (i.e. PCDAI) and biochemical parameters (i.e. CRP, serum albumin, platelet count, erythrocyte sedimentation rate) of disease remis-sion were recorded in both groups for comparison. The remission rate with PEN was significantly lower than that with EEN (15% vs. 42%, p = 0.035). While the PCDAI fell in both groups, the reduction was significantly greater in the EEN group. The authors concluded that the benefits in the PEN group were secondary to symptomatic and nutritional improvements and not to anti-inflam-matory effects.

Solid food-based diets

Specific carbohydrate diet (SCD)

The SCD is a grain-free diet, which is low in sugar. Its design is based on the hypothesis that complex carbohydrates (polysaccha-rides and oligosaccharides) are poorly absorbed and promote gut bacterial overgrowth, which then acts as an inflammatory signal causing mucosal damage, worsening the carbohydrate malabsorp-tion and perpetuating an inflammatory cascade. The SCD restricts carbohydrate intake, and a gluten-free diet would be one exam-ple of such a diet.31 Suskind et al.31 performed a retrospective re-view of 26 children (20 CD, 6 ulcerative colitis) attending an IBD center where patients followed a SCD for between three and 48 months. There was a significant decrease in the disease activity scores (PCDAI and the Pediatric Ulcerative Colitis Activity Index) at 6 months. Mutlu et al.32 reported a case series of 50 adult IBD patients who went into remission when following a SCD over a mean time-period of 35 months and who were able to maintain remission. However, symptom reduction was not correlated with objective markers of gut inflammation, like fecal calprotectin or mucosal healing, and symptomatic response by itself was found to be clearly not adequate for assessment of an anti-inflammatory

Table 1. Practice points on exclusive enteral nutrition in Crohn’s disease

Choice of formula Polymeric

Route of administration Oral/nasogastric tube or combination

Duration of feeds 6 to 8 weeks

Markers of efficacy Symptom reduction, weight gain, normalization of CRP and fecal calprotectin

Reintroduction of regular food 1 to 4 weeks

Partial enteral nutrition in remission Preferable

Abbreviation: CRP, C-reactive protein.



effect of diet.

Fermentable oligo-, di- and monosaccharides and polyols (FOD-MAP) diet

The FODMAP diet is based on the hypothesis that a low FOD-MAP diet would result in reduction of bowel bacterial overgrowth, and thus prevent secondary mucosal damage.33 The FODMAP diet restricts ingestion of vegetables and certain fruits, while the SCD allows for unrestricted fruit and vegetables, with the exception of potatoes and yams. The literature supporting the use of the FOD-MAP diet in IBD is very limited. A retrospective study involving 72 adult patients with IBD showed a decrease in gastrointestinal symptoms, namely abdominal pain, bloating and stool frequency, after starting a FODMAP diet.34 A prospective study concluded that a FODMAP diet was an effective strategy in IBD, in the main, by improving symptoms secondary to superimposed irritable bowel syndrome.35 They showed that 50% of patients (52 CD, 20 ulcerative colitis) on low FODMAP intake responded with signifi-cant reduction in abdominal symptoms, abdominal pain, bloating, wind and diarrhea, and the response showed a direct correlation with dietary adherence in CD patients.36

IgG4-guided exclusion diet

IgG1 and IgG4 are dominating subclasses of antibodies to food antigens, and IgG4 is produced following chronic exposure to the antigen. Patients with CD have significantly higher levels of IgG4 responses to food antigens.37 It has been hypothesized that targeted IgG4-based exclusion diet may reduce the inflammatory response in CD patients and may present a method of personalizing an ex-clusion diet. A sham-controlled randomized trial recruited 145 active CD patients and showed that those who received an IgG4-guided exclusion diet for four weeks, based on exclusion of four food types with highest antibody titers, had significant improve-ment in their quality of life scores, as compared to the sham diet control group.38

Paleolithic diet

This diet is based on the hypothesis that current diseases are a consequence of exposure to processed foods produced by modern agricultural advances and, therefore, dietary treatment should in-volve increasing intakes of lean, non-domesticated meats and non-cereal plant-based foods, like roots, nuts, legumes and fruits. There is no data on use of such a diet in IBD.

Family perceptions of diets

A recent study in a pediatric gastroenterology center looked at the experience of families and children around EEN and their thoughts about potential solid food diet (SFD) alternatives. The majority of families (59%) with experience of EEN were happy to use this treatment again, in the event of a future relapse of CD. This most likely reflects their experience of the efficacy of EEN, improved palatability of polymeric feeds, and the expertise of the pediatric healthcare professionals involved in providing support.39 Many families had already experimented with dietary modification of some sort, as a way of controlling symptoms. The survey supports previously published literature, that, if effective, most families

would prefer to use a SFD than liquid formula for EEN.40

Mechanism of action of EEN

In children with CD, EEN is as effective as corticosteroid therapy but without the side effects. Although the precise mechanism of action remains unknown, there are various hypotheses based on what is known about the pathophysiology of CD. The pathogenesis involves an interaction between a genetic susceptibility, immunol-ogy of the host and its mediation in causing tissue injury or tissue healing, and environmental factors. The hypotheses are: restora-tion of cytokine balance between pro- and anti-inflammatory cy-tokines; a direct effect on gut mucosa; modification of gut flora; change in fat composition of diet influencing the pro- and anti-in-flammatory mediators; and, enhancement of nutritional status and ‘bowel rest’ (including avoidance of multiple food antigens found in normal diet). Here, we review the evidence behind the above postulates on mechanisms of action.

Restoration of cytokine balance

There is evidence that in CD, EEN decreases intestinal permeabil-ity (IP), and that increased IP precedes relapse of CD symptoms and may represent ongoing disease.41–43 Wyatt et al.41 showed that patients who had normal IP could maintain a prolonged remis-sion. The authors measured IP in 72 patients with CD who were in remission, using the lactulose-mannitol test, and found that the permeability index was significantly higher in CD patients than in controls. These patients were followed-up for 1 year. It was found that 70% of those with increased IP relapsed, while only 17% with normal IP did so, suggesting that raised IP represents subclinical disease. These studies did not investigate the relationship between IP and objective markers of inflammation.

Teahon et al.42 showed that 26 out of 37 CD patients with high IP relapsed within a year; however, only a very small proportion of patients with normal IP suffered a clinical relapse. Tumor necro-sis factor alpha (TNFα) has previously been shown to be involved in disruption of cellular tight junctions, and thereby to increase IP.44,45 Nahidi et al.44 demonstrated that the TNF-exposed intes-tinal CaCo-2 monolayers with increased IP showed a complete reversal of the changes induced by TNF when treated with EEN and biologic agents like IFX, including restoration of IP. A similar experiment with corticosteroids showed only partial reversibility of the changes caused by TNF.44

There is now increasing evidence that various proinflammatory cytokines, such as interleukin-1β, interleukin-8 and interleukin-γ, are down-regulated by EEN. An in vitro model used by de Jong et al.46 showed that EEN had a direct action on colonic entero-cytes and reduced production of TNF and interleukin-8 when enterocytes were exposed to proinflammatory cytokines. These anti-inflammatory effects were not affected by boiling or freeze-thawing of the EEN formula, demonstrating that exposing EEN to the above conditions does not alter its effect.

EEN and mucosal healing

There is emerging evidence that mucosal healing improves both short-term and long-term outcomes in CD and may change the natu-ral history of the disease. In the short term, mucosal healing has been associated with reductions in CDAI and reduced steroid use. In the medium and long terms, mucosal healing has been shown to be as-



sociated with longer periods of remission, a reduced complication rate and a decrease in hospitalizations and surgical interventions.47,48

A prospective 10-week randomized, open-label trial in 37 pedi-atric patients comparing polymeric formula (n = 18) with steroids (n = 19) showed reduction in CDAI scores in both groups, with no significant difference in clinical remission rates; however, the number of patients showing mucosal healing was significantly higher in the EEN group (74%) compared to the steroid group (33%).14 Another study looking at the mechanism of action of a specific polymeric formula (rich in transforming growth factor-β2) on mucosal healing demonstrated endoscopic improvement after 8 weeks of EEN in 29 children. Cytokine mRNA in mucosal biopsies before and after treatment with EEN indicated that 79% of children were in remission after 8 weeks of treatment, with macroscopic and histological healing in the terminal ileum and colon. This was associated with a significant reduction in mucosal interleukin-1β mRNA and TNF-α. The ileal mucosa also showed a significant re-duction in interferon-γ mRNA, with an increase in transforming growth factor-β1 mRNA.49

An open label prospective study recruited 34 children using EEN for a minimum of 6 weeks with a clinical, biochemical and endoscopic assessment before and after completion.50 The assess-ment also included disease outcomes at 1 year. The results showed that clinical and biochemical remission was achieved in 84% and 76%, respectively; moreover, 58% had good endoscopic scores and 21% achieved remission of ileal CD on magnetic resonance enterography. The children with good endoscopic scores were found to have reduced rates of disease relapse, anti-TNF use and hospitalizations when they were followed-up at 1 year. This is likely to be a secondary effect that occurs via its effect on cytokine balance or on gut microbiome.

Modification of gut microbiome

Multiple studies have shown that the gut microbiome is different in patients with IBD compared with healthy subjects and is less diverse, especially in relation to the Firmicutes phylum. In addi-tion, there are higher concentrations of certain bacterial species, such as the adherent/invasive strains of Escherichia coli, and these changes may be implicated in the pathogenesis of IBD.51–53 Hans-en et al.54 have shown that in mucosal samples from children with CD there is increased Faecalibacterium prausnitzii and reduced bacterial diversity, indicating dysbiosis. The dysbiosis hypothesis suggests that in IBD, there is an alteration of the balance between the beneficial and harmful microbiota in the gut contributing to gut inflammation.54

The literature has, however, been quite conflicting, especially regarding the type of change in gut microbiota in CD. Leach et

al.55 demonstrated that gut bacterial diversity in children with CD at diagnosis was similar to that of healthy controls, and that changes occurred in bacterial species like Eubacteria, Bacteroides, Clostridium coccoides, Clostridium leptum and Bifidobacterium during and after eight weeks of EEN, proving that EEN can modify the gut microbiome. This change was associated with decrease in the gut mucosal inflammation and was sustained for 4 months after stopping EEN.55 Gerasimidis et al.56 compared stool microbiome in 15 children with CD and 21 healthy subjects showing that the global bacterial diversity and F. prausnitzii concentration both sig-nificantly decreased during EEN; the greatest changes were seen in children who responded clinically to treatment with EEN. All these changes reverted to pre-treatment levels after the regular diet was recommenced. The authors concluded that these results challenge the current perception of a protective role for F. prausnitzii.56

Further publication by the Gerasimidis group confirmed that in pediatric CD, despite improvement in disease activity, EEN made the gut microbiome more dysbiotic, reducing gut microbiome di-versity and decreasing the relative abundance of more than half of the bacterial taxonomic units during EEN (Table 2).56–59 Schwerd et al.,58 in contrast, described an increase in the relative abundance of Firmicutes after EEN therapy. Guinet-Charpentier et al.59 re-cently demonstrated that patients who respond to EEN and in clini-cal remission showed a reduction in Dialister, Blautia, unclassified Ruminococcaceae and Coprococcus compared with patients in re-mission with other treatments, such as anti-TNF and PEN.The limitations of some of these studies are that the analysis had been performed on serial stool samples and emphasizes the impor-tance of studying both mucosal adherent bacteria and stool micro-biota together.

Change in fat composition of diet

Use of high or moderate fat feeds, when comprised of predomi-nantly monounsaturated fats in EEN, previously showed a favora-ble outcome.60 The hypothesis was that depletion of linoleic acid reduces substrate for production of proinflammatory eicosanoids, like leukotreines and prostaglandin E2. A Cochrane meta-analysis of a subgroup in 2007 including 209 patients treated with EEN formula of differing fat content (low fat: <20 g/1000 kCal vs. high fat: >20 g/1000 kCal) found no significant difference in efficacy (OR: 1.13; 95% CI: 0.63–2.01). The use of very low fat content (<3 g/1000 kCal) or the type of fat (long-chain triglycerides) also did not show a difference in efficacy in the treatment of active CD; although, a nonsignificant trend favoring very low fat and very low long-chain triglyceride content was observed. The authors advised that this result should be interpreted with caution, due to signifi-cant heterogeneity and small sample size.5,8,61–64

Table 2. Gut microbiome changes induced by exclusive enteral nutrition

Increased during EEN Decreased during EEN

Firmicutes

Relative abundance of Firmicutes58 Concentration of F. prausnitzii56

Bacteroidetes

Concentration of Alistipes59 Concentration of Bacteroides/Prevotella56

Actinobacteria

Concentration of Bifidobacterium59 Concentration of Bifidobacteria56

Abbreviation: EEN, exclusive enteral nutrition.



Improvement in growth and nutrition

The conventional therapies for IBD (especially combination im-munosuppressive treatment) are generally effective; however, there are significant concerns regarding the impact of disease activity and treatment on growth in children. EEN has been proven to have sig-nificant growth and nutritional benefits apart from its use in inducing remission. Prolonged use of corticosteroids is known to negatively impact linear growth via osteocyte and osteoblast apoptosis, in-creased osteoclastogenesis, decreased osteoblastogenesis, increased autophagy in osteocytes and osteoclasts, and reduced gastrointesti-nal calcium absorption, resulting in reduced bone formation.

Griffiths et al.65 reviewed mechanisms of impaired growth in CD, such as proinflammatory cytokines, causing direct interfer-ence with insulin like growth factor-I and mediation of linear growth, cytokine-mediated anorexia, and mucosal damage lead-ing to protein-losing enteropathy. EEN has been shown to reverse the growth hormone-resistant state induced by proinflammatory cytokines.65 Whitten et al.66 investigated 23 children with a new diagnosis of CD before and after six weeks of EEN. They reported normalization of inflammatory markers, serum markers of bone turnover and bone-specific alkaline phosphatase, concluding that these findings indicated an improvement in bone health. Denne et al.67 showed that in inactive CD, EEN promoted anabolism by suppressing proteolysis and increasing protein synthesis to rates that were similar to those of healthy children.

There is limited literature on micronutrient status in CD pa-tients. A Japanese study that evaluated zinc and selenium status in 31 patients on long-term EEN found these patients to be deficient and recommended zinc and selenium supplementation.68 Howev-er, Akobeng et al.69 found low plasma concentrations of vitamins C and E in childhood CD patients after four weeks of EEN and an increase in selenium concentrations. It is difficult to make any specific recommendations based on the limited literature and con-trasting findings.

Future research directions/perspective

EEN is a simple, safe and effective therapy in pediatric CD. EEN improves nutritional status, growth and bone health. It is associat-ed with mucosal healing, is inexpensive and has no serious side ef-fects, but demands healthcare resources (specifically, dietitians and specialist nurses) and currently there is no agreed exit diet strategy. Although a major advance, anti-TNF therapy is not effective in a significant number of patients. The current treatment strategies in CD are limited and usually linked to ways of increasing immuno-suppression with newer monoclonal antibodies, all associated with potentially significant side effects. There is increasing evidence that IFX appears to be more effective with EEN, as compared to IFX alone, in maintaining remission.70 PEN and SFD are showing promise in management of gastrointestinal symptoms, and RCTs exploring this further are needed. If the mechanisms of action of EEN were fully elucidated, there may be scope for designing more effective EEN formulas or solid food-based diets that would be acceptable across the age range of patients with CD and might be tailored to phenotype.

Conclusions

In children with CD, EEN is a highly effective treatment and in-duces remission in more than two-thirds of newly diagnosed pa-

tients; the efficacy is better than that of corticosteroids. EEN is also efficacious in inducing remission in relapsing CD. There is evi-dence to support on-going use of PEN to maintain remission in the long-term, and there is limited evidence to support the use of PEN in conjunction with modified solid food-based diet at diagnosis of CD to induce remission. In adults, limited evidence suggests that corticosteroids are superior to EEN in inducing remission. Solid food-based diets may be used to decrease concurrent gastrointesti-nal symptoms. There is evidence to support a mechanism of action of EEN via restoration of cytokine homeostasis and its effect on mucosal healing. The data on the mechanism of action via modi-fication of the gut microbiome and alteration of fat content of the diet is limited and conflicting. There is robust data, however, on a direct effect of EEN on promotion of growth and improving nutri-tion and bone health.




Drafting the manuscript with the opportunity to revise or question its contents (RV, JWLP).

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Review Article

Introduction

Diet is an important lifestyle exposure and is the largest modifiable determinant of risk for non-communicable disease, including car-diovascular disease, diabetes and some cancers. Importantly, diet is an ongoing, essential and unavoidable exposure for all people throughout life. Worldwide, non-communicable diseases account for 43% of the burden of disease currently, and the World Health Organization predicts they will represent 60% of the disease bur-den and 73% of all deaths by 2020.1 The number of people with diabetes worldwide rose to 422 million in 2014,2 cardiovascular disease is the leading cause of death,3 and incidence of cancers re-lated to dietary risk factors, such as colorectal and esophageal can-cers, are also rising.4 Today, most of the world’s population lives in a country where overweight and obesity kills more people than underweight.5 Diet is, therefore, considered as a primary preven-tion strategy to reduce the risk for chronic diseases. However, as highlighted by the “Rose prevention paradox”, a lifestyle measure that reduces risk in an entire population may offer little benefit to

the individual.6Current public health initiatives and nutritional guidelines, both

on a global and national scale, are generic recommendations. These recommendations are based on population estimates of required in-takes and the prevention of deficiency.7 However, it is well known that there is considerable variance in how individuals respond to the same nutritional stimuli, and this alters the outcomes in terms of benefits and risks. The broader concept of personalized nutrition is not new, and population guidelines recognize some of this vari-ance in limited cases, with some recommendations tailored to age, sex, or conditions such as pregnancy.8 Ranges of recommended in-take values also account for a portion of this biological variance, as do specific recommendations related to the diagnosis of particular conditions, such as allergies or chronic diseases such as diabetes,9 cardiovascular disease and cancers.10,11

However, population guidelines, even with stratification, may not meet the needs of all individuals equally in terms of optimizing health outcomes and reduction of disease risk.12,13 With improv-ing technology and advances in our understanding of genetics, the concept of personalized nutrition via nutrigenetics has emerged, where dietary recommendations can take into account the variance between individuals by tailoring to each person’s unique genet-ics.13–15 While personalized nutrition based on genetics has sig-nificant future promise, there are many challenges in translating scientific advances into successful strategies for managing dietary intake and diet-related health outcomes on a large scale. These is-sues include the translation of reductionist research outcomes into practice, public perception and the likelihood of uptake, issues of privacy and ethics, commercialization, and the level of evidence required before the transition from traditional approaches is ben-eficial. It is important to consider if these challenges can be met

Nutrigenetics—Personalized Nutrition in the Genetic Age

Emma L. Beckett1*, Patrice R. Jones2, Martin Veysey3 and Mark Lucock2

1School of Medicine & Public Health, University of Newcastle, Australia; 2School of Environmental & Life Sciences, University of Newcastle, Australia; 3Hull York Medical School, UK

Abstract

Diet is an important modifiable determinant of disease, and it is becoming clear that diet and genetic risk factors are interactive in determining risk for diseases such as cardiovascular disease, diabetes and cancers. Advances in technology have improved our understanding of gene-nutrient interactions, and lead to the development of nutri-genetics, personalized nutrition based on genetics. While evidence is strong for some associations, others remain unclear. As such, the implementation of nutrigenetics remains controversial. While some argue it is not ready for clinical use, it has also been argued that nutrigenetics is unfairly held to a higher standard than traditional nutri-tion research. Despite the future promise of nutrigenetic testing for improving health outcomes, several barriers in science, technology, acceptance and ethics exist to its implementation. Gene-nutrient associations have been identified in a number of lifestyle-associated diseases, and better understanding of these relationships may lead to improved health outcomes. However, the success of nutrigenetics is not only dependent on the strength of the science, but in consumer acceptance and uptake. This narrative review provides an overview of the current land-scape for nutrigenetics in relation to key disease states, and addresses the potential barriers to implementation.

Keywords: Nutrigenetics; Personalized nutrition; Genetics; Nutrition.Abbreviations: FTO, fat mass and obesity associated gene; GWAS, genome-wide association studies; TCF7L2, transcription factor 7-like 2 gene.Received: August 01, 2017; Revised: October 23, 2017; Accepted: November 07, 2017*Correspondence to: Emma Beckett, School of Medicine and Public Health, Uni-versity of Newcastle, Chittaway Rd, Ourimbah, NSW 2258, Australia. Tel: (02) 4348 4158, E-mail: [email protected] to cite this article: Beckett EL, Jones PR, Veysey M, Lucock M. Nutrigenet-ics—Personalized Nutrition in the Genetic Age. Exploratory Research and Hypoth-esis in Medicine 2017;2(4):109–116. doi: 10.14218/ERHM.2017.00027.



