Vitamin C metabolites, independent of smoking status, significantly enhance leukocyte, but not...

Adv Ther. 2008;25(10):995-1009.DOI:10.1007/s12325-008-0106-y

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ORIGINAL RESEARCH

Vitamin C Metabolites, Independent of Smoking Status, Significantly Enhance Leukocyte, but not Plasma Ascorbate ConcentrationsMark A. MoyadMaile A. CombsAngelica S. VrablicJanet VelasquezBenilda TurnerSamuel BernalUniversity of Michigan Medical Center, Department of Urology, Ann Arbor, Michigan, USAUniversity of California, Los Angeles, San Fernando Valley Program, Sepulveda VA Medical Center, California, USA

ABSTRACT

Introduction: The objective of this study was to test the effects of acute dos-es of vitamin C alone, calcium ascorbate with vitamin C metabolites, and placebo, on total plasma and leukocyte vitamin C concentrations over 24 hours. Methods: A double-blind, placebo-controlled, four-way crossover study was performed consisting of four separate phases lasting 24 hours each and utilizing one of four oral 1000-mg preparations within each phase (one of vitamin C alone, two separate vitamin C formulations of calcium ascor-bate with vitamin C metabolites, and placebo). There was a 7-day washout between phases, and blood draws at seven time points within each phase of the study for a total of 28 serologic measurements per subject and 420 total measurements for the entire clinical trial. Vitamin C concentration in plasma and leukocytes were measured by high-performance liquid chroma-tography at baseline and at six sequential time periods over 24 hours.

Address correspondence to: Mark A. Moyad, Jenkins/Pokempner Director of Preventive & Alternative Medicine, University of Michigan Medical Center, Department of Urology, Ann Arbor, Michigan 48109-0330, USA. Email: [email protected], [email protected]

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Results: Fifteen healthy males were enrolled, aged 18-39 years; nine were had never smoked and six were chronic smokers. No significant difference in plasma vitamin C levels was observed when comparing the different preparations. However, at 24 hours, calcium ascorbate with metabolites resulted in significantly higher concentrations of vitamin C in leukocytes (P<0.0001) compared with vitamin C alone. These results were similar for both metabolite formulations, and independent of smoking status.Conclusion: Regardless of smoking status, vitamin C metabolites may en-hance leukocyte utilization of vitamin C itself, despite no consistent dif-ference in plasma levels among the different preparations. A larger clinical investigation is warranted to confirm these preliminary findings, and to de-termine the clinical relevance of this impact on overall immune function.

Keywords: ascorbic acid; calcium threonate; immune function; vitamin C; metabolites; white blood cells

INTRODUCTION

Vitamin C’s antioxidant capacity and stability within the plasma milieu has been previously well described.1,2 Past and ongoing randomized trials rely on these specific blood measurements to assist in determining compliance rates and the overall significance or clinical impact of ingesting this specific nutrient compared with other agents.3-5 Vitamin C plasma levels are unique compared with the ma-jority of other nutrients due to their sen-sitivity to physiologic stressors, such as alcohol or smoking, which cause signifi-cant and acute reductions in absorption or bioavailability.6,7 Plasma values obvi-ously provide some insight and value for researchers and clinicians, but finding other blood and tissue markers that pro-vide greater interpretation of the role of vitamin C in humans appears needed due to generally strict plasma concentration

control, and a general lack of therapeuti-cally tangible clinical range limits.8,9

Less understood, but arguably just as critical, is the bioavailability, concentra-tion, and specific function of vitamin C within white blood cells—or leukocytes. Vitamin C concentration in these cells appears critical to their function,10 where the concentration of vitamin C exceeds the amount found in plasma by as much as 100-fold.11 Utilizing vitamin C supple-mentation alone, or alternate formulations of vitamin C, are a plausible initial clinical step into the investigation of ascorbic acid uptake and concentration in cells such as leukocytes, because of its potential role in acute and chronic disease prevention or treatment enhancement.12