Beckett E.L. et al: Nutrigenetics in personalized nutritionExplor Res Hypothesis Med

and whether personalized nutrition can produce improved health outcomes and socioeconomic benefits relative to conventional ge-neric dietary advice.

Gene-nutrient interactions

Dietary factors can interact with the genome in a number of ways. Firstly, genetic variance can influence nutritional status by modu-lating nutrient intake, uptake and metabolism. This is referred to as nutrigenetics.16 Furthermore, nutrients can regulate gene expres-sion in a number of ways. Some nutrients can directly regulate gene expression via the interaction of stimulated receptors with response elements in the genome, acting as nuclear transcription factors. This direct interaction is referred to as nutrigenomics.16 Nutrients can also modify gene expression indirectly, via modula-tion of gene regulatory factors such as epigenetic marks, including the involvement of DNA methylation and miRNA.17 Importantly, nutrigenetics can impact upon the nutrigenomic and epigenetic responses and the sum of these events can modify disease risk.18

With improving technology, and completion of ground-breaking research, such as the human genome project, we are learning more about gene-nutrient interactions. This has led to a dramatic increase in research in this area. However, there are significant challenges to the translation of genetic data into personalized dietary advice, and it is questionable as to whether our level of understanding is suf-ficient for personalized nutrigenetics to progress. The majority of the published data on gene-nutrient interactions stem from observa-tional studies and as such cannot definitively demonstrate cause and effect, and results can often be conflicting. Purpose-specific dietary intervention studies conducted by genotype are needed to achieve this. However, these are complex and expensive, and there are dif-ficulties in considering multiple polymorphisms. Numerous genetic variants that influence nutrient metabolism have been identified, but it can be difficult to conclusively link single variants to the risk for multifactorial diseases, due to interactive and additive effects of multiple variants in defining nutrient metabolism and health out-comes. There is a need for quantitative assessment and mathemati-cal modelling of multiple genetic effects.

Ultimately, the most important question is whether nutrigenet-ics can deliver results superior to population recommendations. It has been argued that the evidence for nutrigenetics is still too im-mature to be used in practice.19,20 However, it has also been argued that nutrigenetics is often held to a higher standard of evidence than generic nutritional advice,21,22 resulting in high-quality evi-dence for several gene-diet interactions potentially being ignored. Given the complexity of gene-nutrient interactions, and the known variance in nutrition-related health outcomes, regardless of genet-ics, we need to consider if we have set this standard too high. Nu-trition is complex and outcomes difficult to assess, with or without a genetic component. It is, therefore, necessary to consider person-alized nutrition in the same context as generic nutrition recommen-dations and not in the same context as clinical genetics.23

Nutrigenetic research often takes a reductionist approach to gene-nutrient interactions, examining the interactions between single polymorphisms and individual disease biomarkers, and how they are modified by single nutrients or food components.23,24 Each individual possesses potentially hundreds of gene variants that may have nutrigenetic consequences, and each consumes their own unique complex and varied diet. Complex statistical and bio-informatics modelling is required to integrate information on nu-merous genes, biomarkers, nutrients and foods for nutrigenetics to deliver on its promise of improving health outcomes.

An early application of nutrigenetics has been the diagnosis of conditions caused by single polymorphisms, such as genetic lac-tose intolerance and phenylketonuria.25 In many cases, genetic testing is not required for monogenic conditions, as the phenotype is sufficient basis for deciding on the appropriate dietary interven-tion. However, for complex polygenic traits, such as cardiovas-cular disease or diabetes, it is much more challenging to find evi-dence for the involvement of genes in disease development. These conditions have genetic risk factors, dietary risk factors, and these risks are modified by the interaction between the two and other lifestyle factors.26,27 As such, it is difficult to elucidate the involve-ment and modifiable component of the interactive factors, and a medical model of nutrigenetics, held to a clinical genetics level of evidence, may not be appropriate.

Cardiovascular disease

Cardiovascular disease has both genetic and dietary risk factors, and risk is likely modified by the interactions between the two.28 The predictive value of single polymorphisms may be small rela-tive to known risk factors, such as family history of cardiovascular disease.29 Multiple minor genetic differences could be modulated by multiple dietary factors, resulting in multiple minor changes in gene expression. Depending on the interactions, these variables could result in negligible changes in final phenotype and therefore disease risk; however, they could also accumulate to significantly alter phenotype and outcomes. We do not yet have enough evi-dence to elucidate the mechanisms and outcomes of these complex interactions, and it is likely that additional interactions remain undiscovered.30 In the future, advances in this understanding may be supported by increased research investment in whole genome sequencing and bioinformatics initiatives, as well as improving technology and reducing associated testing costs.

Homocysteine is accepted as an independent risk factor for car-diovascular disease,31 with homocysteine levels being inversely as-sociated with folate levels. It has been established that the MTH-FR-677T allele results in the enzyme methylenetetrahydrofolate reductase having reduced activity (~35% of the MTHFR-677C variant).32 Low folate status, therefore, impacts homocysteine levels more severely in individuals with the MTHFR-677TT genotype, and the standard recommendations for folate intake have been shown to be insufficient to maintain homocysteine levels below the risk level in this population. It is accepted that increasing folate intake (from 200 µg/day to 400–600 µg/day) reduces the risk for hyperhomo-cysteinemia in most MTHFR-677TT individuals. There is no reli-able evidence that these levels cause harm, and they remain below the upper intake limits found in generic advice.33 As such, this is an example of where personalized recommendations may result in improved outcomes. However, this could also support the increase in population-wide intakes, as homocysteine levels are reduced in populations exposed to folic acid fortification programs.34–37

The response of plasma low-density lipoprotein and triacylg-lycerols (triglycerides) to supplementation with fish oil are clear examples of individual variability in response to an intervention resulting from the influence of both genetic and environmental fac-tors. In a study of fish oil supplementation in 55 males, the mean change in plasma triacylglycerol was a 35% reduction. However, the variance of individual changes ranged from a 114% reduction to an 88% increase, demonstrating that the statistically significant reduction in the mean did not result in an improved outcome for all individuals. Similarly, the mean change for plasma low-density lipoprotein was a 7% increase, but with the variance of change


Beckett E.L. et al: Nutrigenetics in personalized nutrition Explor Res Hypothesis Med

ranging from a 61% increase to a 50% reduction.38,39 In the same cohort,38 and independently,40 it was determined that the APOE2 genotype was related to the magnitude of response, and this re-sponse may also be related to sex.41

Together these data suggest that while fish oil supplementation may improve cardiovascular biomarkers for a subset of individu-als this is not true for all, and in some cases, it may actually be detrimental. As such, this may be an example of where it is not ap-propriate to produce population-based recommendations, whether they are public health guidelines, or used for the marketing of sup-plements. However, there is a lack of evidence to link the variance in responses to genotype,42 and further prospective studies includ-ing genotyping are needed to understand how personalized recom-mendations could be used to overcome the variance in responses and to assess whether this ultimately impacts disease risk, as these interactions remain poorly understood.

Diabetes

It is known that type 2 diabetes has both dietary and genetic risk factors and that outcomes are dependent on the combined and/or interactive influence of these, and other lifestyle risk factors.43–45 The majority of genes implemented in the progression of type 2 diabetes relate to pathways influencing fat distribution and insulin sensitivity. Genetic risk scores for type 2 diabetes are robust pre-dictors of disease and have been shown to interact with western diets (high fat and sugar) to further predict outcomes.46

Genome-wide association studies (GWAS) have identified nearly 100 gene variants associated with modified risk for type 2 diabetes,47 with a recent review suggesting 27 of these inter-act with diet to modify progression of type 2 diabetes.48 Notably, the transcription factor 7-like 2 gene (TCF7L2), involved in Wnt signaling, has the strongest influence on risk for type 2 diabetes. Common variants in this gene have been shown to interact with high intake of both carbohydrate and fiber to modify risk for type 2 diabetes.49,50 Due to the direct association between obesity and type 2 diabetes risk, a number of gene-diet interactions modifying risk of obesity have been identified that are of relevance to type 2 diabetes. Examples include the interactions between genotypes for the fat mass and obesity associated gene (FTO),51 PPARG,52,53 PLIN,54 and MC4R and diet,55 which influence disease-related out-comes, such as insulin sensitivity.

GWAS has also identified more than 40 independent polymor-phisms associated with type 1 diabetes; however, the loci identi-fied do not fully explain the heritability component estimated from familial studies,56 suggesting that dietary and other lifestyle factors are also involved in pathogenesis. Additional studies are needed to fully elucidate the role of gene-nutrient interactions in the etiology of diabetes.

Cancer

A plethora of genes have been linked to cancer risk and outcomes. However, few associations between gene variants and cancer risk remain robust, or reproducible, likely due to the interactive influ-ence of diet and other environmental factors in epidemiological studies.57 Genome instability is a hallmark feature of cancers, with hundreds of genes involved in maintaining genome integrity.58 The interactions between these genes and diet indicate potential mechanisms by which personalized nutrition may influence cancer outcomes.59

Gene variants involved in gene-diet interactions in cancer are namely those involved in detoxifying carcinogens and repairing DNA damage. The majority of these are specific to colorectal can-cer, which has clear links to dietary risk factors.60,61 Increased risk of colorectal cancer has been shown with high red meat consump-tion in combination with variants in CYP2E1, CYP1B1, SULT1A1 and other members of the cytochrome P450 family of detoxifying genes.62–64 Several other gene variants have also been implicated in gene-diet interactions that have been shown to decrease colo-rectal cancer risk. Notably, common variants in the vitamin D re-ceptor gene and MTHFR which codes for an important enzyme in folate metabolism; both have been shown to decrease risk of colorectal cancer when combined with diets high in calcium/vita-min D and folate respectively.65–67 Whilst the majority of reported gene-diet interactions are specific to colorectal cancers, gene-diet interactions have also been reported in progression of gastrointes-tinal, stomach, breast, lung and prostate cancer.57

Another well-studied gene-nutrient interaction involves cruci-ferous vegetables, which have been linked by systematic review with reduced lung cancer in individuals with polymorphisms in the GSTT1 and GSTM1 genes, which code for glutathione s-trans-ferases, but not in individuals without these variants.68,69 However, it is unclear how clinically relevant these findings will prove to be, as the standard public health message to eat less meat and more vegetables applies regardless of genotype and is likely to be in-volved in the reduction of risk of multiple diseases. The benefit of these findings, however, may be found in the development of nutraceuticals for intervention in genetically at-risk individuals, should a mechanism for this interaction be identified. Interac-tions have also been shown between genes and diet in measures of DNA damage,70 prostate cancer risk and levels of the glutathione s-transferase alpha.71,72

Cognitive decline

APOE has been identified as a susceptibility gene for Alzheimer’s disease, with the e4 variant increasing risk for disease. The Risk Evaluation and Education for Alzheimer’s Disease (commonly known as REVEAL) study examined how knowing APOE e4 sta-tus impacted behavior change in the adult offspring of parents with Alzheimer’s disease.73 Having a parent who suffered from the dis-ease, all study participants showed a higher-than-average risk for Alzheimer’s disease, regardless of genotype; however, those car-rying the APOE e4 variant were at higher risk. Participants were given a numerical estimate of their risk and were then randomly allocated into groups. Controls were not given any genotypic data, and the intervention groups were genotyped for APOE e4 and their status revealed to them. Participants who were APOE e4-positive had a higher overall numerical risk score.73,74 One year later, simi-lar proportions of positive behavior changes were reported among controls and among participants who were told that they were APOE e4-negative; however, additional positive behavior changes were reported approximately twice as often among participants who were told that they had the APOE e4 risk variant.73

Obesity

Obesity is related to both dietary and genetic variables, and again the interaction between the two are likely to be important in deter-mining phenotype, both in terms of risk for obesity and risk for dis-eases where obesity is a known risk factor.75 GWAS has revealed



that body fat patterns have large genetic components, with 97 loci related to fat accumulation and an additional 49 loci relating to body fat distribution.76,77 Implicated genes in these loci have di-verse roles and are involved in pathways such as those regulating satiety, food intake, energy metabolism and adipogenesis.76 Mo-nozygotic (i.e. identical) twins have strong similarities in the adap-tation to long-term overfeeding and in terms of weight gain and fat distribution,78 demonstrating genetic concordance.

Insight into genes that influence obesity has led to the calcula-tion of genetic risk scores, the sum of obesity risk conferred by multiple gene variants. Diet (particularly fat and energy intake) has been shown to interact with genetic risk scores to alter the obe-sity risk.79 Genes implemented in diet-gene interactions in obesity namely include FTO, MC4R, APOA2, PLIN and PPARG genes. FTO is regarded as the first identified obesity gene. Common ge-netic variance in FTO is strongly associated with increases in body mass index, with this association shown to be enhanced by diets high in dietary fat and protein.80,81 Similar associations have been found for the MC4R gene, involved in appetite regulation.82,83

Gene-diet interactions in obesity have also been shown between high saturated fat intake and APOA2, which codes for an apolipo-protein,84 and variants in PPARG, which codes for a nuclear tran-scription factor.52,85 Notably, a common polymorphism in PLIN, which is involved in the regulation of lipid storage in adipocytes, has been shown to decrease risk of obesity when combined with a high carbohydrate intake, but increases obesity risk when com-bined with a low intake of this macronutrient.86 These data dem-onstrate the difficulty in the “one-size fits all” approach to weight management, and in particular, weight loss.

In a study conducted on patients with a history of weight loss failures, it was found that nutrigenetic screening resulted in in-creased compliance and longer-term body mass index reductions, when compared with standard weight-loss advice.87 However, it was also shown that the high-risk individuals had lower perceived behavioral control over their eating and hence felt less able to change their dietary habits.88 Another study found that genetic testing increased self-confidence in the participant’s ability to lose weight, regardless of the actual result.89

Consumer acceptance

The utilization and advancement of nutrigenetics, and personal-ized nutrition more broadly, is not just dependent on the strength of the data, elucidating mechanisms and defining the appropriate level of evidence required for implementation but requires public acceptance to facilitate uptake. Motivating individuals to change dietary behaviors is one of the biggest challenges for any nutri-tional intervention.90 There must be ease of use and access, and a perception of benefit. Genetic testing, in general, has a unique set of barriers to uptake, and several of these remain in the context of nutrigenetics, regardless of outcomes.

Numerous studies have been conducted into the acceptance of genetic testing in general.91–95 Whilst testing is well accepted and becoming routine in the cases of high penetrance single poly-morphisms, such as the BRCA1 gene in breast cancer risk, in nu-trigenetics, however, the associations are often weaker and less clear.92,95 Although, attitudes vary by demographics and results have been mixed. It has been reported that men are more will-ing to undergo genetic testing,96,97 but the opposite association has also been reported.98 It has also reported that age is a factor in acceptance of genetic testing; yet, some studies have found that older people are more willing,97,98 while others have reported that

younger adults are the most likely to take part.96

Health has been identified as a primary motivator for undergo-ing genetic testing,96,98 which may facilitate the uptake of nutrige-netics, given that diet is a major modifiable determinant of disease. Acceptance may also be linked to current health; for example, re-spondents with high blood cholesterol or central adiposity were more likely to identify as willing than those without to undergo genetic testing, in general, and specifically for dietary modifica-tion.98 However, several possible outcomes to this are possible. Joost et al.99 suggested that individuals identified as having a higher disease risk through genetic testing may be more motivated to comply with a dietary intervention; however, it was also noted that knowledge of a genetic predisposition may result in a fatalis-tic attitude and reduced compliance. Furthermore, Hunter et al.100 reasoned that a negative result may lead to reduced motivation as individuals become reassured that they will not develop disease. Communicating the nuanced nature of risk and risk modification through nutrigenetics will be vital to ensure responses to informa-tion are not detrimental to outcomes.

Interestingly, in a study of familial hypercholesterolemia, par-ticipants with the at-risk polymorphisms were less likely to believe that eating a lower fat diet would reduce their cholesterol levels, and more likely to believe that medication would.101 Acceptance of genetic testing and personalized nutrition may not depend on the actual science but on the consumers’ understanding of its implica-tions for their personal health. However, following a systematic re-view, Marteau et al.101 argued in addition to significant gaps in the relevant science, there is limited evidence that nutrigenetic dietary advice will motivate appropriate behavior changes.

Technology and regulation

Web-based and smartphone technologies increase product reach and ease of delivery, but also raise issues of data security and accu-racy of information. However, the technology for offering direct-to-consumer genetic testing has out-paced regulation for its provi-sion, at times out-pacing the scientific evidence. Therefore, there needs to be a progression of regulation and technology to ensure consumer privacy is protected, data is appropriately stored, and that consumers are not misled. Early practitioners of nutrigenetics need to be mindful that inaccurate information offered prematurely can damage the reputation of the field. This may be exacerbated by the delivery of genetic information outside of a clinical setting.

Several studies have documented concerns regarding the tech-nology used to provide nutrigenetic information. These include on-line privacy concerns, and the potential for information to be mis-used by insurers, employers, governments or other entities for profit or exploitation.102–104 Advances in technology have driven the rise of personalized nutrition in general and nutrigenetics specifically. This includes the advances in genetic testing technology and the reduction in cost, as well as the use of technology for the collection and dissemination of information. The expansion of internet deliv-ery systems needs to be considered in personalized nutrition. As oversight and regulation varies by jurisdiction, it is reasonable for consumers to be cautious about their privacy and future use of their data. Additionally, provision of results directly to the consumer may be harmful without the input of a genetic counselor.105

Ethical considerations for implementation

There is significant debate surrounding whether or not the current



knowledge base is sufficient for ethically responsible implementa-tion of nutrigenetic testing.23,25,106,107 While there is still much to be discovered, we need to consider if we are holding nutrigenetics to a higher standard than generic population recommendations.23 The ethics of implementation may need to be considered on a case-by-case basis. For some gene-nutrient interactions, there is significant evidence that for some individuals with particular genotypes there could be a benefit from following dietary patterns other than those recommended by the standard guidelines; however the strength of evidence varies depending on the association in question. The key question is how to proceed in the face of uncertainty. Gorman et al.23 argue that a precautionary approach should be adopted, sug-gesting that personalized dietary advice should be offered only in cases where there is strong scientific evidence for health benefits, followed by continued stepwise evaluation to identify unforeseen behavioral and psychological effects.23

Future research predictions

The use of genotypic information in personalized nutrition offers considerable future promise, but significant barriers exist to suc-cessful implementation, independent of scientific knowledge. These include consumer acceptance, ethical, technological and regulatory considerations. Research into nutrigenetics has produced inconsist-ent results; however, the same could be said for conventional nutri-tion studies. This is not necessarily due to the overall quality of the research and the magnitude of the body of knowledge, rather it is due to the complexity interactions between nutrition, genetics, and long-term health. Improved frameworks are required to translate nutrigenetic studies into usable guidelines to direct practicing nutri-tion and medical professionals. This will require an interdiscipli-nary approach, including geneticists, bioinformaticians, nutrition-ists, dietitians and other biomedical professionals.108

Furthermore, additional research is needed not only into the gene-nutrient interactions themselves but also into the public atti-tudes and acceptance on nutrigenetics and the associated risks and benefits of uptake. Without a holistic approach to implementation, it is unlikely that nutrigenetics will deliver on its early promise to improve health outcomes.

Conclusions

The ongoing reduction in costs of genetic testing, and the im-proved technology available to collect and disseminate informa-tion will lead to ongoing improvements in both quality and quan-tity of relevant data. There is significant evidence of gene-nutrient interactions in a number of chronic conditions, such as cardiovas-cular disease, diabetes and obesity, and nutrigenetics could lead to improved outcomes for patients and consumers. However, imple-mentation of nutrigenetic testing in the mainstream will depend on numerous factors, including regulation, technology and consumer acceptance, and not just availability of scientific evidence. It is also necessary for the public and implementing authorities to decide what level of evidence is required before nutrigenetics is no longer considered to be controversial.

Acknowledgments

Emma Beckett is supported by an NHMRC Early Career Fellow-ship (APP1129430). Patrice Jones is supported by a Research

Training Program (RTP) Scholarship from the Australian Govern-ment.




Manuscript writing (EB, PJ), design and revision (ML, MV).

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Review Article

Introduction

There is little doubt that type 2 diabetes is one of the major causes of death worldwide, with only heart disease, stroke and respira-tory conditions having a higher mortality rate.1 Diabetes is also the fastest growing global chronic disease.2 According to the In-ternational Diabetes Federation, 415 million people worldwide have diabetes, most of whom have type 2 diabetes. This number is expected to rise to 642 million by 2040.3 Indeed, a recent article by Paul Zimmet suggests diabetes may be the largest epidemic in human history and presents the greatest challenge to health.4 There are a number of risk factors associated with type 2 diabetes,

including being overweight or obese, hypertension, family his-tory, low high-density lipoprotein, sedentary lifestyle and depres-sion.5

Australia has a population of around 24.4 million as of the end of 2016.6 The number of Australians with diabetes totals around 1.7 million, or 7% of the population. This figure contains 1.2 mil-lion diagnosed, with an estimated 500,000 undiagnosed, with type 2 diabetes making up 85–90% of cases. Combined, the annual cost of treating and managing diabetes in Australia is AU$14.6 billion.7 What is more disturbing is the disparity between Australian Abo-riginal and Torres Strait Islanders and non-Aboriginal Australians, with the former having a diabetes prevalence around 4–5 times that of non-Aboriginal Australians.8 This figure, coupled with the re-duced access to adequate health care facilities, represents a major health issue in Australia.

This imbalance in the incidence and prevalence of type 2 diabe-tes is not just observed in Australian Aboriginal and Torres Strait Islander populations. Significantly higher numbers are seen in Af-rican American, Alaska Native, American Indian, Asian American, Hispanic/Latino, Native Hawaiian and Pacific Islander people.5 This is a fairly recent phenomenon, with some of the highest prev-alence of diabetes discovered amongst Australian Aborigines,9 Pima Indians and Pacific Islanders.10,11 More recently, diabetes has been reported to affect around 8.7% of Asian Indians,12 and increasing from 2.6% in 2002 to 9.7% in 2012 in the rapidly-mod-

Maternal Undernutrition and Type 2 Diabetes in Australian Aboriginal and Torres Strait Islander People: History and Future

Direction

Dean V. Sculley1* and Mark Lucock2

1School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2258, Australia; 2School of Environmental & Life Sciences, University of Newcastle, NSW 2258, Australia

Abstract

Type 2 diabetes is one of the most common chronic disease conditions, accounting for the majority of the 415 mil-lion diabetes cases worldwide. Australia currently has 1.7 million diabetics, with a prevalence among Australian Aboriginal and Torres Strait Islander populations 4–5 times that seen in non-indigenous Australians. The finan-cial burden amounts to AU$14.6 billion per year. Known risk factors for type 2 diabetes are being overweight or obese, hypertension, a sedentary lifestyle, low concentration of high density lipoprotein, depression and family history. Nutrient restriction during pregnancy can program alterations to organs and systems in the developing fetus due to intrauterine growth restriction. This plasticity, known as the ‘thrifty phenotype’, has been implicated in a wide range of adult disease conditions, including type 2 diabetes. Developmental programming via epigenetic mechanisms has resulted in a reduction of pancreatic beta cell mass, disruption of glucose transport proteins and signaling, and earlier onset of glucose intolerance of offspring, and is transgenerational in nature. Indigenous populations around the world appear to be at greater risk of programming effects, thought to be a consequence of rapid dietary and lifestyle changes. Interventions aimed at ensuring adequate maternal nutrition may reduce the extent of the deleterious epigenetic modifications and reduce the prevalence of type 2 diabetes in Australian Aboriginal and Torres Strait Islander populations.

Keywords: Maternal diet; Low protein; Developmental programming; Type 2 dia-betes.Abbreviations: DNMT, DNA methyltransferase; IUGR, intrauterine growth restric-tion.Received: August 01, 2017; Revised: November 9, 2017; Accepted: November 14, 2017*Correspondence to: Dean V Sculley, School of Biomedical Sciences and Pharmacy, University of Newcastle, Brush Rd, Ourimbah, NSW 2258, Australia. Tel: (0)2 4349 4596, E-mail: [email protected] to cite this article: Sculley DV, Lucock M. Maternal Undernutrition and Type 2 Diabetes in Australian Aboriginal and Torres Strait Islander People: History and Future Direction. Exploratory Research and Hypothesis in Medicine 2017;2(4):117–121. doi: 10.14218/ERHM.2017.00028.



Sculley D.V. et al: IUGR and T2D in indigenous AustraliansExplor Res Hypothesis Med

ernizing economy of China.12,13 Current figures from the World Health Organization show that 10% of the population, or 110 mil-lion people, suffer with diabetes in China.14 These data suggest there must be specific reasons, possibly genetic or epigenetic, why certain populations endure higher rates of diabetes.

Change of lifestyle

To better understand why these indigenous populations, includ-ing Australian Aboriginal and Torres Strait Islander peoples, are at greater risk of developing diabetes we need to look at what environmental and lifestyle changes have accompanied the in-creased incidence and prevalence, together with an examination of the specific genetic risk factors. It had been generally assumed that Australia was populated somewhere between 40,000–100,000 years ago and that it remained isolated, barring occasional visits from Asian fishermen and European seafarers, until the late 18th century.15–17 However, more recent discoveries in excavations in northern Australia now indicate human occupation at around 65,000 years ago.18

Whilst data pertaining to the health and condition of Australian Aborigines prior to European settlement is difficult to establish, a study of isolated populations living the traditional hunter-gatherer lifestyle around 50 years ago reported an average height of 167.1 cm and average weight below 56 kg.19 Foods eaten were dictated by the locality and seasonal availability and included kangaroo, wallaby, possum, bandicoot, snakes, turtles, goanna, bush turkey, fish and other seafood, in addition to a variety of fruits, nuts and seeds.17 By the 1960s, the way of life for a large number of remote Australian Aborigines had changed dramatically. Many were liv-ing in camps and missions, with the available food containing large amounts of refined flour, milk powder, fat and sugar, whilst being deficient in key micronutrients, including vitamin A and C, folate and calcium.17,20

Milk powder is of specific interest, as studies have found lac-tose intolerance figures of between 84–95% in children from the Northern Territory, with these children being in the lowest percen-tile for height and weight.21 In a more recent study, the poor and restrictive diet was found to result in low birth weight, followed by rapid ‘catch-up growth’ to around 6 months of age with a sub-sequent reduction in growth to 5 years, after which records are difficult to find.22 This is of great significance for future disease susceptibility, including type 2 diabetes.

The rapid loss of the active hunter-gatherer existence, coupled with an extensive change of diet and societal structure, has had both a severe physiological and psychological impact. Australian Aborigine and Torres Strait Islander peoples aged 15 years or over were found to be more overweight or obese than non-indigenous people (1.2 times), with 29% being classified as overweight and 37% obese. They are also 1.2 times more likely to have high blood pressure (≥149/90 mmHg) than non-indigenous people, with 20% of Australian Aborigines and Torres Strait Islanders being hyper-tensive.

Blood lipid analysis found that 25% of Australian Aborigi-nes and Torres Strait Islanders had high total cholesterol (≥5.5 mmol/L), 25% had high low-density lipoprotein cholesterol (≥3.5 mmol/L), 40% had low levels of high-density lipoprotein choles-terol (males <1.0 mmol/L, females <1.3 mmol/L) and 25% had high blood triglycerides (≥2.0 mmol/L). The same study found 42% of Australian Aborigines and Torres Strait Islanders over the age of 15 years smoked regularly, 2.6 times more than non-indig-enous people. From a dietary perspective, an inadequate intake of

fruit and vegetables was reported, with 93% of Australian Abo-riginals and Torres Strait Islanders over the age of 2 years failing to reach the minimum fruit and vegetable intake; a figure rising to 97% in over 15 year-olds.23,24

Developmental programming

This array of detrimental physiological markers represents key risk factors for the development of insulin resistance and type 2 diabetes and goes some way towards explaining the higher preva-lence of these disorders in Australian Aboriginal and Torres Strait Islander peoples. Certainly, when lifestyle and nutritional intake were re-established to more traditional conditions (i.e. bush living and eating foods including crocodile, kangaroo and native plants), individuals displayed a reduction in adiposity and beneficial ef-fects with respect to glucose tolerance, insulin sensitivity, blood lipid profile and blood pressure.25 However, we may not be look-ing at the complete picture.

While there is little doubt that improving diet and exercise reduces the risk of developing type 2 diabetes, the effects of in-adequate nutritional intake and disease during pregnancy may predispose offspring to a greater risk of diseases, including type 2 diabetes, in later life. Research pioneered by David Barker in the 1990s initially found the link between low birthweight and an increased prevalence of coronary heart disease in populations in northern England in the early 20th century.26 This was followed up by more detailed analysis of records from Hertfordshire, UK (the Hertfordshire Cohort Study), where low birthweight was associ-ated with an increased risk of death by circulatory diseases.26,27 Further study revealed insulin resistance and type 2 diabetes to be strongly correlated to low birthweight.28

Periods of famine have provided additional insight into the effects of nutrient restriction and development of the fetus. The Dutch Hunger Winter, or Dutch Famine, occurred towards the end of 1944 and was the result of a Nazi food blockade to western regions of the Netherlands. The fate of offspring born to mothers who suffered severe nutrient restriction during this period has been extensively studied.29 Offspring exposed to nutrient restriction in utero showed increased blood glucose concentrations,30 hyperten-sion and coronary heart disease.31,32 A similar pattern emerged in China after the 1958–62 famine. Prior to 1980, diabetes was al-most non-existent in China; however, over 120 million Chinese now suffer from diabetes.33 A parallel study on the same popula-tion found an increased prevalence of hypertension.33,34

This phenomenon became known as the ‘thrifty phenotype hy-pothesis’, and its basic premise is that in a suboptimal in utero environment, the fetus’ metabolic development changes in order to maximize survival in post-natal nutritional insufficiency.35 How-ever, if the nutritional availability is not limited and offspring have access to a normal or obesogenic diet, the physiological adapta-tions during gestation can predispose it to developing a variety of metabolic disorders, such as diabetes and hypertension.36,37 This branch of research now falls under the title of Developmental Ori-gins of Health and Disease, as early life factors can also impact later disease status.38

Further research has identified other factors that can influence fetal and early-life development, including smoking, maternal stress, gestational diabetes and maternal obesity.39 These disrup-tions to fetal development and future disease risk do not appear to be confined to the first generation. Programming effects have also been found in the second generation, indicating a trans-generation-al process and signaling longer term plastic adaptations.40


Sculley D.V. et al: IUGR and T2D in indigenous Australians Explor Res Hypothesis Med

Epigenetic effects of developmental programming

So, just how does a disruption to maternal nutrition cause such a deleterious effect on offspring and increase their risk of develop-ing type 2 diabetes? This article will focus on one of the main nutritional factors believed to confer these effects on a developing fetus—maternal low-protein diet. This is arguably one of the most relevant scenarios due to the change in diet of Australian Aborigi-nes and Torres Strait Islanders from a high-protein, high-fiber, low saturated fat diet to one favoring large quantities of highly-refined carbohydrates.41

It is generally accepted that the key mechanism driving devel-opmental programming of fetal tissues and organs is epigenetic re-modeling. Epigenetics encompass heritable factors that alter gene expression rather than the genetic code and includes DNA meth-ylation, post-translational histone modification and non-coding RNAs, such as microRNAs.42 DNA methylation occurs at cytosine bases (CpG sites), where they are converted via DNA methyltrans-ferase (DNMT) enzymes to 5-methylcytosine. DNA methylation serves to silence genes by inhibition of gene promoter activity and plays a vital role in embryonic development, chromosomal stabil-ity and X-chromosome inactivation.43 This was demonstrated in a study where DNMT-knockout mice died early in development.43,44 Post-translational histone modification also affects gene expression early in mammalian development via histone acetylation and dea-cetylation. Acetylation by histone acetyltransferase enzymes adds an acetyl group and reduces the bond between DNA and histones, thereby generally increasing transcription rates. Histone deacety-lase enzymes produce a more condensed form of chromatin and reduce transcription rates.45 MicroRNA is a class of non-coding RNA typically between 20–25 nucleotides long, which have the capacity to reduce gene expression at the post-transcriptional level and also to inhibit gene expression of enzymes, including DNMT and histone deacetylase.46

Programming effects on the pancreas and insulin resistance

If the Developmental Origins of Health and Disease hypothesis relates to the development of type 2 diabetes, we should be able to observe epigenetic effects in two key areas—pancreatic islet β cells, and insulin sensitivity in key organs and tissues such as the liver, skeletal muscle and adipose tissue. Fetal undernutrition, particularly that induced by a maternal low-protein diet, has been demonstrated to cause intrauterine growth restriction (IUGR), low birthweight and an increased adult prevalence of diseases includ-ing type 2 diabetes.28

More specifically, IUGR in rats via a maternal low-protein diet resulted in a reduction in pancreatic weight and mean islet β cell area, thought to be due to a combination of reduced proliferative capacity and vascularity in the pancreas. After being fed a nor-mal diet post-weaning, these same animals maintained a reduced islet β cell mass in addition to a lower insulin content.47 Another study found similar results, with caloric restriction and low-protein models both producing a 20–40% reduction in islet β cell mass at birth.48 This results in glucose intolerance at around 4 months of age and insulin resistance and type 2 diabetes by 17 months.49,50

These results are mirrored in human studies, where offspring exposed to IUGR demonstrate an increase in glucose intolerance in adulthood.51 Interestingly, the timing of maternal undernutrition is crucial, with both rat and human studies indicating a more del-eterious effect if induced later in pregnancy.52,53 This is also in line with other studies investigating nephron number and renal func-

tion using a similar IUGR model.54

Insulin functions to remove glucose from the blood when con-centrations are high and promote glucose uptake into the liver, adi-pose tissue and skeletal muscle. Insulin resistance describes the sit-uation where glucose uptake is inhibited. This results in prolonged hyperglycemia and/or hyperinsulinemia.55 As with pancreatic de-velopment, a poor maternal diet and IUGR results in an increased risk of developing insulin resistance in later life.56 In a study using both rats and humans, IUGR resulting in low birth weight lead to a significant reduction of glucose transporter 4 gene expression, thereby inhibiting blood glucose transportation into adipose tissue and skeletal muscle.57

Protein kinase C-zeta is another protein that plays a major role in insulin-mediated glucose transport. Analysis of muscle taken from offspring subjected to IUGR and of low birth weight dis-played a reduction in protein kinase C-zeta concentration, indicat-ing a decreased capacity to absorb glucose from the blood. This was coupled to a significantly higher blood insulin concentration after administration of an intravenous infusion of glucose.58 These data suggest a strong influence of IUGR on key mechanisms re-sponsible for glucose absorption from the blood to peripheral tis-sues and, in conjunction with the programming effects on pancre-atic islet β cells, implicate IUGR and low birth weight as major risk factors in the development of type 2 diabetes.

Implications and perspective

Whilst maternal undernutrition is only one determinant involved in programming and developmental plasticity, it remains a key modifiable risk factor. The shift from a high-protein to high car-bohydrate diet looks to be a major driver of type 2 diabetes in off-spring, brought about by inherited epigenetic modifications. These changes remain in future generations and could result in a habitual increase in disease risk. This effect seems to be more pronounced in Australian Aboriginals and Torres Strait Islanders, possibly due to the rapid change in diet and lifestyle.

With the colonization of Australia by Europeans and recent ex-posure to a potentially obesogenic diet, populations accustomed to a more restrictive diet may be more prone to its deleterious effects, as they have not been exposed to centuries of gradual dietary shift towards a high carbohydrate diet. A similar pattern has already been observed in other aboriginal populations around the world that may go some way to proving this point.59 Certainly, Austral-ian Aboriginal populations with higher levels of European genetic admixture had a reduced prevalence of diabetes and glucose in-tolerance, which may indicate an additional genetic component to diabetes risk.60

Specific genetic variability between different populations is out-side the remit of this article but further highlights the importance of the role of developmental programming in Australian Aboriginals and Torres Strait Islanders and goes some way towards explain-ing the higher prevalence rates of diabetes in these populations. If these indigenous populations with little to no European genetic admixture naturally present with a reduced ability to handle high carbohydrate load in their diet, the additional developmental pro-gramming effects on the pancreas and glucose transport proteins, as discussed earlier, would certainly exacerbate the situation.

Appropriate dietary intake during pregnancy, including an ad-equate protein component, may help to reduce the potentially dam-aging epigenetic changes and mediate the risk of type 2 diabetes in future generations. This is especially important considering the increase in both availability and affordability of an obesogenic


Sculley D.V. et al: IUGR and T2D in indigenous AustraliansExplor Res Hypothesis Med

diet to these offspring. A coordinated effort to provide suitable nutritional advice, together with the easy availability of essential nutrients, can potentially help to limit cases of type 2 diabetes in Australian Aboriginal and Torres Strait Islander peoples and re-duce the burden of disease and the socio-economic disadvantage it entails. Ideally, this will be in conjunction with other potential risk factors, such as stress, gestational diabetes, obesity and smoking. Data collected regarding maternal nutritional status, fetal growth rates, birthweight and on-going growth rates and physiological markers of disease in offspring would provide valuable informa-tion and help to elucidate the role of developmental programming in type 2 diabetes in the Indigenous Australian and Torres Strait Islander populations.




Study conception (DVS, ML), review of the literature and writing of the article (DVS, ML).

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http://www.healthinfonet.ecu.edu.au/health-risks/nutrition/plain-language/our-review



Review Article

Introduction

Sports-related concussion (SRC), like other acute traumatic brain injuries (TBIs), involves mechanical forces that shear neuronal, glial and endothelial membranes, inducing a complex pathophysi-ologic process that adversely affects the brain.1,2 Collectively, these forces induce a primary response involving neuronal and tissue damage, which is subsequently amplified by a ‘secondary

injury’ phase in response to membrane and cellular damage.3 This encompasses a cascade of biochemical changes that impairs func-tion of the blood–brain barrier, compromises oxygenation and po-tentiates further tissue damage.4

Targeted patient education and improved protective equipment has led to some decrease in primary injury.5 However, to date, the adverse health effects of secondary injury (e.g., sleep disruption, depression, impulsivity, decline in cognitive function, migraine, impaired vision) have not been met with effective treatment. It is generally considered that decline in neuropsychological function-ing is transient, resolving to pre-injury benchmarks within 7–10 days,6 with the timeline paralleling the acute neurometabolic events triggered by the concussive impact.7 However, reports link-ing the injury to long-term declines in cognitive functioning,8,9 de-pression and mild cognitive impairment has propagated substantial scrutiny of the “transient nature” of SRC,10–12 particularly during adolescence.

The injured adolescent brain poses a unique complexity to un-derstanding the pathophysiology of concussion from the incident to the recovery process which extend investigation beyond adult trauma,13 rodent models and cell culture towards longitudinal stud-ies assessing the short- and long-term intricate cognitive, emotion-al, behavioral, neurobiological and neuropathological consequenc-es of concussions that can also identify predictors and modifiers

Nutrition as Medicine to Improve Outcomes in Adolescents Sustaining a Sports-related Concussion

Krista Casazza1* and Erin Swanson2

1University of Alabama at Birmingham, Department of Pediatrics, Division of Adolescent Medicine, Birmingham, AL 35233, USA; 2University of Alabama at Birmingham, Department of Pediatrics, Division of Rehabilitative Medicine, Birmingham, AL 35233, USA

Abstract

Recognition and diagnosis of sports-related concussion (SRC) among adolescents has significantly increased. In, fact, among high school adolescents, SRC incidence has more than doubled from 2007 to 2014, with recent es-timates at approximately 2 per 100 athletes. SRC-related research has also increased; recognition of symptoms that may prolong recovery have been examined, potential biomarkers have been scrutinized, return-to-learn and return-to-play protocols have been developed and honed. However, to date, clinicians and researchers have struggled to find effective interventions to mitigate the significant symptoms after SRC and shorten recovery times. Despite the understood role of the brain as the primary regulator of metabolism, and the well-established metabolic impairments evoked after a concussion, nutrition is often ignored as a core complement to the re-covery and rehabilitation process. In this article, we will identify deficiencies and/or inadequacies in nutrients post-concussion and provide support for potential exacerbation of injury and delayed recovery due to inadequate intake of nutrients prior to sustaining an SRC. Additionally, we will discuss the effect of derangement of the metabolic cascade post-concussion, and identify key nutrients, that if supplemented immediately post-injury, could increase neuroprotection, and improve recovery outcomes. Animal and cell culture studies have provided substantial evidence for not only the interrelationship of nutrient adequacy and the adaptation in the metabolic cascade post-concussion on neuroprotection, but also key nutrients that if supplemented immediately post-injury could enhance standard of care with minimal risk.

Keywords: Sports-related concussion; Adolescent; Athlete; Nutrition.Abbreviations: SRC, sports-related concussion; TBI, traumatic brain injury; PUFA, polyunsaturated fatty acid; DHA, decosahexanoic acid; ATP, adenosine triphosphate; BCAAs, branched chain amino acids; CNS, central nervous system; ADP, adenosine diphosphate; CRP, C-reactive protein; ALA, alpha-linoleic acid; VDR, vitamin D re-ceptor; BBB, blood brain barrier; NADPH, nicotinamide adenine dinucleotide phos-phate; TCA, tricarboxylic acid; PARP-1, poly-adenosine diphosphate-ribose polymer-ase-1; GH, growth hormone.Received: August 14, 2017; Revised: September 25, 2017; Accepted: October 07, 2017*Correspondence to: Krista Casazza, University of Alabama at Birmingham, Depart-ment of Pediatrics, Division of Rehabilitative Medicine, 1601 4th Ave S, CPPI 310, Birmingham, Al 35233, USA. E-mail: [email protected] to cite this article: Casazza K, Swanson E. Nutrition as Medicine to Improve Outcomes in Adolescents Sustaining a Sports-related Concussion. Exploratory Re-search and Hypothesis in Medicine 2017;2(4):122–130. doi: 10.14218/ERHM.2017. 00029.