The objective of this study was to de-termine plasma and leukocyte vitamin C concentrations over 24 hours after an acute morning 1000-mg dose was administered in the form of vitamin C alone, calcium

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ascorbate with vitamin C metabolites (Es-ter-C®, NBTY, Inc., Bohemia, NY, USA; calcium ascorbate as two different formu-las – metabolite 1 [M1] with approximate-ly 3% calcium threonate and metabolite 2 [M2] with approximately 1% calcium threonate), or placebo in a double-blind, randomized, four-way crossover study in human subjects. A secondary endpoint of this investigation was to compare vitamin C leukocyte concentrations in subjects who had never smoked versus chronic smokers. Smokers harbor significantly lower concentrations of vitamin C in plas-ma,6 which can further negatively impact immune function,4,5 but an adequate de-termination of leukocyte concentrations needed to be further defined, regardless of smoking status.

MATERIALS AND METHODS

Population and Study Design

Inclusion criteria were as follows: males aged 18-50 years with no acute or chronic disease (as determined by labora-tory results, medical history, and physical examination). Exclusion criteria were as follows: females; subjects with history of use of prescription or over-the-counter products known to interact with vitamin C within 1 week of randomization or during the trial; subjects who used mul-tivitamins and other natural health prod-ucts containing vitamin C within 1 week of randomization or during the trial; and subjects with a history of alcohol or drug abuse within the past year. Fifteen adult males aged 18-39 years were tested for

plasma and leukocyte absorption and re-tention of vitamin C from three different preparations and placebo at baseline and from multiple time points after receiving a computer-randomized sequence of each tablet. Females were excluded in this clini-cal study to avoid gender-specific varia-tions in vitamin C pharmacokinetics that have been observed in a past clinical inves-tigation, mostly due to the impact of spe-cific but variable menstruation time points on vitamin C status that could potentially confound our investigation, regardless of multivariate analysis.13 The protocol, which included the consent form, was ap-proved by the institutional review board at the University of California, Los Angeles/San Fernando Valley Program, Sepulveda VA Medical Center. Written informed consent was subsequently obtained from each subject.

Of the 15 subjects, nine had never smoked and six were chronic smokers. The nonsmoking group reported that they had no history of ever smoking, but the chronic smokers averaged 10 cigarettes per day, for at least 2 consecutive years, ranging from 6 to 14 cigarettes per day, for at least 2 weeks before and throughout the duration of the study. Detailed self-reports were part of the design to improve self-report reliability.14 Past smokers were not included due to previous studies that have suggested an under- or overestima-tion of the time period since smoking ces-sation,15,16 which could theoretically have compromised the objectivity of this pre-liminary study, somewhat similar to the past and ongoing issue in regards to self-re-ported studies of weight.17 Both nonsmok-


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ing and smoking groups were matched in terms of age, weight, anthropometric vari-ables and blood glucose levels, Charlson Comorbidity Status (no comorbidities or a mandatory score of zero),18 moderate al-cohol intake, and up-to-date vaccinations. These normal baseline values were also re-quired during recruitment to improve the validity or objectivity of our comparisons, since past,19-22 and especially recent find-ings,23 have suggested our investigation and serologic analysis could be potentially compromised in participants with abnor-mally large mean values in these specific baseline characteristics.

Three groups of five subjects each were randomized in a double-blind, four-way crossover design. The three groups were rotated according to the order of tablets (one per day), which consisted of vitamin C alone (1000 mg), M1, M2 (each containing 1000 mg of vitamin C as determined by iodometric titration), and placebo. All interventions had a similar appearance, size, and lack of odor.

To further minimize potential plasma and leukocyte baseline vitamin C bias, subjects were instructed to follow identi-cal vitamin C-deficient diets, which con-sisted of avoiding foods and beverages with high vitamin C content to achieve intake of less than 5 mg/day ascorbic acid, as de-scribed in previous depletion investiga-tions.1,24 Subjects were also instructed to refrain from taking supplements, including vitamin C, for 5 days prior to and through-out the study duration and not to take any other over-the-counter tablets (including analgesics such as aspirin) throughout the study, which could have compromised

the plasma and leukocyte analysis.25 Adverse events were recorded throughout the study.