Casazza K. et al: Nutrition in sports-related concussion Explor Res Hypothesis Med

of outcomes.14 Clinical studies have focused predominantly on descriptive or observational investigations into qualitative symp-toms and/or semi-quantitative analysis of cognitive impairments.15 As such, important elements of the underlying pathophysiology of concussion have been delineated through experimental models and reports of the long-term effects of concussion in adult athletes (e.g., the National Football League).

Data collected in adults and animals, however, are unable to ac-count for developmental differences and changes in mental status, respectively. For example, Kerr et al.16 highlighted a difference in return-to-play times in adolescents demonstrating the unique adap-tions and recovery during this critical developmental period and highlighting the need for enhanced management in this popula-tion. Given the unique cascade of series of adaptive neurometa-bolic responses that take place in the brain following a head injury, optimizing treatment strategies—particularly during the critical adolescent brain developmental period—are needed.

We review research evidence for potential benefits of nutrition as medicine in SRC and explore the contention of how nutrient in-adequacies affect such. According to this concept, neurometabolic consequences contribute to the development and persistence of post-concussive symptoms after SRC in some youth. We hypoth-esize that a) nutrition supplementation initiated immediately post-SRC represents a key component of rehabilitation with particular relevance in adolescents; and b) the poorer outcomes in females can be explained at least in part by greater inadequacies of nutri-ents in females relative to their male counterparts prior to sustain-ing a concussion. Addition of nutrition as medicine to guidelines that emphasizes prompt supplementation is encouraged.

A key pathophysiological phenomenon after a mechanical trauma to the brain is the complex cascade of neurochemical and neurometabolic events and consequent alterations in cerebral me-tabolism and blood flow, which may result in an energy crisis.7,17,18 Two major alterations of glucose metabolism have been described: hyperglycolysis (and thus hyperglycemia) and oxidative dysfunc-tion (rampant activation of the inflammatory cascade). Particular-ly, glutamate release from damaged cells leads to excitotoxicity, mounting an adverse response including the influx of Ca2+ through glutamate receptors and voltage-gated Ca2+ channels.19,20 This in turn activates calcium-dependent proteases, such as calpains and lipases, which degrade membrane phospholipids, leading to the release of fatty acids.7,17,18 Fatty acids (primarily arachidonic acid, the precursor of eicosanoids) are released from membranes and transformed into pro-inflammatory eicosanoids which exacerbate tissue injury by inducing vasoconstriction and platelet aggrega-tion, increased inflammation, and production of free radicals.21 Within hours, there is also activation of complex dynamics of local cytokine and chemokine production. Concomitantly, synthesis and release of anti-inflammatory factors such as the ω-3 polyunsatu-rated fatty acid (PUFA) docosahexanoic acid (DHA) are oxidized as fuel; thus, their role as structural components of membranes needed for cellular repairs is attenuated.22

The stress response is also known to induce gluconeogenesis and glycogenolysis, as well as the release of cortisol, further exac-erbating hyperglycemia and as a consequence augmenting cellular injury and secondary response.7,17,18 Increased glutamate release and subsequent increased lactate production (and acidosis) are also associated with hyperglycemia. Although a general characteriza-tion of the post-concussion adaptive responses exists, each concus-sion is unique, with developmental stage, sex and race differences observed in addition to brain region that was affected.

We contend supplementation-specific dietary components alone and/or in combination have the potential to positively influence and to effectively treat the neuroinflammation associated with

SRC. Despite the understood role of the brain as the primary regu-lator of metabolism and the well-established metabolic impair-ments evoked after a concussion, nutrition is often ignored as a core complement to the recovery and rehabilitation process. Here-in, we will identify deficiencies and/or inadequacies in nutrients post-concussion and provide support for potential exacerbation of injury and delayed recovery due to inadequate intake of nutrients prior to sustaining an SRC.

Specifically, among high school adolescents, SRC incidence has more than doubled from 2007 to 2014,23 with recent estimates at approximately 2 per 100 athletes.24 Rapid changes in brain or-ganization and development occur in adolescence, with profound reshaping of the prefrontal, parietal temporal-associated, and sen-sorimotor cortex prior to achieving adult-like connections by the mid-20s. These areas, which do not fully mature until approxi-mately 18 years of age, play a substantial role in executive func-tions such as problem solving and decision making.14 Paralleling the structural changes are changes in cerebral glucose metabolism and cerebral blood flow.25

Although post-concussed adolescent athletes often display no gross neural pathologies and have no immediate threat to their life, profound biochemical changes induced by injury in immature, highly-sensitive structures (e.g., the prefrontal cortex, hippocam-pus, hypothalamus) during this critical developmental stage can increase risk for several neuropsychiatric conditions, with far-reaching acute and chronic systemic effects. Brain injury, of any severity, in the developing brain is complicated by ongoing cer-ebral maturation.26 Of crucial importance in prognosis is the rapid increase in reactive stressors that heighten the metabolic demand on the brain.27

Concussion induces inflammatory sensory stimulus that is preferentially transmitted to brainstem and specific hypothalamic nuclei which have direct connection to the hypothalamic paraven-tricular nucleus. Specifically, activation of the inflammatory cas-cade, increased protein catabolism and altered energy metabolism have been well-established as inducing long-term adverse effects on the developing brain via altered neuroendocrine and physiologic processes.28 Increased inflammation markedly affects neurotrans-mission within emotional regulatory brain circuits and can dys-regulate the hypothalamic-pituitary-adrenal axis. Consequently, a strategy that enhances the brain’s response against harmful effects of inflammation and protein catabolism may potentiate damaging post-concussive effects in the stress-sensitive brain structures of the adolescent athlete.

Energy (macronutrient) and micronutrient (vitamin and min-eral) needs during adolescence are relatively high, as compared to in adulthood. While few studies have evaluated the dietary pat-terns of young athletes, particularly those participating in sport at the community/high school level and especially based on sex, race/ethnicity and socioeconomic status, it has been reported that dietary intakes in young athletes are reportedly superior to their non-athletic counterparts.29–32 However, the increased demands of competition in the context of growth and development imply that the potential consequences of a deficiency may be more detrimen-tal in athletes.

To get a better understanding of how certain nutrients may aid in the treatment of SRC, it is important to understand the neuromo-lecular cascade that occurs in the brain after a concussion, which has been best studied in animal TBI models.33 Observational data consistently show that during the initial post-concussed state, ener-gy and protein deficits are apparent and are associated with worse outcomes. Insufficient micronutrient intakes in the general popula-tion adolescent population can profoundly impact energy and ma-cronutrient metabolism, particularly for the B vitamins, vitamin D


Casazza K. et al: Nutrition in sports-related concussionExplor Res Hypothesis Med

and iron. In this review, we will demonstrate specific nutrients can be utilized for post-SRC supplementation based on their putative roles in the mechanisms of brain injury.33

Energy crisis

In an effort to restore cellular homeostasis and membrane potential, hyperactive adaptive responses of adenosine triphosphate (ATP)-requiring ion pumps occur, causing hyperglycolysis and concomi-tant low intracellular energy reserves.3 In the context of reduced cerebral blood flow (due to edema), a mismatch between energy supply and demand ensues. Disturbance in intracellular calcium flux, manifests into sequestration of calcium into mitochondria, which can then result in mitochondrial dysfunction, exacerbate oxidative metabolism and worsen the cellular energy crisis. Af-ter an initial period of hyperglycolysis and metabolic uncoupling, glucose metabolic rates go into a state of hypoglycolysis. Energy crisis is associated with behavioral impairments in spatial learning as well as altered gene expression and enzyme/transporter regula-tion, which may underlie long-term sequelae.

Glucose

The post-injury energy crisis and glucose metabolic disturbance is exacerbated by increased flux of glucose through the pentose phos-phate pathway and inhibition of key glycolytic enzymes. Emerg-ing data suggest that under these post-brain injury conditions of impaired glycolytic metabolism, glucose may not be the best fuel for the injured brain.34–36 While the neuroprotective benefit of a ketogenic diet after SRC has yet to be investigated systematically, some studies in animals have demonstrated improvement in contu-sion volume and behavioral outcomes with a ketogenic diet.37

Protein

Protein and its amino acid building blocks play critical roles in various metabolic and energetic pathways. As a substantial amino acid pool does not exist, protein homeostasis depends upon a func-tioning system of protein degradation and recycling.38 The effects of energy crisis trigger protease activation and apoptotic cell death, limiting protein synthesis. Further, the profound increases in rest-ing energy expenditure in the injured brain and associated negative nitrogen balance induce transient protein inadequacy.39

The lack of availability of essential amino acids due to sec-ondary inflammatory cellular damage not only adversely affects cell survival and function in the injured brain but can also induce system-wide detrimental effects. Increased protein catabolism is observed within 24 hours of injury, and low levels of branched-chain amino acids (BCAAs), histidine and methionine have been shown to persist 2 months after injury.40–43 Protein catabolism and subsequent hypoalbuminemia can also disrupt mineral availability (e.g., zinc, iron),44 further exacerbating oxidative stress.45 Restora-tion of protein and nitrogen balance may reduce oxidative stress secondary to concussive injury and facilitate recovery.46

Beyond protein synthesis, essential amino acids, provided only through the diet, serve as a substrate for key molecules. For exam-ple, BCAAs are integral in overall energy metabolism, regulation of gluconeogenesis and protein synthesis, as well as functioning as a major source of nitrogen for glutamine and nitric oxide pro-duction. As such, inadequate protein and BCAA availability in the post-concussed state impacts a number of biological processes,

including cytoprotection and gene expression.47 While it has been suggested that protein inadequacy may be related to a decreased overall intake following a brain injury, a study of healthy fasted volunteers versus individuals with a traumatic brain injury, in which both groups had not eaten for 7 hours demonstrated signifi-cantly greater protein catabolism in TBI. Given that the adolescent athlete requires approximately 1.2–1.5 g/protein/day to maintain lean body mass and normal protein synthesis, protein supplemen-tation post-concussion is a reasonable strategy to improve out-comes following a SRC.

BCAAs

In the brain, BCAAs serve as important metabolic precursors for synthesis of proteins and neurotransmitters, including dopamine, serotonin and norepinephrine.48 Following brain injury, BCAAs become readily oxidized, potentially contributing to the metabolic crisis post-SRC. Lower BCAAs in TBI patients relative to controls has been observed in various studies.40–43,49 BCAA supplementa-tion in rodents has been shown to restore net synaptic efficiency and hippocampal function and consequent restoration of cognitive function.50–52

Creatine

Creatine is well-known for its use in athletes for purported benefits regarding muscle hypertrophy. However, as a biochemical effect in the central nervous system (CNS) via phosphorylation of adeno-sine diphosphate (ADP) to make ATP, and theoretically decreasing hyperglycolysis and oxidative damage, creatine has emerged as a potential treatment of concussions.53 The primary role of creatine in the body involves energy homeostasis via maintenance of con-stant cellular ATP levels in tissues with highly fluctuating energy demands (i.e. muscle and brain).54,55 The creatine-ATP energy system enables rapid, efficient ATP regeneration in the absence of oxygen. The system decreases glycolysis and the consequential synthesis and accumulation of lactic acid.

Assessment of creatine pre- and post-concussion by magnetic resonance spectroscopy has shown decreased levels in the brain after sustaining a concussion.56 A potential antioxidant effect has also been hypothesized. In animal models, creatine supplementa-tion pre-TBI has demonstrated a neuroprotective effect on mito-chondrial function and secondary injury.57 Human studies, though limited to severe TBI, have shown increased cognitive function and decreased headache and dizziness.58

DHA

PUFAs, specifically ω-3, are essential for the rapid neurogenesis during brain development as well as neuroprotection across the lifespan.59 Dietary depletion has been shown across species to impair structural and neuronal development and function in early life.60 In addition to the structural role, ω-3 PUFAs, particularly DHA, are involved in multiple brain functions, including cell membrane fluidity, receptor affinity and modulation of signal transduction molecules.61–63 The mechanisms involved in ω-3 PUFAs’ neuroprotection over the life course (including injury) include decreased neuroinflammation and oxidative stress, neuro-trophic support and activation of cell survival pathways. The level of total ω-3 PUFAs in plasma is inversely correlated with the level of pro-inflammatory markers (IL-6, TNFα, IL-1 and C-reactive



protein (CRP)) and the ω-6/ω-3 PUFA ratio is negatively corre-lated with IL-10 anti-inflammatory marker.

ω-3 PUFAs are synthesized through desaturation and elongation reactions from their precursor, an 18-carbon-atom fatty acid, alpha-linolenic acid (ALA), which is available in the diet.64 However, the ω-3 PUFA required by the CNS is essential, and not produced de novo in mammals. Under pathological conditions, including SRC, phospholipases rapidly release PUFAs from membranes.59 The accumulation of free fatty acids activates inflammatory path-ways through cell-specific receptors and activation of protein kinases. In turn, a large number of lipid mediators augment oxi-dative stress and induce mitochondrial dysfunction.65 DHA, the primary structural ω-3 PUFA present in the brain, preferentially accumulates in the frontal cortex and hippocampus, constituting up to 97% of the ω-3 PUFAs in the brain. It is converted to ox-ylipin intermediates, thus rendering high CNS demands for DHA unattainable.65 The unavailability of DHA in such instances as TBI, affects physical properties of membranes via alteration of transmembrane enzymes and binder receptor proteins as well as neurotransmitter synthesis and release.66 Substantial evidence for animal studies investigating ω-3 PUFA supplementation before or after TBI supports a protective effect of ω-3 PUFA on the brain by limiting structural damage to the axon and attenuating events inducing neuronal apoptosis.

While an adequate supply of DHA is recommended, worldwide consumption is low, with most adolescents having an inadequate intake of ω-3 PUFA.67 Dietary sources of DHA are limited, with cold-water algae being the primary producers of DHA and EPA. Fish are also rich sources of DHA due to a diet consisting of algae. Dietary intake of the essential FA and precursor to DHA α-linolenic acid (ALA; 18:3n-3) is generally much higher.68,69 Although ALA can be metabolically converted to DHA, the conversion rate is low. Further, diets higher in ALA seem to limit the conversion rate by increasing the rate of ALA oxidation,70 and contemporary food manufacturing has dramatically altered the ω-3/ω-6 PUFA ratio in the Western diet. There has been an increase in ω-6 PUFA intake and concomitant decrease in ω-3 PUFA intake.

In humans, dietary supplementation with DHA against acute and chronic inflammation within the CNS was first reported as case studies (i.e. lone survivor of the Sago Mine disaster, TBI after a motor vehicle accident in an adolescent), in which recovery ap-peared to be associated with supplementation of PUFAs. Supple-mentation of DHA has been shown to reduce glutamate-induced excitotoxicity and both axonal and neuronal injury through mod-ulation of ion channels. In embryonic hippocampal and cortical neuron cultures, supplementation with DHA has been shown to increase neurite number and length.71 In rodent models, DHA sup-plementation post-moderate TBI was shown to decrease number of axon apoptosis and to increase hippocampal neurons as well as decrease pro-inflammatory cytokines and markers of oxidative stress.71–76

Rodent models have also been fairly consistent in demonstrat-ing a beneficial effect of DHA supplementation through multiple signaling pathways in brain injury, spinal cord injury and cardiac ischemia-reperfusion.65 Specifically in models of head injury, prophylactic supplementation with DHA attenuates white matter damage, as evidenced by fewer beta amyloid precursor-positive axons, enhanced preservation of myelin, and protection of neu-rofilament morphology.73,77–80 Further, DHA supplementation has been shown to allay glutamate cytotoxicity, suppress mitochon-drial dysfunction and the eventual development oxidative stress, decrease calcium influx, and down-regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits.81 While the preservation of white matter undoubtedly aids in maintaining

neurocognitive function following injury, the blunting of injury-induced reductions in molecular elements important for learning may also play a role.82

While the precise mechanism by which DHA confers a neuro-protective effect is multifaceted and not completely understood, it likely includes a series of mutual mechanisms that enhance the structural integrity of neurons, thereby diminishing the secondary pathological sequelae (i.e. neuroinflammation, impaired energy metabolism, glutamate release) that occurs post-injury. In summa-ry, ω-3 PUFAs have been shown to address several of the hallmark pathologic features of brain injury, such as excitotoxicity, oxida-tive stress, and inflammation. A dose-dependent relationship ex-ists, whereby plasma phospholipid DHA concentrations increase up to a dosage of approximately 2 g/day, after which any further increase in dose negligibly increases plasma phospholipid concen-tration.67 However, despite substantial evidence from rodent mod-els of TBI, human-based studies examining the neuroprotective effects of DHA have been limited to clinical case studies.

Vitamin D

Vitamin D, most notably studied for its role in calcium homeosta-sis, has emerged as a significant contributor to a broad range of physiologic activities, including muscle function, metabolic con-trol and immune modulation. More recently, it has been identified as a substance with substantial involvement in brain development, health and function.83 The vitamin D receptor (VDR) protein is reportedly expressed broadly in the brain, including the neurons and glial cells.84 Further, 1,25(OH)D appears to influence neuronal cell differentiation and exert neuroprotective actions against cyto-toxicity. Vitamin D deficiency exacerbates inflammatory response and cell death.83

The role of vitamin D supplementation post-TBI and/or inves-tigation of the role of vitamin D deficiency on outcomes following TBI was recently reviewed.83 A significant reduction in phospho-rylation of nuclear factors, which in turn stimulate downstream genes involved in the inflammatory cascade, were shown to be reduced by vitamin D treatment post-TBI.85 In separate studies, Tang et al.86 and Cekic et al.87 demonstrated that vitamin D defi-ciency was associated with more adverse pathophysiological out-comes following TBI. Of note, an estimated 40% of adolescents have insufficient vitamin D.88 Further, in a clinical sample, Hua et al.89 observed improved functional outcomes with vitamin D sup-plementation post-TBI.

Despite its emergence as an integral cofactor in brain health, there have been no clinical trials to assess the efficacy of vitamin D as a treatment for mild TBI. Studies conducted in other aspects of neurocognition (i.e. stroke, Alzheimer’s, dementia) have es-tablished an association with vitamin D insufficiency and suggest prevention of vitamin D deficiency may serve a valuable role as a neuroprotective therapy.45,90,91

Thiamin

Restoration of energy metabolism and membrane homeostasis may reduce oxidative stress secondary to concussive injury and facilitate recovery. “Much of the role in neuronal excitability has been derived from the evaluation of effects of alcohol on thiamine diphosphate in the context of the brain and Wernike-Korsakoff syndrome. The synergistic effects of thiamin deficiency and alco-hol-induced neurotoxicity from excessive glutamate release elicit acute cerebral damage and subsequent irreversible neuronal dam-



age.”92 It is biochemically analogous that, in TBI, changes due to inadequate thiamin primarily involve decreased activity of the enzymes, a process that leads to energy deficits as a consequence of impairment of the tricarboxylic acid (TCA) cycle which can ultimately lead to neuronal apoptosis.

Altered energy metabolism, secondary to unavailability of thia-min co-factors, also results in the development of lactic acidosis in response to impaired TCA cycle activity. This acidosis can con-tribute to cytotoxic brain edema, a contributor to excitotoxicity, as well as a net increase in free radical production and oxidative stress along with development of inflammatory processes. Because thiamin represents an integral cofactor involved in processes asso-ciated with the metabolism of lipids, glucose, amino acids and neu-rotransmitters, inadequate levels can lead to severe complications in the nervous system.93 Diminished availability of thiamin in the brain profoundly limits enzyme activity, leading to alterations in mitochondrial activity, impairment of oxidative metabolism, de-creased energy status, and exacerbation of secondary injury path-ways.93

Accumulating evidence indicates that deficiency in thiamin in-duces mild chronic impairment of oxidative metabolism of micro-glia, astrocytes and endothelial cells, as well as abnormalities of cerebral glucose metabolism. This in turn, increases neuronal apo-ptosis. In animal models, thiamin inadequacy is associated with pathology of brain injury.94 Compromised mitochondrial function, is also apparent and leads to increased free radical production that contributes to neuronal dysfunction and apoptosis. Further, inad-equate thiamin dysregulates glucose metabolism, which induces alterations in cerebral blood flow, along with compensatory stimu-lation of lactic acidosis, which contributes to impairments in the blood–brain barrier (BBB) breakdown. BBB disruption then leads to decreased fatty acid synthesis, which itself can lead to demyeli-nation and decreased nucleic acid synthesis.93

Niacin

Niacin (nicotinamide; vitamin B3) is regarded as a broad-spectrum neuroprotectant. Its role is as a precursor for NAD+, the coenzyme commonly known for its role in the electron transport chain, al-lowing for the production of ATP.45 Activation of microglia in TBI causes up-regulation of nicotinamide adenine dinucleotide phos-phate (NADPH) oxidase and activation of the inflammatory cas-cade and consequent cytotoxicity. Nicotinamide supplementation has been shown to attenuate secondary damage in TBI.95

The mechanistic underpinnings of the purported benefits have been linked to increased bioavailability of ATP to cells following the neural insult, an inhibition of injury-induced poly-adenosine diphosphate-ribose polymerase-1 (PARP-1) and sirtuin-1 activa-tion, both of which act to deplete NAD+ and reductions in apoptot-ic and necrotic death. The decrease in NAD+ is that which is often linked to apoptosis. NAD+ theoretically provides cytoprotection through pathways that involve poly(ADP-ribose) polymerase, Akt, mitochondrial membrane potential and cysteine protease activity, and prevents apoptosis by maintaining DNA integrity as well as protects against microglial activation with subsequent phagocytic destruction of cells.96 Niacin is also actively involved in redox re-actions, modulates the mitochondrial permeability transition pore and replenishes nicotinamide adenine dinucleotide phosphate lev-els with resultant increases in glutathione reducing free radicals produced following injury.

Direct and indirect inhibition of PARP-1, has also been re-ported to have beneficial effects on tissue and behavior following injury, while activation of the PARP-1 pathway has been shown

to be detrimental. In clinical trials, nicotinamide supplementation has been reported to significantly reduce injury volume, decrease glial fibrillary acidic protein activation, reduce the blood brain barrier breach, reduce acute edema, reduce behavioral impair-ments, and improve outcomes.97 Additionally, niacin treatment has been shown to counteract gene expression changes due to TBI via down-regulation of genes activated by injury.

Iron

The increased need for iron in adolescents to support growth, ex-pansion of red blood cell volume and addition of lean body mass have been long appreciated. Iron is important to maintain adequate hemoglobin concentrations as well as total iron stores during growth. Furthermore, it is well documented that iron deficiency anemia can lead to fatigue, performance, and learning ability.98,99 Brain injury is associated with iron deficiency and has been shown to exacerbate symptoms of fatigue and muscle weakness.100 In turn, iron deficiency impairs erythropoiesis and enhances erypto-sis, resulting in anemia. Improper distribution of iron within the body has been suggested to contribute to the iron deficit in the erythropoietic system and iron overload in the substantia nigra in neuroinflammatory conditions, such as Parkinson’s disease.101 Moreover, iron modulates dopamine synthesis and re-uptake (as does vitamin C at the level of dopamine β-hydroxylase); tyros-ine hydroxylase, an enzyme responsible for dopamine synthesis, is iron-dependent. Iron deficiency has also been shown to impair dopamine re-uptake in rodents.102

Sex differences in concussion outcomes

Although arguably confounded by across-sport participation num-bers and inconsistencies in baseline measures, sex differences in both the incidence and outcomes of concussions have been re-ported.103,104 Collectively, in evaluations of sports played by both sexes (e.g., soccer, baseball/softball, basketball), concussions rep-resented a greater proportion of total injuries in females, with a greater number and severity of symptoms, poorer outcomes, and longer duration to recover.103,105–111 The mechanisms underlying this sex difference are unclear, and may in part relate to biological differences that are compounded by cultural distinctions in how concussions are reported and managed. Proposed differences in neck strength, movement biomechanics and symptom reporting prevalence in females compared with males have been suggested. However, we contend dietary factors likely play a substantial role.

Nutrition parameters in adolescent athletes are sparse, with the majority focused at the college level. In female collegiate athletes, Sato et al.112 reported blood thiamin concentration decreased sig-nificantly during training, with a greater magnitude in females. Iron balance of the female adolescent athletes, beyond the tradi-tional explanation of dietary intake/iron loss balance, has emerged as a significant concern. A recent study among collegiate athletes observed 2.2% iron deficiency anemia and 30.9% iron deficien-cy without anemia in females, as compared to 1.2% and 2.9%, re-spectively, in males.113 Further, Sandstrom and colleagues recently reported lower serum iron and elevated hepcidin in the athlete group relative to that in the non-athlete group.114

Impaired iron balance also influences metabolic fuel availabil-ity and associated growth hormone (GH) function.114 In addition, inadequate iron suppresses ghrelin concentrations. Moreover, iron deficiency coupled with impaired GH function observed in SRC may reduce free fatty acid availability to further lower serum glu-



cose concentrations and enhance muscle protein degradation.115

Key points

● Although impaired glucose metabolism is characteristics of SRC under post-brain injury conditions of impaired gly-colytic metabolism, glucose may not be the best fuel for the injured brain.

● Protein is essential for normal brain energy metabolism. Inadequacy leads to a wide-ranging cascade of events, many of which overlap, and may interact, ultimately re-sulting in neuronal apoptosis.

● Given the consistent reporting of depressed endogenous levels and absence of adverse outcomes from supplemen-tation of BCAAs, supplementing appears feasible.

● A potential synergistic effect of creatine supplementation post-SRC on muscular and cognitive function warrants in-vestigation.

● Animals supplemented with ω-3 PUFAs consistently ex-hibit enhanced resilience to TBI, with functional outcomes mirroring those biological indicators of injury, even fol-lowing multiple mild TBIs, similar to that which would be observed in repetitive sports-related concussive injuries over a lifetime of play.

● Thiamin is essential for normal brain energy metabolism. Inadequacy leads to a wide-ranging cascade of events, many of which overlap, and may interact, but ultimately result in neuronal apoptosis.

● Evidence for improved outcomes using vitamin D supple-mentation, particularly in those who are deficient, lends credence to the potential for use to limit complications.

Future directions/prospective/prediction

Because of the complex and multifactorial nature of the adverse effects of the TBI process, identifying a single strategy that will be sufficient to address the sequelae of physical, behavioral and cognitive manifestations is unlikely. However, capitalizing on the multitude of beneficial effects of dietary components represents a cost- effective strategy for reducing both the cognitive and behav-ioral deficits. Further study in humans is needed to demonstrate the utility of post-concussive supplementation for cognitive and behavioral recovery after mild TBI.

While nutritional intervention has been shown to change meta-bolic function in cell culture, brain function in animal models of mild TBI as well as other neurological populations, some impor-tant unanswered questions remain. For example, with similar se-verity of injury, why are complications more severe among some individuals? Will baseline nutritional status prior to concussion in-fluence response to treatment? We hypothesize that the differences in response to and recovery from concussion include physiologic as well as environmental factors.

Though improving treatment post-concussion has received recent attention, lack of baseline data describing the underlying processes limits identification of a successful approach to improve long-term outcomes and elucidate differences in complications be-tween sexes as well as emerging differences among racial groups. Future studies will require a pre-participation baseline assessment in student athletes. Given the intense consequences of lack of fuel availability for recovery of the injured brain, with little informa-tion on the nutritional needs in the still developing adolescent

brain, evaluation of baseline nutritional status (i.e. energy metabo-lism, essential nutrient adequacy) is required.

Conclusions

Treatment strategies of adolescents sustaining an SRC often in-clude principles, guidelines and recommendations developed for adults. A number of unique concerns remain with respect to the developing brain, including the greater susceptibility to long-term sequelae. The sudden and profound “neurotoxicity” induced by biochemical changes following SRC manifest energy crisis, mi-tochondrial dysfunction, activation of the inflammatory cascade, and metabolic vulnerability to nutritional inadequacies. Prompt initiation of nutritive therapy represents an integral facets of con-cussion management. Concussive outcomes may differ in terms of the precise underlying biochemical anomalies and functional impairments, and by sex.

Current best-practice guidelines recommend a period of cogni-tive and physical rest, followed by a gradual return to school and activity, with little (if any) focus on restoring metabolic sufficien-cy. The concept of physical and cognitive rest as the cornerstone of concussion management asserts that during the acute (1–7 days, which are arguable longer in youth) post-injury period of increased metabolic demand and limited ATP reserves, non-essential activity draws oxygen and glycogen away from injured neurons. This “shut down” or “dark closet” approach following concussion ignores nu-trition as a key component of therapy.

Nutrient adequacy is essential for compensatory metabolic reg-ulation, yet is often overlooked as a critical component to metabol-ic recovery after concussion. Sufficiency of endogenous substrates is a strong determinant of recovery and rehabilitation; thus, nutri-tion supplementation represents a viable and valuable component of treatment, particularly in adolescents post-SRC.


The authors have no conflict of interest(s) related to this publica-tion.


Conception and/or the acquisition of literature reviewed (KC, ES), interpretation of the overall concepts (KC, ES), drafting of the manuscript (KC, ES), revising or questioning its intellectual content (KC, ES), and giving approval of the final version of the manuscript for publication (KC, ES).

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Original Article

Vitamin D-related Nutrigenetics and Cognitive Decline in an Elderly Population

Charlotte Martin1, Zoe Yates2, Martin Veysey3,4, Katrina King4, Suzanne Niblett4 and Mark Lucock1*

1University of Newcastle, Faculty of Science & IT, School of Environmental & Life Sciences, Ourimbah, New South Wales, Australia; 2University of Newcastle, Faculty of Health, School of Biomedical Sciences & Pharmacy, Ourimbah, New South Wales, Australia;

3Teaching & Research Unit, Central Coast Local Health District, Gosford, New South Wales, Australia; 4University of Newcastle, Faculty of Health and Medicine, School of Medicine and Public Health, Callaghan,

New South Wales, Australia

Abstract

Background and objective: Vitamin D has been linked to brain function. To date, there have been limited studies investigating vitamin D receptor (VDR) genetic polymorphisms and cognition. The objective of this study was, there-fore, to examine whether any relationships exist between VDR polymorphisms and cognitive decline in an elderly population.

Methods: Six hundred and fifty participants aged ≥ 65 years were recruited from the Central Coast, New South Wales, Australia, and were genotyped for 8 VDR polymorphisms (VDR-ApaI, VDR-BsmI, VDR-TaqI, VDR-FokI, VDR-Tru91, VDR-Cdx2, VDR-A1012G, and VDR-NIaIII). Gene variants were identified using polymerase chain reaction, followed by restriction fragment length polymorphism analysis and gel electrophoresis. Cognitive decline was measured using the mini-mental state examination (MMSE), while a self-administered food frequency question-naire was used to estimate participants’ dietary intake of vitamin D.

Results: Odds ratio (OR) analysis found that VDR-BsmI and VDR-TaqI polymorphic alleles were both associated with increased risk of cognitive decline (OR = 1.55 and OR = 1.49, respectively). VDR-TaqI was also found to be significantly associated with MMSE score, following adjustment for age and sex (p = 0.0005). Examination of the distribution of VDR-TaqI genotypes showed that a greater proportion of participants with the homozygous reces-sive tt genotype had some degree of cognitive decline (24%). As might be predicted, a significant association was also observed between age and MMSE score (p = 0.015). When examined by sex, a significant relationship was found between age and MMSE for females (p ≤ 0.0001) but no relationship was observed in males. Dietary intake of vitamin D did not influence MMSE outcomes in this cohort.

Conclusions: The VDR-BsmI and VDR-TaqI genetic polymorphisms are associated with cognitive decline in an elderly population.

Keywords: Vitamin D; Cognition; Aging.Abbreviations: AD, Alzheimer’s disease; CI, confidence interval; FFQ, food fre-quency questionnaire; MCI, mild cognitive impairment; MMSE, mini mental state examination; OR, odds ratio; PD, Parkinson’s disease; PCR, polymerase chain reac-tion; RFLP, restriction fragment length polymorphism; RHLS, Retirement Health and Lifestyle Study; SNP, single nucleotide polymorphism; UVR, ultraviolet radiation; VDR, vitamin D receptor.Received: March 27, 2017; Revised: June 21, 2017; Accepted: July 19, 2017*Correspondence to: Mark Lucock, School of Environmental & Life Sciences, Uni-versity of Newcastle, P.O. Box 127, Brush Rd, Ourimbah, NSW 2258, Australia. Tel: +61 43484109, Fax: +61 2 4348 4145, E-mail: [email protected] to cite this article: Martin C, Yates Z, Veysey M, King K, Niblett S, Lucock M. Vitamin D-related Nutrigenetics and Cognitive Decline in an Elderly Population. Ex-ploratory Research and Hypothesis in Medicine 2017;2(4):131–138. doi: 10.14218/ERHM.2017.00006.

Introduction

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and participates in a number of diverse biological ac-tions, due to its distribution in almost all organs and tissues. This expression pattern includes the brain, where VDR expression has been detected in the hypothalamus, hippocampus, cortex and sub-cortical regions,1,2 which are essential for cognition. The active form of vitamin D, 1,25(OH)2D3, plays an important role in neu-ronal differentiation and maturation by controlling the synthesis of neurotrophic agents,1,3 and also by regulating the expression of numerous genes involved in neurotransmitter synthesis, predomi-



Martin C. et al: Vitamin D and cognitive declineExplor Res Hypothesis Med

nantly in the hippocampus (Fig. 1).4,5

The VDR is well known for modulating the transcription of genes encoding proteins that execute the “classic” genomic functions of vitamin D for skeletal and mineral homeostasis. However, it also regulates the expression of several genes that mediate “non-classi-cal” actions in non-calcemic tissues. Many of these “non-classical” actions are associated with decreased risk for disorders associated with ageing, including those linked to the nervous system.6,7 The elderly are particularly at risk for a number of degenerative disorders related to low serum calcidiol (25(OH)D3) levels. This is due to a decrease in ultraviolet radiation (UVR) exposure as a consequence of changes in lifestyle, particularly a decline in outdoor activity.8

Diet can also become less varied with age, resulting in low intake of vitamin D content.9 Most significant, however, is a de-crease in the ability to synthesise cutaneous vitamin D after UVR exposure–a result of atrophic changes in the skin and a reduced level of the vitamin D precursor, 7-dehydrocholesterol.10 Investi-gation of the impact of vitamin D deficiency on the ageing brain

is critical, with evidence linking low vitamin D status with poorer outcomes on one or more cognitive function tests,11,12 or a higher frequency of dementia,13 mood disorders,14 cognitive decline and Alzheimer’s disease (AD).15–17

The VDR gene is located on chromosome 12 (12q13.11) and con-tains several known polymorphisms. VDR-ApaI (G>T substitution) and VDR-BsmI (A>G substitution) are restriction fragment length polymorphisms (RFLPs) located at the intron between exon 8–9, and are considered to be silent single nucleotide polymorphisms (SNPs) as they do not change the amino acid sequence of the encoded VDR protein.15,16 They may, however, alter gene expression through reg-ulation of messenger RNA.16,17 VDR-TaqI (T>C substitution) is a RFLP in exon 9, while the VDR-FokI polymorphism (T>C substitu-tion) is situated at the start of the codon in exon 2 of the VDR gene.18 The VDR-Tru91 (G>A substitution) located within the intron 8 re-gion has been studied to a lesser extent.16,19 Other polymorphisms in the 1A promoter region, including VDR-Cdx2, VDR-A1012G and VDR-G1520C, have also recently been reported.20

The objective of this study was to examine the relationship be-tween eight vitamin D-related genetic polymorphisms and cogni-tive decline in an elderly cohort. Furthermore, dietary intake of vitamin D and other related markers were analysed to determine their influence on cognition.

Methods

Study design

A total of 650 participants (287 males and 363 females, aged 65–95 years) living independently in either a retirement village or within the community on the Central Coast, New South Wales were assessed for the prevalence of eight VDR genetic polymor-phisms, dietary intake of vitamin D, and cognitive ability using the mini-mental state examination (MMSE). Following screening, all dementia participants were excluded from the study.

Informed consent was obtained from all participants prior to study participation. This cross-sectional study was approved by the University of Newcastle Human Research Ethics Committee (H-2008-0431), and the Occupational Health and Safety Commit-tee (28.2009). The data used in the present study formed part of a larger Retirement Health and Lifestyle Study (RHLS) funded by an Australian Research Council Linkage Project Grant.

DNA analysis

VDR gene variants were examined using polymerase chain reac-

Table 1. Summary of the PCR reagents and thermal cycling conditions for each VDR polymorphism

Gene variant GoTaq® Green Master Mix, µL

Primer in 5 pmol, µL DNA, µL H2O, µL Thermal cycling conditions

VDR-A1012G 10 4 2 4 95°C x 2 min; 35 x [95°C x 30 sec; 58°C x 30 sec; 72°C x 30 sec]; 72°C x 7 min; hold at 15°C

VDR-TaqI, VDR-ApaI, VDR-BsmI, VDR-FokI, VDR-Tru91***

10 4 2 4 95°C x 3 min; 35 x [95°C x 30 sec; 61°C x 30 sec; 72°C x 30 sec]; 72°C x 7 min; hold at 15°C

VDR-Cdx2 G*VDR-Cdx2 A*

1010

2.41.6

22

44

95°C x 3 min; 35 x [95°C x 30 sec; 56°C x 45 sec; 72°C x 30 sec]; 72°C x 7 min; hold at 15°C

VDR-NIaIII 10 4 2 4 95°C x 2 min; 35 x [95°C x 30 sec; 58°C x 30 sec; 72°C x 30 sec]; 72°C x 7 min; hold at 15°C

Fig. 1. Interaction of calcitriol in the target cell in regulating metabolic processes such as brain health.


Martin C. et al: Vitamin D and cognitive decline Explor Res Hypothesis Med

tion (PCR) to amplify blood DNA, followed by RFLP analysis and gel electrophoresis. Initially, a QIAamp DNA blood mini-kit was used to extract DNA from whole blood using the QIAamp blood and body fluid spin protocol.21 The optimal PCR reaction mixture and temperature varied for each polymorphism examined. Table 1 provides the thermal cycling conditions for each VDR polymorphism, whilst Table 219,22–26 gives the primer sequence details for VDR-ApaI, VDR-BsmI, VDR-TaqI, VDR-FokI, VDR-Tru91, VDR-Cdx2, VDR-A1012G and VDR-NIaIII. RFLP was performed following PCR amplification, as described in Table 3. VDR-Cdx2 requires a nested primer allele-specific strategy for genotype scoring, eliminating the need for enzyme digestion.

Examples of each genotype for each VDR polymorphism can be seen in Figure 2.

MMSE

The MMSE is a screening tool used to assess dementia, and fo-cuses solely on the cognitive characteristics of mental functions, excluding questions involving mood, abnormal mental experi-ences and the form of thinking.27 The MMSE is comprised of 11 questions, covering: orientation to time; orientation to place; registration of three words; language; and visual construction. A

Table 2. Primer sequences for each VDR polymorphism

Gene variant Forward primer, 5′→3′ Reverse primer, 5′→3′ Fragment length, bp Ref

VDR-A1012G CCT CCT CTG TAA GAG GCG AAT AGC GAT GGA CAG GTG AAA AAG ATG GGG TTC 177 [22]

VDR-TaqI, VDR-ApaI*** ACG TCT GCA GTG TGT TGG AC TCA CCG GTC AGC AGT CAT AG 211 [23]

VDR-BsmI CAG TTC ACG CAA GAG CAG AG ACC TGA AGG GAG ACG TAG CA 236 [24]


AGG ATA GAG AAAA TAA TAG AAA ACA TTTCC TGA GTA AAC TAG GTC ACA A

AAC CCA TAA TAA GAA ATA AGT TTT TACACG TTA AGT TCA GAA AGA TTA ATT C

297 [22]

VDR-FokI TGC AGC CTT CAC AGG TCA TA GGC CTG CTT GCT GTT CTT AC 157 [25]

VDR-NIaIII TGC AGA GAA TGT CCC AAG GT GTC CTG CCA GTC TGA TGG AT 236 [26]

VDR-Tru91 GCA GGG TAC AAA ACT TTG GAG CCT CAT CAC CGA CAT CAT GTC 177 [19]

Table 3. Summary of the restriction enzyme digestion reactions and electrophoresis conditions for each VDR polymorphism

Gene variant Restriction endonuclease

PCR product, µL

Digestion buffer Other Incubation length & temperature

Gel conditions - Agarose %

Fragment of gel, bp

VDR-A1012G 20U EcoRV 5 10x Buffer 4 (1 µL) 3.8 µL H2O 6 hr @ 37°C 3 AA: 150, 27AG: 177, 150, 27GG: 177

VDR-TaqI 20U TaqI 5 10x Buffer 4 (1 µL) 1 µL BSA2.8 µL H2O

3 hr 20 min @ 65°C 3 TT: 211Tt: 211, 172, 39tt: 172, 39

VDR-ApaI 50U ApaI 5 10x Buffer 4 (1 µL) 1 µL BSA2.9 µL H2O

3 hr @ 25°C 3 AA: 211Aa: 211, 121, 90aa: 121, 90

VDR-BsmI 10U BsmI 5 10x Buffer 4 (1 µL) 3.5 µL H2O 3 hr 20 min @ 65°C 3 BB: 236Bb: 236, 197, 39bb: 197, 39


Not applicable 3 GG: 297, 110AG: 297, 235, 110AA: 297, 235

VDR-FokI 4U FokI 5 10x Buffer 4 (1 µL) 3 µL H2O 3.5 hr @ 37°C 3 FF: 157Ff: 157, 121, 36ff: 121, 36

VDR-NIaIII 10U NIaIII 5 10x Buffer 4 (1 µL) 2.5 µL H2O 6 hr @ 37°C 3 GG: 236GC: 236, 197, 39CC: 197, 39

VDR-Tru91 4U MseI 5 10x Buffer 4 (1 µL) 3 µL H2O 3.5 hr @ 37°C 3 UU: 177Uu: 177, 91, 86uu: 91, 86



score of more than 25 (out of 30) is classified as normal, whilst a score of 25 or less is indicative of some degree of cognitive decline.28

Food frequency questionnaire (FFQ)

Participants completed a self-administered FFQ, enabling an es-timation of daily vitamin D intake. The FFQ was comprised of 38 questions relating to diet, covering 205 food items and all food groups. Participants also provided a full list of supplements they were taking, enabling an estimation of total dietary intake of vitamin D. Each FFQ was analysed in FoodWorks™ (version 6.0.2562) nutritional analysis software program (Xyris Software, Brisbane, QLD, Australia), providing a breakdown of vitamin D intake measured as an average per day. This software package is comprised of a number of different food databases, covering the majority of foods consumed by Australians. These include: Abbott

products, Ausfoods (brand food) 2007, and Aus Nut (all foods) 2006.