Biological Measurements

An in-dwelling venous catheter (with heparin lock) was inserted for multiple repeated sampling of blood, thus avoiding concern over reliability and discomfort of repeated venipunctures by both research staff and participants. After the initial washout period, subjects fasted for 9-12 hours overnight. Baseline blood samples were drawn, and subjects were adminis-tered treatment according to the random-ization. Subjects received study tablets with 250mL water in the controlled laboratory setting to further eliminate bias and com-pliance issues. Otherwise, subjects were instructed to drink water as needed dur-ing the entire study period. Subsequent blood samples were taken at 1, 2, 3, 4, 8, and 24 hours postdose in order to approx-imate a variety of past pharmacokinetic studies with a modified schedule.1,24,26,27 This sequence was repeated for all four arms of the crossover, which were separat-ed by 7-day washout periods for a total of 28 blood measurements per subject, and 420 total measurements for the entire clinical trial.

Blood samples for plasma and leuko-cyte vitamin C measurements were col-lected from the subjects at the specified times after ingestion of test tablets, into 5-mL heparinized Vacutainer (BD Diag-nostics, Franklin Lakes, NJ, USA) tubes. The tubes were labeled with the subject’s number and the time of blood collection.

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The codes and the samples were collected in the laboratory, without any identifica-tion by the laboratory staff. Blood samples were kept in ice throughout the collection period and during white cell separation.28

An aliquot from each blood sample was collected in a citrated tube for determi-nation of blood volume and white blood cell counts. After centrifugation at 1000 r.p.m. for 15 minutes, the plasma in the upper layer was pipetted into a new tube. The white layer or buffy coat layer was then pipetted into fresh tubes and diluted threefold with a buffered saline solution. The cell suspension was layered onto two volumes of Ficoll-Hypaque (Amersham Pharmacia Biotech, Inc, Piscataway, NJ, USA) in conical centrifuge tubes. After centrifugation at 2000 r.p.m. for 15 min-utes, the white cell layer was pipetted into a fresh centrifuge tube. The white cell sus-pension was diluted with 5 mL cold saline and centrifuged at 2000 r.p.m. for 5 min-utes. The pelleted cells were resuspended in 2 mL cold saline for counting and extrac-tion. The cell suspension was repelleted and suspended in 1 mL 90% methanol containing 1.0 mM EDTA and stored at –70°C until study completion. The frozen suspensions of white blood cells were dis-rupted by thawing and sonication for a to-tal of 15 seconds. These suspensions were centrifuged and the supernatant separated from the insoluble sediment. All samples underwent precolumn deproteination us-ing a hydroxyapatite cartridge (PCPure cartridge, Koken Company, Tokyo, Japan) eluted with 10 mM sodium phosphate buffer (pH 6.8) containing 2.5 mg/mL L-cysteine.29 After preparation, the eluent

solution was stored frozen, then thawed and kept on ice prior to use. The solution was stable at 5°C for 3 days.

A high-pressure liquid chromatog-raphy (HPLC) system (Waters Corpo-ration; Milford MA, USA) with a UV detector was used to analyze the vita-min C levels in the plasma and leukocyte samples. Separations were carried out on C18 columns (5 μm bead; 4.6 mm diam-eter) with a 3-cm C18 guard column. The mobile phase consisted of 5% (by volume [v/v]) acetonitrile, 1% (v/v) acetic acid, 1.8 mM tetrabutylammonium hydroxide, at pH 4.8 (adjusted with 1.0 N NaOH). Absorbance at 265 nm by the UV detector was used to detect and quantify the ascor-bate in the effluent. At this wavelength, there was no significant absorbance by several metabolites of ascorbate, including dehy-droascorbate. A range of 500 to 2000 pmol vitamin C solubilized in the EDTA/metha-nol was used as standards, and checks were performed each time samples were run.