Statistics

Statistical analysis was conducted using Microsoft Excel 2010 and JMP for Windows (version 11; SAS Institute Inc., Cary, NC, US). Age quartiles, sex and dietary vitamin D data distributions were examined, with mean, standard deviation (SD), median and in-terquartile range (IQR) reported as appropriate. Participants were stratified by sex and by cognitive decline based on the cut-off score of the MMSE (≤ 25 = cognitive decline; > 25 = no cognitive de-cline). Genotype prevalence (%), allele number (frequency) and carriage of polymorphic allele (%) were ascertained and tabulated according to cognitive decline. The degree and significance of an allele as a risk factor for a given biochemical/clinical phenotype was ascertained using an odds ratio (OR) and associated 95% con-

Fig. 2. Examples of each genotype (wild type, heterozygote, homozygous recessive) for the VDR polymorphisms examined in this study. A: VDR-ApaI; B: VDR-BsmI; C: VDR-A1012G; D: VDR-FokI; E: VDR-TaqI; F: VDR-Tru91; G: VDR-Cdx2. See Table 3 for further information on banding patterns.



fidence interval (CI).The cognitive decline phenotype was defined as nominal data,

and stepwise regression was used to create the best model, for sub-sequent nominal logistic regression analysis. Stepwise regression was performed in a mixed direction with significant probability [0.250] for a parameter to be considered as a forward step and en-tered into the model or considered as a backward step and removed from the model. Mallow’s Cp criterion was used for selecting the model where Cp first approaches p variables. R2 is reported and is the proportion of the variation in the response that can be attributed to terms in the model as opposed to random error. While an initial alpha level of 0.05 was set, Bonferroni corrections for multiple comparisons were also applied. Data is reported after adjusting for age and sex in analyses where stepwise regression modelling was used.

Results

Descriptive data for all subjects with and without cognitive decline is provided in Table 4, while the distribution of cognitive decline by age quartiles for each sex is given in Table 5. Seventy-three participants (11%) were found to exhibit some degree of cogni-tive decline based on their MMSE score. When examined by age quartiles, the proportion of participants with cognitive decline was highest in males in the 65–72 year-old group (15.2%) and the 73–80 year-old group (11.3%), and highest in females in the 81–88 year-old group (15.9%) and 89–95 year-old groups (28.6%) (Table 5). Total vitamin D intake (diet + supplements) was similar for participants with or without cognitive decline.

Table 6 presents the genetic distribution of each vitamin D-re-lated polymorphism for the cognitive decline phenotype, and gives the genotype prevalence (%), allele number (frequency) and car-riage of polymorphic allele (%). An OR and associated 95% CI were calculated to assess the degree of significance of risk for each of the vitamin D-related gene polymorphisms in relation to cogni-tive decline. Table 6 also shows that the VDR-BsmI and VDR-TaqI polymorphic alleles were both associated with an increased risk of

cognitive decline (OR = 1.55, 95% CI: 1.08–2.22 and OR = 1.49, 95% CI: 1.04–2.14, respectively).

Multivariate analyses of all vitamin D-related genetic polymor-phisms using stepwise regression showed a significant relationship between VDR-TaqI and MMSE (p = 0.0014; R2 = 0.0292). When adjusted for age and sex, the relationship between VDR-TaqI and MMSE remained significant (p = 0.0004; R2 = 0.0473). Both re-sults upheld their significance following a Bonferroni correction. To further examine the significant relationship between VDR-TaqI and MMSE, the percentage of participants with cognitive decline for each genotype was calculated. Figure 3 shows that the highest percentage of participants with some degree of cognitive decline was amongst those with the homozygous recessive tt genotype (24%).

Nominal logistic regression analysis was used to examine the relationships between other variables and MMSE score, independ-ent of genetic influence. Results showed a significant inverse as-sociation between age and MMSE score (p = 0.0145; R2 = 0.0131; slope estimate = −0.0180), and when examined by sex, a signifi-cant relationship was shown between age and MMSE for females (p ≤ 0.0001; R2 = 0.0865; slope estimate = −0.1160) but not males.

Discussion

The present study examined the relationship between VDR pol-ymorphisms and cognition in 650 elderly participants using the MMSE to assess their degree of cognitive decline. An OR analy-sis showed that both the VDR-BsmI and VDR-TaqI polymorphic alleles increased the risk of cognitive decline and that the VDR-TaqI genetic polymorphism was also significantly associated with MMSE score. The percentage of participants with some degree of cognitive decline was highest amongst those with the VDR-TaqI homozygous recessive tt genotype (24%).

Vitamin D is important for brain and other physiological func-tions, and plays an important role in the biosynthesis of neuro-transmitters,4 neuroprotection,5 immunomodulation and detoxi-fication.4 Consequently, some of these biological effects suggest

Table 4. Descriptive data for all subjects and for those participants with and without cognitive decline, based on MMSE (≤25 = cognitive decline; >25 = no cognitive decline)

All subjects Cognitive decline No cognitive declineNumber of participants 650 73 (11%) 575 (89%)Sex Male 287 33 (12%) 253 (88%) Female 363 40 (11%) 322 (89%)Age, x ± SD; median (IQR) 77. 8 ± 7.0;

78 (72–83);79.6 ± 7.6;80 (74–86);

77.5 ± 6.9;78 (72–83);

Total dietary vitamin D in µg/d,x ± SD; median (IQR)

7.76 ± 11.54;2.65 (1.77–7.93)

8.41 ± 10.78;2.87 (1.9–12.24)

7.65 ± 11.61;2.65 (1.75–7.41)

Table 5. Distribution of cognitive decline by age quartiles for each sex, based on MMSE (≤25 = cognitive decline; >25 = no cognitive decline) with percent-age of participants with and without cognitive decline for each age quartile

65–72 yr 73–80 yr 81–88 yr 89–95 yr

Males Cognitive declineNo cognitive decline

12 (15%)67 (85%)

12 (11%)94 (89%)

8 (9%)77 (91%)

1 (6%)15 (94%)

Females Cognitive declineNo cognitive decline

3 (3%)85 (97%)

12 (9%)127 (91%)

17 (16%)90 (84%)

8 (29%)20 (71%)



that vitamin D may influence cognitive function and mood disor-ders. Evidence surrounding vitamin D and the human brain have revealed individuals are increasingly vulnerable to mood disorders during the winter months,29,30 and a vitamin D deficiency may con-tribute to seasonal affective disorder. Additionally, evidence has shown a link between low vitamin D status and mood disorders, accompanied with poor cognitive function.31 Balion et al.32 con-ducted a systematic review and meta-analysis to examine the as-sociation between vitamin D, cognitive function and MMSE score; overall, it was found that lower vitamin D concentrations were as-sociated with poorer cognitive scores and higher AD risk. In the

present study, no associations were found between dietary vitamin D intake and MMSE score.

Research surrounding VDR polymorphisms and cognition have mostly focused on AD,33,34 and to a lesser extent cognitive decline.35 In one study, a meta-analysis found associations be-tween VDR polymorphisms and AD and Parkinson’s disease (PD) susceptibility.36 Other findings include a significant association between the VDR-TaqI “T” allele and AD susceptibility (OR = 0.735, 95% CI: 0.596–0.907),36 while in a further study the VDR-ApaI A allele and AA genotype increased the risk of mild cognitive impairment (MCI) (OR = 1.62, 95%CI: 1.13–2.31 and OR = 3.49, 95% CI: 1.570–7.740, respectively).24 The results also showed that the variant B allele of VDR-BsmI increased the risk of MCI (OR = 1.94, 95% CI: 1.240–3.050).37

Najmi Varzaneh et al.38 found the VDR-FokI was significantly associated with cognitive function following assessment using the MMSE. Examination of cognitive function amongst FokI geno-types showed “FF” participants had a higher cognitive score com-pared with “ff” participants.38 Similar findings were shown for FokI in a recent longitudinal study by Gatto et al.39 This study ex-amined VDR polymorphisms with PD using the MMSE, and found that for each additional copy of the FokI-A allele (also referred to as the “f” allele) an associated decrease in the total MMSE score occurred. Furthermore, their results indicated that participants with the AA genotype (also referred to as “ff” genotype) had a faster decline in cognitive function than participants with other VDR-FokI genotypes. No significant association was found between any other VDR polymorphism and PD in this study based on a level of significance of p < 0.05.39

Another study involving elderly subjects found significant asso-ciations between VDR-BsmI, VDR-TaqI and a low composite cog-nitive score (calculated by averaging the scores of other cognitive function tests) but not with a low MMSE score.40 Additionally, a significant association was observed between VDR-ApaI and less

Table 6. Occurrence of cognitive decline phenotype, based on MMSE (≤25 = cognitive decline; >25 = no cognitive decline), for genotype prevalence, allele number and carriage of polymorphic allele for vitamin D-related genetic polymorphisms

Polymor-phism Phenotype

Genotype prevalence (%)a

Allele number (frequency)

Carriage of poly-morphic, allele %

Odds ratio

CI (Yates cor-rected p value)

Wildtype Hete-rozygous

Reces-sive Wildtype Polymor-

phic

VDR-A1012G Cognitive declineNo cognitive decline

22 (31)185 (33)

40 (56)272 (48)

10 (13)108 (19)

84 (0.58)642 (0.57)

60 (0.42)488 (0.43)

6967

VDR-ApaI Cognitive declineNo cognitive decline

14 (19)120 (21)

30 (41)298 (53)

29 (40)148 (26)

58 (0.40)538 (0.48)

88 (0.60)594 (0.52)

8179

VDR-BsmI Cognitive declineNo cognitive decline

22 (31)207 (37)

29 (40)277 (49)

21 (29)79 (14)

73 (0.51)691 (0.61)

71 (0.49)435 (0.39)

6963

1.55 1.075–2.220 (0.0180)

VDR-Cdx2 Cognitive declineNo cognitive decline

12 (50)87 (50)

11 (46)71 (41)

1 (4)15 (9)

35 (0.73)245 (0.71)

13 (0.27)101 (0.29)

5050

VDR-FokI Cognitive declineNo cognitive decline

3 (12)29 (17)

10 (40)85 (49)

12 (48)61 (34)

16 (0.32)143 (0.41)

34 (0.68)207 (0.59)

8883

VDR-NlaIII Cognitive declineNo cognitive decline

10 (40)56 (32)

13 (52)87 (50)

2 (8)32 (18)

33 (0.66)199 (0.57)

17 (0.34)151 (0.43)

6068

VDR-TaqI Cognitive declineNo cognitive decline

25 (34)203 (37)

27 (37)280 (51)

21 (29)66 (12)

77 (0.53)686 (0.62)

69 (0.47)412 (0.38)

6663

1.49 1.039–2.142 (0.0290)

VDR-Tru91 Cognitive declineNo cognitive decline

59 (81)414 (73)

13 (18)138 (24)

1 (1)16 (3)

131 (0.90)966 (0.85)

15 (0.10)170 (0.15)

1927

aRounded to the nearest whole number.

Fig. 3. Percentage of participants with each VDR-TaqI gene variant (wild type TT, heterozygote Tt, homozygous recessive tt) and cognitive decline, based on an MMSE score of ≤ 25.



depressive symptoms based on the Geriatric Depression Scale.40

Since vitamin D was the nutrient of particular interest, blood 25(OH)D3 measurements would have provided further details sur-rounding the vitamin D status of participants. This is therefore a limitation in the present study.


Further research is necessary to either confirm or refute the pres-ently observed associations of VDR gene variants with cognitive decline as measured using the MMSE scale. By obtaining a more thorough understanding of VDR variants and their influence on risk of cognitive decline, new insights into the underlying patho-physiology of cognitive decline and development of possible intervention and treatment strategies will emerge. These may in-clude the screening of particular VDR polymorphisms as part of a routine health check and the use of supplemental vitamin D at a younger age. Based on the preliminary results of this study, we hypothesize that the use of vitamin D as a potential preventative agent in cognitive decline will reduce the impact this degenerative disorder currently has on our health system.

Conclusions

Our results show that the VDR-BsmI and VDR-TaqI polymor-phisms are associated with cognitive decline in an elderly popu-lation. Since the elderly are particularly at risk for a number of degenerative disorders related to low serum 25(OH)D3 levels, in-cluding cognitive decline, further exploration of the influence of VDR gene variants is necessary to reveal the full extent of poten-tial interactions.

Acknowledgments

Part of the research on which this paper is based was conducted as part of the Retirement Health and Lifestyle Study, The University of Newcastle. We are grateful to the Australian Research Council, Central Coast Local Health District Public Health Unit, Uniting-Care Ageing NSW/ACT, Urbis Pty Ltd, Valhalla Village Pty Ltd, and Hunter Valley Research Foundation for funding the initial study and to the men and women of the Central Coast region who provided the information recorded. The authors would also like to thank the researchers and RHLS clinic staff based at Gosford Teaching Unit, including Jenny Marriott, Marie Mazaroli, Eliza-beth Death, Jodi Humphreys, and Louise Lambeth. This research was supported by an Australian Research Council linkage grant (G0188386) awarded to Martin Veysey (lead CI).




Study design (CM, ML), performance of experiments (CM), anal-ysis and interpretation of data (CM, ML), manuscript writing (CM, ZY, MV), critical revision of the manuscript (CM, ZY, KK, SN,

ML), statistical analysis (CM), providing critical funding (MV).

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Original Article

Introduction

Pancreatic cancer (PC) is the fourth most lethal form of cancer in

Western society, with mortality closely mirroring incidence and an overall 5-year survival of less than 7%.1,2 Upon diagnosis, only 20% of patients are eligible for surgery, the only potential cure to date, with the remainder in advanced stages of the disease.

Surgery is invasive due to the physical location of the pancreas and is often used as a treatment option in combination with chemo-therapy. Despite this, a majority of patients who undergo surgery suffer a relapse from metastatic disease or experience comorbidi-ties such as venothromboembolic disease.3–5 Chemotherapy for PC patients involves treatment with agents such as the standard chemotherapeutic anti-metabolite drug gemcitabine (a pyrimidine analogue), or adjuvant therapy with additional agents such as the anti-tubulin agent nab-paclitaxel that can block nuclear division, causing cell death.3,6,7 Combination drug 5-fluorouracil/folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) is also a common treat-ment option but despite better response and survival rates than with single-agent gemcitabine treatment, FOLFIRINOX has greater lev-els of toxicity.3 Chemotherapy outcomes are varied due to physical and genetic obstacles of PC that cause complications, as they can

Keywords: Bitter melon; Pancreatic cancer; Herbal medicine.Abbreviations: AE, aescin equivalent; BMJ, bitter melon juice; CCK-8, Cell Count-ing Kit-8; CE, crude ethanol extract; CuB, Cucurbitacin B; F1, fraction 1; F2, fraction 2; F3, fraction 3; FBS, fetal bovine serum; GI50, minimum 50% growth inhibitory concentration; HPDE, human pancreatic ductal epithelial; HPLC, high-pressure liq-uid chromatography; L-Glu, L-glutamine; PBS, phosphate-buffered saline; PC, pan-creatic cancer; SE, saponin-enriched extract.Received: September 06, 2017; Revised: November 14, 2017; Accepted: November 24, 2017*Correspondence to: Rebecca A Richmond, Pancreatic Cancer Research Group, School of Environmental & Life Sciences, University of Newcastle, Ourimbah, NSW, Australia. Tel: 419121535, E-mail: [email protected] to cite this article: Richmond RA, Vuong QV, Scarlett CJ. Cytotoxic Effect of Bitter Melon (Momordica charantia L.) Ethanol Extract and Its Fractions on Pancreatic Cancer Cells in vitro. Exploratory Research and Hypothesis in Medicine 2017;2(4):139–149. doi: 10.14218/ERHM.2017.00032.

Cytotoxic Effect of Bitter Melon (Momordica charantia L.) Ethanol Extract and Its Fractions on Pancreatic Cancer Cells in vitro

Rebecca A. Richmond1*, Quan V. Vuong2 and Christopher J. Scarlett1

1Pancreatic Cancer Research Group, School of Environmental & Life Sciences, University of Newcastle, Ourimbah, NSW, Australia; 2Nutrition, Food & Health Research Group, School of Environmental & Life Sciences, University of Newcastle, NSW, Australia

Abstract

Background and objectives: The incidence of pancreatic cancer (PC) closely matches mortality, with current therapies ineffective often due to late diagnosis and difficulties in drug delivery. Bitter melon (Momordica charantia, Cucurbitaceae) has been traditionally used as an herbal medicine, particularly for the treatment of diabetes, in South East Asian countries. The aim of this study was to investigate the anti-PC potential of a crude ethanol extract (CE) and its enriched fractions.

Methods: The CE was used to prepare the saponin-enriched extract (SE) using n-butanol: water extraction. CE was also used for preparation of fractions 1, 2 and 3 (F1, F2 & F3) with a semi-preparative high-pressure liquid chromatography system. Cucurbitacin B (CuB), a triterpenoid present in many Cucurbitaceae species was also investigated for its effect on PC cells. The cytotoxicity was assessed in the PC cells MiaPaCa2, BxPC3 and CFPAC-1, and normal pancreas cells (HPDE) using the Cell Counting Kit-8 viability assay. Cell cycle analysis and induction of apoptosis in cells treated with F3 or CuB was determined using the Muse™ flow cell analyzer.

Results: The CE reduced the viability of MiaPaCa2 cells without affecting the normal cells, but only at 1,000 µg/mL. The SE reduced viability of all cells; however, the GI50 was significantly lower for the HPDE cells (72h: 72.1 µg/mL HPDE vs. 350.8 µg/mL MiaPaCa2). F3 and CuB appeared to arrest the cell cycle at G1/0 and G2/M, respec-tively; however, only CuB induced apoptosis via increased expression of caspase 3/7.

Conclusions: The CE, SE and three fractions elicited a weak cytotoxic effect on PC cells. Further research into bitter melon is recommended to isolate and identify any active components in F3 and further investigate their potential as novel agents against PC.



Richmond R.A. et al: Cytotoxicity of bitter melon for pancreatic cancerExplor Res Hypothesis Med

prevent or impede drug effectiveness and create chemotherapeutic resistance.3,8 These obstacles include barriers caused by the tumor microenvironment and pancreatic stem cells.9,10 Herbal medicine may hold the key to overcoming these barriers for PC sufferers.

Herbal medicine in the prevention and treatment of PC is cred-ited with substantial advantages, such as tumor suppression, im-proving radio- and chemotherapy efficacies, reducing side effects from treatment, improving immune function and sensitizing cells to chemotoxins.11 Bitter melon, as an herbal remedy, has demon-strated success in a variety of ailments, particularly in the treat-ment of diabetes, and has also been shown to prevent proliferation and growth of cancer cells, and as such is a novel candidate in the development of a treatment for PC. To date, bitter melon extracts and some purified isolated components have been assessed in vari-ous in vitro and in vivo studies as potential cancer therapies for prostate, liver, colon and breast cancers, and has been shown to induce cell death mechanisms, promoting apoptosis.

In PC, bitter melon juice (BMJ) increased necrotic cell death in gemcitabine-resistant pancreatic adenocarcinoma cells by enhanc-ing autophagy and inhibiting AKT, ERK1/2, PI3K and PTEN phos-phorylation, molecules involved in the prevention of apoptosis and also thought to be involved in drug resistance.12 Another identified mechanism of action of BMJ in PC cells is activation of adenosine monophosphate kinase, which is an enzyme involved in cellular metabolism and the signaling pathways that suppress growth.13 In PC cells, BMJ also induced intrinsic apoptosis by increasing lev-els of the pro-apoptotic molecule Bak and reducing levels of the anti-apoptotic molecules XIAP and survivin, both of the inhibitors of apoptosis protein (IAP) family, able to inhibit caspases 3/6/7.13 Bitter melon has also been linked with prevention of tumor for-mation and increasing life expectancy, and in reducing the size, weight, volume and proliferation of tumors in vivo for mouse and rat models of pancreatic, breast and prostate cancers.8,13–16

The extracts and juice of bitter melon contain high levels of glycosides (saponins), alkaloids, reducing sugars, resins, phenolic constituents, fixed oils and free acids.17,18 The bioactive compo-nents contributing these effects have so far been identified as ribo-some inactivating proteins, other proteins (identified as MAP30, MCP30 and momordin), fatty acid alpha-eleostearic acid, and vari-ous triterpenoid compounds of the Cucurbitaceae family. The cyto-toxic effect of various saponin compounds have also been explored in an array of different cancer cell lines in vitro, including colon, leukemia and breast cancers, with proposed mechanisms of action including inhibition of cell cycle signaling processes, initiation of apoptosis and degradation of the cytoskeleton.19–27 Bitter melon has potential as an anti-PC agent, inhibiting pancreatic cell growth by causing cell cycle arrest, apoptosis and reducing the metastatic potential of this disease.

Due to the high anticancer potential of saponin compounds and the additional cytotoxic effects of bitter melon extracts in various cancer cell lines, the aim of this study was to assess the anticancer activity of a saponin-enriched Big Top Medium bitter melon extract on pancreatic cell lines in vitro. There is currently no data available on the use of the Big Top Medium variety of bitter melon in this type of research, with studies favoring the Chinese or Thai varie-ties. Recently, Tan and colleagues reported optimized conditions for preparing an extract of Big Top Medium bitter melon with an in-creased saponin yield.28 The mechanisms involved in cytotoxicity against cell lines of different genetic profiles were examined as well as isolating the bioactive component/s responsible for the effects on these cells. It was hypothesized that saponin compounds from the enriched bitter melon extract are the bioactive components, which contribute to the cytotoxic action against PC cells in vitro and would give a greater cytotoxic effect than the crude extract.

Methods

Preparation of bitter melon extracts

Crude ethanol extract (CE) and saponin-enriched extract (SE) prepared from Big Top Medium bitter melon (Momordica char-antia L.) as described by Tan and colleagues was provided by Dr. Quan V Vuong (University of Newcastle, Faculty of Science, Australia).28 The extracts were kept at −20 °C until time of analy-sis. In brief, the SE was prepared from the CE by first dissolving in water (10 mg/mL) and then extracting three times (1/1 v/v) with water-saturated n-butanol. The SE were then combined, washed twice with deionized water and concentrated to dryness under reduced pressure (rotary evaporator at 40 ± 2 °C). The re-maining solid was then redissolved in 50% aqueous methanol and again evaporated to dryness to obtain the saponin enriched frac-tion (i.e. the SE).

At the time of publication, there were no commercially avail-able saponins from the Cucurbitaceae species, and Cucurbitacin B hydrate (Catalogue Number C8499-5MG; Sigma Life Sciences, St Louis, MO, USA), a commercially prepared cucurbitane triter-penoid was used to observe the effects on PC cells. The CE was used to prepare three enriched fractions with a preparative high-pressure liquid chromatography (HPLC) system (Shimadzu Aus-tralia, Rydalmere, NSW, Australia). The extract was dissolved in 50% methanol (10 mg/mL), filtered twice using a 0.45 µm nylon membrane syringe filter, and auto-injected at 300 µL with a flow rate of 3 mL/min. The column was a Synergi 4u Polar-RP 80A column (250 × 10 mm) connected to a PDA detector, scanning all wavelengths. MilliQ Water containing 0.2% formic acid was Solvent A and acetonitrile was Solvent B. Solvent B concentra-tion increased with a linear gradient from 0–40% between 0 to 15 min, increasing further to 100% Solvent B from 15 min to 25 min, then decreasing to 0% Solvent B from 25 min to 35 min. Peaks are identified with their retention time, the first peak eluting at 9.76 min. Three fractions were collected using an auto-collector, with the elution time for Fraction 1 (F1) = 4.50–12.60 min, Fraction 2 (F2) = 20.30–30.00 min and Fraction 3 (F3) = 34.20–41.80 min (Fig. 1). These fractions were evaporated, freeze-dried and stored at −20 °C for further analysis.

Total saponin content

The total saponin content of CE and SE extracts was measured according to the method of Hiai and colleagues (1976) with slight modifications. For the extracts, they were diluted to 1 mg/mL dis-solved in deionized water and 0.5 mL was mixed with 0.5 mL of 8% (w/v) vanillin solution, cooled in an iced water bath and 5 mL of 72% (v/v) sulphuric acid added. After 10 m, the cooled mix-ture was incubated at 60 °C for 15 m and then cooled on ice for a further 10 m. The absorption of the mixture was measured at 560 nm using a spectrophotometer (Cary 60 Bio; Varian Pty. Ltd., Mul-grave, Vic, Australia) against a reagent blank. Aescin was used as a standard and results were expressed as aescin equivalents (AEs) per gram of the dry weight of the bitter melon preparation used in the extraction (mg AE/g).

Cell culture of pancreatic cells

MiaPaCa2 primary pancreatic adenocarcinoma cells were maintained in Dulbecco’s modified Eagle’s medium contain-


Richmond R.A. et al: Cytotoxicity of bitter melon for pancreatic cancer Explor Res Hypothesis Med

ing 10% heat-inactivated fetal bovine serum (FBS), 2.5% horse serum and 1% L-glutamine (L-Glu). CFPAC-1 PC cells, from a metastatic liver lesion, were kept in Iscove’s modified Dulbecco’s medium with 10% heat-inactivated FBS and 1% L-Glu. BxPC3 primary pancreatic adenocarcinoma cells were kept in Roswell Park Memorial Institute medium containing 1% L-Glu and 10% heat-inactivated FBS. A comparatively normal human pancreatic ductal epithelial (HPDE) cell line was maintained in keratinocyte serum-free medium containing 5 ng/mL epidermal growth factor and 50 µg/mL bovine pituitary extract. All cell lines were obtained from the American Type Culture Collection (ATCC Laboratories, Manassas, VA, USA). Cells were split once 90% confluence was reached, using 0.25% trypsin to dissociate cells, and growth me-dium was changed every 2 days. Incubator conditions were main-tained at a humidified 37 °C atmosphere containing 5% CO2 in air. Growth media and supplements were obtained from Ther-moFisher Scientific (formerly LifeTech), Australia.

Determination of extract effect on cell viability

To determine the effect of the two extracts, three fractions (F1, F2 & F3) and Cucurbitacin B (CuB) on cell viability, MiaPaCa2 (3 × 103 cells per well), BxPC3 (7 × 103 cells per well), HPDE (1 × 104 cells per well) and CFPAC-1 cells (7 × 103 cells per well) were cul-tured and seeded into 96 well plates. After seeding, the cells were incubated for 24 h prior to treating, to ensure adequate adhesion. A stock solution of the extracts and fractions was made to 400 mg/mL in MilliQ and diluted in growth media, as required. Gemcit-abine, dissolved in phosphate-buffered saline (PBS) to 1 mM and stored at −80.0 °C, was thawed and diluted in fresh growth media on the day of use.

The cells were treated with doses of SE, CE and Fractions 1–3 ranging from 7–1,000 µg/mL, and CuB doses ranging from 7–1,000 nM. Cells were incubated with treatment doses for 24, 48, 72 or 96 h. The treatments were compared to a positive control containing 50 nM of gemcitabine in complete media and a nega-tive (vehicle) control. Cell viability was determined using a col-orimetric Cell Counting Kit-8 (CCK-8) assay (Dojindo Labora-

tories, Inc, Rockville, MD, USA) and microplate reader at 450 nm (Benchmark Plus™; Bio-Rad, Hercules, CA, USA). A blank consisting of media and CCK-8 reagent was used and subtracted from all measurements prior to data analysis.

Determination of apoptotic cell population

To determine the apoptotic cell population after treatment with F3 and CuB, a caspase 3/7 kit was used (Catalogue Number MCH100108; Merck Millipore, Australia). Firstly, cell samples were prepared by seeding MiaPaCa2 cells at a density of 3 × 105 cells per well, and HPDE cells at a density of 1 × 106 into a 12-well plate, treating cells with 500 and 750 µg/mL of F3, and 25 nM and 50 nM of CuB for 24 h. A positive control of gemcitabine at 50 nM and negative (vehicle) control of MilliQ was used for F3. DMSO was used as the vehicle for CuB. The cells were dissociated from the flask using 0.25% trypsin and resuspended in 50 µL 1X assay buffer ‘BA’. The cell suspension was mixed with 5 µL of Muse Caspase-3/7 Reagent diluted 1:8 in PBS and incubated at 37 °C in 5% CO2. After 30 m of incubation, 150 µL of Muse™ Caspase 7-AAD Solution, consisting of 1.3% 7-AAD reagent in 1X As-say buffer BA, was added and incubated at room temperature in the dark for 5 m. The sample was then loaded into the Muse™ Cell Analyzer and analyzed by Muse™ software (V1.4), as per the user’s guide.

Determination of cell cycle arrest

To determine the effect of the bitter melon F3 and CuB on cell cy-cle progression, MiaPaCa2 and HPDE cells were treated with 500 µg/mL and 750 µg/mL of F3, and 25 nM and 50 nM CuB for 24 h. After the 24 h period, the cells were fixed by resuspending 1 × 106 cells with ethanol (70%; ice cold) and incubating at −20 °C for 3 h. A 200 µL aliquot of the fixed cell suspension was then centrifuged at 300xg for 5 m, washed with PBS, pelleted and resuspended with 200 µL of Muse™ Cell Cycle Stain (Muse™ Cell Cycle Kit, Cata-logue Number MCH100106; Merck Millipore) before incubating

Fig. 1. HPLC chromatogram of CE and the three fractions. Absorbance was at 254 nm versus elution time (min). Abbreviations: CE, crude ethanol extract; HPLC, high-pressure liquid chromatography.



in the dark at room temperature for 30 m. The sample was then analyzed by a Muse™ Cell Analyzer.

Statistical analysis

All data was analyzed using GraphPad Prism (v7) software. Data are expressed as the arithmetic mean ± standard deviation of a minimum three replicates. Statistical differences in viability be-tween dose groups and negative control group were determined us-ing multiple comparison one-way ANOVA, with p < 0.05 deemed significant. ‘Viability %’, a reflection of cell viability of treated compared to untreated cells, was calculated by dividing the mean absorbance of each treatment group (less the absorbance blank) by the mean absorbance of the negative control (less the absorbance blank) and multiplying by 100. Dose response was analyzed using nonlinear regression of the log of the concentration with a defined bottom equal to 0.0 and constrained hillslope of −1.0 to deter-mine the minimum 50% growth inhibitory concentration (GI50), a measure of drug potency. Statistical differences between treatment groups and negative controls of the cell cycle and apoptosis assays were calculated using multiple comparison two-way ANOVA, with p < 0.05 deemed significant.

Results

Assessment of bitter melon extracts on pancreatic cell viability

Total saponin content

Total saponin content of the CE was 1.12 mg AE/g dry weight and its n-butanol fraction was considerably higher in saponins, with 345.62 mg AE/g dry weight.

Effect of CE on pancreatic cell viability

Pancreatic cell viability after a 24, 48, 72 and 96 h treatment with CE was assessed using a CCK-8 assay. The CE showed cytotoxic activity towards MiaPaCa2 cells dosed with the highest concen-tration (1,000 µg/mL) for longer time periods (>48 h). Similarly, CFPAC-1 cells only reduced viability to 1,000 µg/mL. MiaPaca2 and HPDE cells did not decrease in viability after the 24 h treat-ment with concentrations of CE between 0–1,000 µg/mL. GI50 values were calculated for these treatments and were all >500 µg/mL (Table 1). MiaPaCa2 cells treated for 48 h with 1,000 µg/mL

of CE decreased viability to a greater extent than the cells treated with gemcitabine (see Supplementary Fig. 1F). BxPC3 cells treat-ed with the CE showed reduced viability after 48 and 96 h at doses above 125 µg/mL but no change was observed for the 24 or 72 h treatments. GI50 values for CE were not able to be calculated for the CFPAC-1 and BxPC3 cell lines (Table 1). The CE had poor cytotoxic potential overall but at its highest dose, affected only cancerous MiaPaCa2 and CFPAC-1 cell lines.

Effect of SE on pancreatic cell viability

Pancreatic cell viability after 24, 48, 72 and 96 h treatment with SE was assessed using a CCK-8 colorimetric assay. MiaPaCa2 and HPDE cells treated with the SE for 24, 48, 72 and 96 h exhibited decreasing cell viability with increasing concentrations (>125 µg/mL) for all time points. The 96 h GI50 for MiaPaCa2 cells was 338.2 µg/mL, approximately 80% less than the GI50 for the 24 h treatment of 1,669.0 µg/mL (Table 2). The GI50 value for the SE in HPDE cells was 88.1 µg/mL for 24 h, which decreased to 72.05 µg/mL for 72 h, but at 96 h increased again to 164.0 µg/mL (Table 2).

No decrease in viability of CFPAC-1 cells and BxPC3 treated with the SE for 24 h were observed. SE only reduced viability of CFPAC-1 cells after 72 h from 1,000 µg/mL. After 96 h, CF-PAC-1 cells demonstrated a dose-dependent reduction in viability, increasing the GI50 from 599.7 µg/mL to 776.8 µg/mL from the 72 to 96 h treatments. BxPC3 cells treated with the SE for 48–96 h observed a dose-dependent reduction in viability with GI50 values decreasing 2-fold, from 1,010.0 µg/mL at 48 h to 553.4 µg/mL at 96 h (Table 2). The SE showed minimal to slight cytotoxic activ-ity overall; however, the lowest doses (<100 µg/mL) affected the HPDE cells within the first 24 h, and low doses (<500 µg/mL) only showed a cytotoxic effect in MiaPaCa2 cells and only after 72 h (Table 2).

Cytotoxicity of CE fractions on pancreatic cell lines in vitro

The CE was semi-purified by HPLC into Fractions 1–3 (F1, F2 and F3) and their cytotoxicity was assessed on first HPDE and MiaPaCa2 cell viability using a 24 h CCK-8 assay. There was no reduction in MiaPaCa2 cell viability observed in response to 24 h treatment with F1 (0–1,000 µg/mL), while HPDE cell viability de-creased following treatment with F1 (>125 µg/mL; Fig. 2A, E). F2 produced a dose-dependent reduction in viability of HPDE cells at 24 h with 1,000 µg/mL, while MiaPaCa2 cells did not show any decrease in viability (Fig. 2B, F). F3 was the only fraction to de-

Table 1. GI50 values of CE treatment (µg/mL) in MiaPaCa2, CFPAC-1, BxPC3 and HPDE cell lines calculated for 24, 48, 72 and 96 h

CE GI50, µg/mL

CELL LINE 24 h 48 h 72 h 96 h

MiaPaCa2 ND 1,475.0 711.6 821.8

CFPAC-1 ND ND ND ND

BxPC3 ND ND ND ND

HPDE ND ND ND ND

Abbreviations: CE, crude ethanol extract; GI50, minimum 50% growth inhibitory con-centration; HPDE, human pancreatic ductal epithelial; ND, not detected. Viability not reduced by more than 50%.

Table 2. GI50 values of SE treatment (µg/mL) in MiaPaCa2, CFPAC-1, BxPC3 and HPDE cell lines calculated for 24, 48, 72 and 96 h

SE GI50, µg/mL


MiaPaCa2 1,669.0 783.5 350.8 338.2

CFPAC-1 ND ND 599.7 776.8

BxPC3 ND 1,010.0 859.9 553.4

HPDE 88.1 87.4 72.1 164.0

Abbreviations: GI50, minimum 50% growth inhibitory concentration; HPDE, human pancreatic ductal epithelial; SE, saponin-enriched extract; ND, not detected. Viability not reduced by more than 50%.



crease the MiaPaCa2 cell viability in 24 hours and produced GI50 values of 939.1 µg/mL and 516.5 µg/mL in MiaPaca2 and HPDE cells, respectively, and as such F3 was further investigated (Fig. 2C, D, G, and H).

F3 was assessed for its effect on cell viability for more than 24 h and in the additional two cancer cell lines. At 48 and 72 h, F3 at high doses (<500 µg/mL) reduced viability of HPDE cells (see Supplementary Fig. 4). Doses above 250 µg/mL reduced Mia-PaCa2 cell viability at 24 h, while only doses greater than 500 µg/mL showed an effect at 48 h. Interestingly, no reduction in Mia-PaCa2 viability at any dose was observed after 72 h. The reduction of viable MiaPaCa2 cells in 48 h was not greater than 50% and, thus, no GI50 was able to be calculated (Table 3). A dose-depend-ent reduction in viability of CFPAC-1 and BxPC3 cells treated with F3 for 24 h was observed. This reduction in viability contin-ued only for BxPC3 cells with the 48, 72 and 96 h treatments, and the GI50 value reduced from 1,883.0 µg/mL at 24 h to 547.8 µg/mL at 96 h (Table 3). Of the three fractions made from CE, only F3 held cytotoxic potential towards cancer cells but none of the GI50 values were below 500 µg/mL, even after 96 h, indicating its cytotoxic action is not strong.

Cytotoxicity of the commercially prepared triterpenoid CuB

The cytotoxicity of the commercially prepared triterpenoid CuB was assessed on the four pancreatic cell lines using CCK-8 vi-ability assay. MiaPaCa2 PC cells treated with CuB (0–1,000 nM) showed a dose-dependent reduction in viability with increasing doses of CuB. After 24 h, cell viability reduced from concentra-tions above 15.81 nM with no further reduction observed at con-centrations higher than 62.50 nM. After 96 h, reduction in cell viability was observed at doses of 31.25 nM or higher (see Sup-plementary Fig. 5).

HPDE cells treated with CuB responded similarly to MiaPaCa2 cells, with the 72 h treatment producing the lowest GI50 of 15.30

nM, at which there was close to 100% reduction in viability of HPDE cells treated with gemcitabine (Fig. 3C, D). CFPAC-1 cells had reduced viability after 72 h at a CuB dose of 62.50 nM. The GI50 of BxPC3 cells treated with CuB for 24 h was not able to be calculated, however, after 48 h the viability of BxPC3 cells started to decrease. The BxPC3 GI50 was 9.38 nM after 48 h and 15.63 nM after 96 h (Table 4). All CuB GI50 values for all cell lines were less than 100 nM after 96 h, suggesting strong cytotoxic action of this triterpenoid.

Effect of bitter melon extracts on pancreatic cell cycle arrest

MiaPaCa2 and HPDE cells were treated with F3 at 500 and 750 µg/mL for 48 h to determine if progression of the cells through the cell cycle could be stalled. HPDE cells treated with F3 for 48 h demonstrated an increased number of cells in the G1/0 phase, few-er cells in the S phase and significantly less cells in the G2/M phase compared to the untreated cells (Fig. 4A). HPDE cells treated with gemcitabine also demonstrated a greater quantity of cells in the G1/0 phase than cells with no treatment, but to a greater extent

Table 3. GI50 values of F3 treatment (µg/mL) in MiaPaCa2, CFPAC-1, BxPC3 and HPDE cell lines calculated for 24, 48, 72 and 96 h

F3 GI50, µg/mL


MiaPaCa2 939.1 ND ND NA

CFPAC-1 1,860.0 ND ND ND

BxPC3 1,883.0 1,168.0 651.5 547.8

HPDE 516.5 942.6 857.1 NA

Abbreviations: F3, fraction 3; GI50, minimum 50% growth inhibitory concentration; HPDE, human pancreatic ductal epithelial; ND, not detected. Viability not reduced by more than 50%; NA, not available. Assay not performed.

Fig. 2. Viability of HPDE and MiaPaCa2 cells treated for 24 h with Fractions 1–3 ranging from 0–1,000 µg/mL and gemcitabine 50 nM (G 50 nM), as deter-mined by CCK-8 colorimetric assay. HPDE cells treated for 24 h with A: Fraction 1, B: Fraction 2, C: Fraction 3, D: GI50 for Fraction 3 in HPDE cells at 516.5 µg/mL, MiaPaCa2 cells treated with E: Fraction 1, F: Fraction 2, G: Fraction 3, MiaPaCa2 cells; H: GI50 for Fraction 3 in MiaPaCa2 cells 939.1 µg/mL. Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p<0.05, ‘**’ = p<0.01, ‘***’ = p<0.005, ‘****’ = p<0.001. Abbreviations: CCK-8, Cell Counting Kit-8; HPDE, human pancreatic ductal epithelial.



than seen with F3. The MiaPaCa2 cells treated with F3 had more cells in the G1/0 phase and less in the S phase for both treatment concentrations compared to untreated cells (Fig. 4B). This was similar to the gemcitabine-treated cells. HPDE cells treated with 25 and 50 nM CuB for 24 h revealed that treatment with 50 nM significantly increased the population of cells in the G2/M phase compared to untreated cells (Fig. 4C). F3 appeared to induce G1/0 phase cell cycle arrest in both MiaPaCa2 and HPDE cells, while CuB appeared to induce G2/M arrest.