Statistical Measurements

Area under the curve for vitamin C levels in plasma and leukocytes were de-termined for each group using the trap-ezoidal rule.30 The area under the curve was normalized, dividing by 24 to cre-ate a time-weighted average in the origi-nal units. The data were analyzed using repeated-measures analysis of variance (ANOVA). A P value of <0.05 was con-sidered statistically significant.

A multivariate ANOVA, incorporat-ing the crossover design, was performed with product (vitamin C, M1, M2, and


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placebo), and smoking status (yes or no) as two factors (and the method, plasma or leukocyte, as two response variables). Least-square means were calculated with standard errors, as estimated by the ANO-VA model. A series of a-priori contrasts were calculated and are presented with P values testing for (statistically significant) differences from zero.

RESULTS

Figures 1 through 4 represent the vi-tamin C concentration kinetics in plasma (Figures 1 and 2) and leukocytes (Figures 3 and 4), and for nonsmokers (Figures 1 and 3) and smokers (Figures 2 and 4) as a func-tion of time. In order to convert μmol/L

vitamin C to mg/dL, multiply μmol/L by 0.0176. To convert mg/dL vitamin C to μmol/L, multiply mg/dL by 56.8.

Mean age, and clinic-recorded weight, body mass index, and waist-to-hip ratio for both groups were similar: 28 years, 79 kg, 25 kg/m2, and approximately 0.9, respec-tively. Vitamin C plasma concentrations of smokers differed from those of nonsmokers. Plasma levels of smokers demonstrated an acute reduction of vitamin C between 4 to 8 hours after intervention, compared with a gradual decrease in nonsmokers, between 2 to 8 hours after intervention. No signifi-cant differences in plasma levels, regardless of posttreatment time monitoring period, were observed between the different vita-min C interventions (Figures 1 and 2).

Figure 1. Plasma vitamin C concentrations in subjects who had never smoked, after four treatments: vitamin C alone (AA), calcium ascorbate with 3% calcium threonate (M1), calcium ascorbate with 1% calcium threonate (M2), and placebo (PL). Error bars represent SE.

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In contrast to the similar kinetic pro-files for plasma vitamin C concentration among the various formulations, a differ-ence was observed in white blood cell ki-netics. The retention of vitamin C from the M1 and M2 preparations resulted in 1.3 to 1.7 times the level of vitamin C in leukocytes relative to ascorbic acid alone at 24 hours posttreatment; this difference was evident regardless of smoking status. Additionally, return to baseline vitamin C status in the leukocytes of smokers was less acute with the metabolite formulas compared with ascorbic acid by itself. Least-square means and a-priori contrasts, when tabulated, confirmed the observa-tions from Figures 1 through 4, which demonstrate the following specific find-

ings, independent of smoking status, when comparing the different interventions (vi-tamin C, M1, M2, and placebo):

Vitamin C is not significantly (P=0.3761) different from M1 in plasma, but it is significantly (P<0.0001) different in leukocytes compared with M1.Vitamin C is not significantly (P=0.3254) different from M2 in plasma, but it is significantly (P<0.0001) different in leukocytes compared with M2.Vitamin C is significantly differ-ent compared with placebo in plasma (P<0.0001) and leukocytes (P<0.0001).M1 and M2 are not significant-

Figure 2. Plasma vitamin C concentrations in chronic smokers after four treatments: vitamin C alone (AA),calcium ascorbate with 3% calcium threonate (M1), calcium ascorbate with 1% calcium threonate (M2), and placebo (PL). Error bars represent SE.

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ly different from each other in plasma (P=0.9262) or leukocytes (P=0.0652).M1 is significantly different from placebo in both plasma (P<0.001) and leukocytes (P<0.001).M2 is significantly different from placebo in both plasma (P<0.0001) and leukocytes (P<0.0001).