Effect of bitter melon extracts on apoptosis of pancreatic cells in vitro

The caspase 3/7 assay was performed to determine if F3 could induce caspase 3/7-dependent apoptosis in the HPDE and Mia-PaCa2 cells at 24 h. Despite the appearance of apoptotic popula-tions increasing in HPDE cells treated with 500 and 750 µg/mL of F3 (Fig. 5A), there were no significant differences between cells treated with F3 compared to the negative control. Similarly, there

appeared to be increasing populations of dead cells, increasing in MiaPaCa2 cells with doses of F3 (Fig. 5B), yet these were statisti-cally insignificant. As expected, the positive control gemcitabine revealed significant increases in apoptotic and dead populations in both cell lines (Fig. 5A, B). CuB at 50 nM increased the total apo-ptotic and dead populations but only in HPDE cells (Fig. 5C, D). F3 did not appear to cause apoptosis in the MiaPaCa2 and HPDE cells, while CuB induced apoptosis at 50 nM but only in HPDE cells.

Discussion

PC is a lethal disease. Natural sources, such as bitter melon, offer novel options for therapeutic development that may overcome the present challenges impeding current treatment efficacy. Previous studies have reported that the juice and extracts of bitter melon inhibit the growth of specific cancer cells by causing cell cycle arrest and apoptosis, and reducing the metastatic potential of this disease. Due to the anticancer potential of bitter melon, which con-tains high levels of saponin compounds, this study investigated the cytotoxic potential of a bitter melon ethanol extract and its saponin enriched n–butanol fraction on pancreatic cells in vitro.

The crude bitter melon extract only reduced viability in Mia-PaCa2 cells (at 1,000 µg/mL; >48 h) and did not appear to af-fect the HPDE cells. To understand if a selective cytotoxic action existed, it was explored further. Whole fruit aqueous extracts of a Chinese bitter melon have displayed preferential cytotoxicity towards breast cancer and leukemia cells, without effecting the viability of normal cells.29,30 Similarly, saponin-rich extracts se-lectively induced apoptosis in hepatoma cells, without causing the same effect in their normal counterparts.31 In this study, the SE did not discriminate in its cytotoxic activity and, in fact, was more potent to the HPDE cells. The lower doses (125 and 250 µg/mL) required longer treatment times, of 72 or 96 h, before any observed

Fig. 3. Viability of HPDE cells treated with CuB ranging from 0–1,000 nM and gemcitabine 50nM (G 50 nM), as determined by CCK-8 colorimetric assay. HPDE cells treated with CuB for A: 24 h, B: 48 h, C: 72 h, D: 96 h. GI50 values of HPDE cells are E: 25.47 nM for 24 h, F: 17.27 nM for 48 h, G: 15.30 nM for 72 h, H: 23.38 nM for 96 h. Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p<0.05, ‘**’ = p<0.01, ‘***’ = p<0.005, ‘****’ = p<0.001. Abbreviations: CCK-8, Cell Counting Kit-8; CuB, Cucurbitacin B; HPDE, human pancreatic ductal epithelial.

Table 4. GI50 values of Cucurbitacin B treatment (nM) in MiaPaCa2, CF-PAC-1, BxPC3 and HPDE cell lines calculated for 24, 48, 72 and 96 h

CuB GI50, nM


MiaPaCa2 14.76 39.22 35.21 66.69

CFPAC-1 ND ND 52.65 60.81

BxPC3 ND 9.38 15.32 15.63

HPDE 25.47 17.27 15.30 23.38

Abbreviations: GI50, minimum 50% growth inhibitory concentration; HPDE, human pancreatic ductal epithelial; ND, not detected. Viability not reduced by more than 50%.



reduction in cancer cell viability, specifically for the CFPAC-1 and BxPC3 cells, which showed no response to SE in the first 24 h. This may be due to differences between the mechanisms and kinet-ics of cell death, such as the cells’ ability to absorb the compounds in a given time.32

Over time, the effect of the same concentration of SE appeared

to reduce, with more viability of HPDE cells after 96 h than at 72 h. This could indicate that the bioactive compound/s in the SE had been fully utilized by the HPDE cells in the 72 h timeframe, with remaining cells proliferating in the final 24 h to give the observed increase in viability at 96 h. Despite this, it was clear that there was no specificity of action by the saponin extract. This might sug-gest that compounds other than saponins may confer this effect, that the responsible compound/s might have been excluded from the preparation of the extract via the butanol fractionation step, or that there may have been other reasons for only the MiaPaCa2 and CFPAC-1 cells being affected by the CE.

The crude extract was subjected to partial purification by HPLC in an attempt to elucidate the phytochemical profile of the bitter melon. The dose-dependent effect of F3 on BxPC3 cells was not observed in CFPAC-1 or MiaPaCa2 cells, indicating a sustained ef-fect of F3 in the BxPC3 PC cells. This infers that the BxPC3 cells may be more sensitive to F3 than the other cell lines. The GI50 for both MiaPaCa2 and HPDE cells treated with F3 increased with time, and only reduced in MiaPaCa2 cell viability in re-sponse to F3 in the first 48 h, leaving cells apparently unaffected at 72 and 96 h. It is possible that re-administering the drug after the first 24 h may be necessary for MiaPaCa2 and CFPAC-1 cells.

After testing SE and the three fractions, it is clear that no selec-tive cytotoxic action was present. There may have been synergism between compounds to cause the CE to only reduce MiaPaCa2 and CFPAC-1 cells that was lost when partially purifying to obtain the fractions. Synergistic effects of saponin compounds with ther-apeutic compounds have been reported to result in an increased cytotoxic capability.33 BMJ has also targeted multiple signaling pathways in a gemcitabine-resistant PC PanC cell line and increase their susceptibility to the effects of the drug.12

Gemcitabine is the standard treatment for PC; however, in the first 24 h, MiaPaCa2 cells are not responsive to the gemcitabine treatment but were slightly responsive to F3, but only minimally. Even though there was a short-lived action of F3 in HPDE and Mi-aPaCa2 cells in these instances, F3 may work to weaken the cells and may sensitize them to other agents. Further work is needed to elucidate and isolate any synergistic potential of the extract and its active components.

Genetic variation between the cell lines may account for their differences in response to F3. The MiaPaCa2 and BxPC3 cells are both sourced from primary pancreatic adenocarcinomas, however they have different genetic profiles. MiaPaCa2 cells have muta-tions with oncogene KRAS, and tumor suppressor genes CDKN2A and TP53, while BxPC3 cells have a wild-type KRAS and also possess mutations in CDKN2A and TP53 but have further muta-tions in SMAD4 and MAP2K4.34 CFPAC-1 cells, by contrast, are cells derived from a metastatic site in the liver and have muta-tions in KRAS, SMAD4 and TP53.34 Further work is necessary to explore if inducing specific mutations; for example, KRAS in BxPC3 cells, gives these cells increased resistance to the effect of F3, as seen in the response to this fraction by KRAS-mutated MiaPaCa2 and CFPAC-1 cells. The synergistic capabilities of pu-rified bitter melon agents could also then be explored in combina-tion with gene knockouts or forced expressions to understand the relationship between genetic profile, drug interactions and drug responsiveness.

To understand how F3 reduced cell viability, caspase 3/7 apop-tosis and cell cycle assays were performed. Apoptosis is a transient cellular process that involves numerous intermediate cellular signals to initiate the self-destruction of the cell. Caspase levels are likely to change with time as cells undergo necrosis, which is secondary to in vitro apoptosis.32 In MiaPaCa2 cells treated with F3, there were slight increases in the population of dead cells compared to

Fig. 4. Cell cycle analysis of HPDE and MiaPaCa2 Cells treated with F3 (0–750 µg/mL) and CuB (0–50 nM) for 48-hours as determined using Muse™ Cell Cycle Kit. A: HPDE cells treated with F3, B: MiaPaCa2 cells treated with F3, C: HPDE cells treated with CuB. Statistical differences between treat-ment groups and negative control determined by one-way ANOVA. p<0.05 represented by ‘*’, ‘∧’ and ‘#’ for G1/0, S and G2/M phases, respectively. p<0.01 represented by ‘**’, ‘∧∧’, ‘##’ for G1/0, S and G2/M phases, re-spectively. p<0.005 represented by ‘***’, ‘∧∧∧’ and ‘###’ for the three cell cycle phases. Similarly, p<0.001 represented by ‘****’, ‘∧∧∧∧’ and ‘####’, respectively. Abbreviations: CuB, Cucurbitacin B; F3, fraction 3; HPDE, hu-man pancreatic ductal epithelial.



the negative control, though statistically insignificant. HPDE cells treated with the same doses had slight increases in total apoptotic populations, but again were not found to be significant. As such, further experiments with adjustments in treatment times will need to be explored to delineate the effects of bitter melon extracts on apoptosis of pancreatic cells. BMJ has been shown to increase cas-pase 7 and caspase 3 activity at 48 h in MCF-7 and MDA-MB-231 breast cancer cells.35 Indeed, further purification and concentration of purified components may increase their cytotoxic potential.

Following a 48 h treatment of both HPDE and MiaPaCa2 cells with F3, an increased number of cells in the G1/0 phase was ob-served, indicating that cell cycle arrest in the G1/0 phase may be induced by the bitter melon fraction. Considering that the cells do not continue to decrease in viability at the 72 h treatment, it is unlikely that cell cycle arrest is sustained. Cycle arrest can occur if the cell’s internal checkpoints are not passed.36 The fate of the cell after mitotic cell cycle arrest may either be cell death, if sufficient signals are given to the cell to undergo apoptosis, such as with caspase signaling, or the cell may have adapted and will then go on to continue proliferating, as seen in cases of drug

resistance.37

The longer the cell remains in cell cycle arrest, whereby mitosis occurs until the cell breaks down the agent responsible for causing the arrest (cyclin B1), the less likely it is to survive.38 This poten-tially explains why the cells continue to proliferate after 72 h of treatment. Despite indications that cell cycle arrest may be occur-ring in these cells with treatment of F3, there was no confirmation of apoptosis, which could explain why the cells continue to prolif-erate. The accumulation of cells in the G1/0 phase was unexpected, as BMJ has been shown to increase populations in the G2/M phase although G1/0 arrest has also been induced by bitter melon, but in a bitter melon leaf ethanol extract.16,35,39

The cytotoxic potential of CuB, a commercially prepared triter-penoid was also investigated. CuB elicited a strong dose response in BxPC3 PC cells, comparable to the gold standard chemothera-peutic agent gemcitabine. This was also confirmed in MiaPaCa2 PC cells, while interestingly ‘preserving’ HPDE cell viability to a greater extent than gemcitabine. CuB at 200 nM has previously been found to induce apoptosis in a time-dependent manner in MiaPaCa2, Panc-1 and PL45 PC cells.40 However, in this study,

Fig. 5. Dead, live and total apoptotic HPDE and MiaPaCa2 cells after 24-hour treatment with F3 (0–750 µg/mL) and CuB (0–50 nM) as determined using Muse™ Caspase 3/7 kit. A: HPDE cells treated with F3, B: MiaPaCa2 cells treated with F3, C: HPDE cells treated with CuB, D: MiaPaCa2 cells treated with CuB. Statistical differences between treatment groups and negative control determined by one-way ANOVA. P< 0.05 represented by ‘*’, ‘∧’ and ‘#’ for G1/0, S and G2/M phases, respectively. P<0.01 represented by ‘**’, ‘∧∧’, ‘##’ for G1/0, S and G2/M phases, respectively. Similarly, P<0.001 represented by ‘****’, ‘∧∧∧∧’ and ‘####’, respectively. Abbreviations: CuB, Cucurbitacin B; F3, fraction 3; HPDE, human pancreatic ductal epithelial.



CuB was tested at 50 nM and induced caspase 3/7 apoptosis in the HPDE cells but not in the MiaPaCa2 cells. Cell cycle arrest in the G2/M phase by 50 nM CuB was also observed in the HPDE cells. G2/M phase arrest was previously observed at 200 nM in PC MiaPaCa2 and Panc-1 cells.40 Despite CuB appearing to ‘preserve’ HPDE to a greater extent than gemcitabine in these studies, CuB clearly has a more potent effect to these normal pancreatic cells than to the cancerous cell lines.


While the cytotoxicity of the extracts was not strong, there is much more work that can be done, such as further purification of the extracts, which may increase their cytotoxic potential. Isola-tion and concentration of components that make up F3 may unveil an alternate bioactive agent, perhaps a cucurbitane triterpenoid or saponin, with the potential to induce significant cytotoxicity in PC cells. The synergistic capabilities of purified bitter melon agents could also then be explored in combination with gene knockouts or forced expressions to understand the relationship between ge-netic profile, drug interactions and drug responsiveness. Similar-ly, changes to protein levels and/or gene expression in the cells, such as in cyclin, caspase or Bak/Bax, to confirm the mechanisms of action are warranted. Further work is also necessary to explore if inducing specific mutations, e.g., KRAS in BxPC3 cells, gives these cells increased resistance to the effect of F3, as seen in the response to this fraction by KRAS-mutated MiaPaCa2 and CF-PAC-1 cells.

Conclusions

Overall, both crude and saponin-enriched fractions were not ef-fective at eliciting a cytotoxic response towards PC cells in vitro. Semi-purification of the crude extract, which displayed potential for selectively reducing viability of cancerous cells without the same effect to a comparatively normal pancreatic cell line HPDE, provided three fractions for further assessment. Of the three frac-tions, only F3 reduced the viability of MiaPaCa2 PC cells, and no further selective action was observed. The cytotoxic action of F3 was short-lived, with cells decreasing viability initially, and then increasing viability again with time. Investigations into the mechanism of action of F3 indicated that it potentially causes G1/0 cell cycle arrest, without appearing to induce apoptosis. In con-trast, treatment with CuB suggests that a G2/M cell cycle arrest occurred, as well as caspase 3/7-dependent apoptosis. Further re-search may assist in investigating these agents and why they have minimal effect in the treatment for PC while other bitter melon preparations have positive outcomes.

Acknowledgments

The authors would like to acknowledge the University of New-castle and the Pancreatic Cancer Research Group for supporting this project.




Experimental conduct (RR), minor editing and supervisory role (QV), main editor of manuscript and main supervisor (CS).

Supporting information

Supplementary material for this article is available at https://doi.org/10.14218/ERHM.2017.00032.

Supplementary Figure 1. Viability of HPDE and MiaPaCa2 cells treated with CE ranging from 0–1,000 µg/mL and Gemcitabine 50 nM determined by CCK 8 colourimetric assay. HPDE cells treated with CE for A: 24 hours, B: 48 hours, C: 72 hours, D: 96 hours; MiaPaCa2 cells treated with CE for E: 24 hours F: 48 hours (GI50 1,475 µg/mL), G: 72 hours (GI50 711.6 µg/mL), H: 96 hours (GI50 821.8 µg/mL). Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p < 0.05, ‘**’ = p < 0.01, ‘***’ = p < 0.005, ‘****’ = p < 0.001.

Supplementary Figure 2. Viability of MiaPaCa2 cells treated with Saponin fraction (SE) ranging from 0–2,000 µg/mL and Gemcit-abine 50 nM determined by CCK8 colourimetric assay. MiaPaCa2 cells treated SE for A: 24 hours, B: 48 hours, C: 72 hours, D: 96 hours. GI50 values of MiaPaCa2 cells are E: 1,669.0 µg/mL for 24 hours F: 783.5 µg/mL for 48 hours G: 350.8 µg/mL for 72 hours and H: 338.2 µg/mL for 96 hours. Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p < 0.05, ‘**’ = p < 0.01, ‘***’ = p < 0.005, ‘****’ = p < 0.001.

Supplementary Figure 3. Viability of HPDE cells treated with SE ranging from 0–1,000 µg/mL and Gemcitabine 50 nM determined by CCK8 colourimetric assay. HPDE cells treated with SE for A: 24 hours, B: 48 hours, C: 72 hours, D: 96 hours. GI50 values of SE cells E: 88.1 µg/mL for 24 hours F: 87.35 µg/mL for 48 hours G: 72.05 µg/mL for 72 hours and H: 164.00 µg/mL for 96 hours. Signif-icance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p < 0.05, ‘**’ = p < 0.01, ‘***’ = p < 0.005, ‘****’ = p < 0.001.

Supplementary Figure 4. Viability of HPDE and MiaPaCa2 cells treated with Fraction 3 (F3) ranging from 0–1,000 µg/mL and Gemcitabine 50 nM as determined by CCK8 colourimetric assay. HPDE cells treated with F3 for A: 24 hours (GI50 516.5 µg/mL), B: 48 hours (GI50 942.6 µg/mL), C: 72 hours (GI50 857.1 µg/mL), MiaPaCa2 cells treated with F3 for D: 24 hours (GI50 939.1 µg/mL) E: 48 hours, F: 72 hours. Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p < 0.05, ‘**’ = p < 0.01, ‘***’ = p < 0.005, ‘****’ = p < 0.001.

Supplementary Figure 5. Viability of MiaPaCa2 cells treated with Cucurbitacin B (CuB) ranging from 0–1,000 nM and Gemcitabine 50 nM as determined by CCK8 colourimetric assay. MiaPaCa2 cells treated with CuB for A: 24 hours, B: 48 hours, C: 72 hours, D: 96 hours. GI50 values of MiaPaCa2 cells E: 14.76 nM for 24 hours F: 39.22 nM for 48 hours G: 35.21 nM for 72 hours and H: 66.69 nM for 96 hours. Significance between treatment groups and the negative control calculated using one-way ANOVA represented over treatment columns by ‘*’ = p < 0.05, ‘**’ = p < 0.01, ‘***’ =





p < 0.005, ‘****’ = p < 0.001.

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Reviewer Acknowledgement

Belgin Akan TurkeyMajid Anushiravani IranAlessandra Arcolaci ItalyMarianne Besnard FranceTatiana Betakova SlovakiaIgor Burstyn United StatesJavier Camacho MexicoBohao Chen United StatesSzu-ta Chen China (Taiwan)Jianding Cheng ChinaHsueh-Ling Cheng China (Taiwan)Salvatore Chirumbolo ItalyAhmet Cumaoglu TurkeyIna Danquah GermanyAndrew Stewart Day New ZealandBhavtosh Dedania United StatesSrdjan Denic United Arab EmiratesJianqiang Ding ChinaIliana B. Doycheva BulgariaMehmet Ali Ergun TurkeyAhmet Eroglu TurkeyYuchen Fan ChinaShirley Steffany Muñoz Fernández Brazil

Clara Gabás-Rivera SpainXueqin Gao United StatesShanzhong Gong United StatesOsamu Handa JapanRichard Hansen United KingdomLokesh Jain IndiaWan Fariza Wan Jamaludin MalaysiaCagatay Karaaslan TurkeyAynur Karadag TurkeyA. Kauppinen FinlandAbbas Khani SwitzerlandChao Li United StatesDong Li ChinaJunxia Li ChinaJosé Manuel Lou-Bonafonte SpainMark Lucock AustraliaCharlotte Martin AustraliaTracey A. Martin United KingdomGerald Martone United StatesDavid Miller United KingdomJohn H. Miller New ZealandRosiane A. Miranda BrazilJavier A. Miret SpainSlávka Mrosková SlovakiaAshesh Nandy India

Nenad Naumovski AustraliaYoshikazu Ogawa JapanBarbara Olendzki United StatesKeith Pecor United StatesPetrick Periyasamy MalaysiaAline Valeska Probst FranceEdward V. Quadros United StatesAleksei V Rakov United StatesRama Sankar Rath IndiaSamiran Ray United KingdomJonathan Roth IsraelIan Russell United KingdomMiroslav Simunic CroatiaKeerti Singh BarbadosSurajit Sinha United StatesH Skaltsa GreeceJames Stamey United StatesDeborah Sundin AustraliaBartlomiej Szulczyk PolandPawel Szulczyk PolandJonathan E. Teitelbaum United StatesGeorgios Tsoulfas GreeceTuxun Tuerhongjiang ChinaAlexander M. Vaiserman UkraineGuojun Wang United States☆DOI: 10.14218/ERHM.2017.000RA

2017 Reviewer Acknowledgement

Editorial Office of Exploratory Research and Hypothesis in Medicine

We thank the following reviewers for their contribution and support in 2017.


DOI: 10.14218/ERHM.2017.000RA | Volume 2 Issue 4, December 2017 151

2017 Reviewer Acknowledgement Explor Res Hypothesis Med

Lili Wang United StatesToru Watanabe JapanEwa Widy-Tyszkiewicz PolandJennifer M. Windt AustraliaQichao Wu ChinaGuogang Xu China

Yongkang Yang United StatesŞenay Görücü Yılmaz TurkeyJuliana Yordanova GermanyChengyue Zhang United StatesSarah Zhang ChinaHuantian Zhang China

Chong Zhang ChinaWeilin Zhang ChinaLanjing Zhang United StatesLei Zhong UruguayXiaohong Zhou United States

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