No drop-outs occurred among any of the participants (nonsmokers or smokers) and all serologic collections over 24 hours were obtained, regardless of the time pe-riod and specific group rotational assign-ment. No significant adverse effects were reported in the different interventions during the various time periods.

DISCUSSION

Vitamin C has been the subject of discussion as of late, with controversy over whether or not this nutrient impacts the common cold.31 Missing from this discourse are the other, and in our opin-ion more pressing, clinical attributes of this vitamin beyond the common cold debate.32 It has been one of the primary components in combination products utilized in a variety of large-scale positive clinical trials from macular degeneration,33 to general health prevention.34 These stud-ies have demonstrated significant findings that have even changed clinical practice guidelines.35 Vitamin C is a standard com-ponent of prenatal vitamins and serves to

Figure 3. Leukocyte vitamin C concentrations in subjects who had never smoked, after four treatments: vitamin C alone (AA), calcium ascorbate with 3% calcium threonate (M1), calcium ascorbate with 1% calcium threonate (M2), and placebo (PL). Error bars represent SE.

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significantly increase iron absorption and reduce the risk of anemia in pregnancy.36,37 Additionally, vitamin C has been the sole component in some positive and large randomized trials that may change other disciplines of medicine, such as orthope-dics, due to its potential to prevent com-plex regional pain syndrome in individuals who have recently suffered from wrist frac-tures.38,39 Vitamin C is also currently a part of one of the largest primary prevention trials in human history that seeks to deter-mine its clinical impact on cardiovascular disease, cancer, and eye disease by itself or as part of a combination of supplements.5

Thus, improving and utilizing other tangible blood and tissue markers of vi-tamin C to predict clinical outcomes in

patients seems imperative, especially since plasma and red blood cell levels of this vi-tamin are tightly controlled.1,40 Vitamin C levels have been known to vary as much as 100 times between plasma and a variety of tissues.8 A critical novel clinical and physi-ologic issue surrounding vitamin C is its role outside of tightly controlled plasma and red blood cell levels. For example, a re-cent clinical study of adrenal tissue found localized tissue or paracrine concentrations in excess of what was expected with tightly regulated plasma levels suggesting multiple roles for this vitamin beyond what plasma levels are able to predict.41 Perhaps part of the strict control of the human body in regards to plasma vitamin C can be con-strued as a method of allowing greater tis-

Figure 4. Leukocyte vitamin C concentrations in chronic smokers after four treatments: vitamin Calone (AA), calcium ascorbate with 3% calcium threonate (M1), calcium ascorbate with 1% calciumthreonate (M2), and placebo (PL). Error bars represent SE.

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sue or other cell-enhancing concentrations of this same nutrient. One of the ideal methods of pursuing this hypothesis is to further delineate the role of vitamin C in leukocytes,42,43 which in some cases have a potential dual function of circulating in blood or extravasating into and around tis-sue sources.44 Past studies of vitamin C in leukocytes have been inconclusive,43 but most suggest they harbor higher concen-trations except when subject to acute stres-sors such as alcohol and smoking.

In our preliminary clinical investiga-tion, we decided to utilize multiple time points over 24 hours to provide some clarity on cell concentration issues, and to recruit subjects who had never smoked as well as chronic smokers to provide in-sight into any potential behavioral con-centration differences. We also attempted to determine the role of vitamin C me-tabolites in terms of leukocyte vitamin C concentrations, because of the large differ-ences observed consistently in past stud-ies of these compounds,45 and conflicting past clinical data on the importance of metabolites or other potential absorption mediators.26,27 Our data confirm that de-spite tight regulation of plasma vitamin C levels with and without the use of oral me-tabolites, there were significant increases in leukocyte levels for plain ascorbic acid and both vitamin C metabolite sources, regard-less of smoking status.

The enhanced absorption and perhaps bioavailability over 24 hours seen in this study suggests something mechanistically novel. Vitamin C gradient-driven transport into leukocytes primarily occurs through an initial oxidation into dehydroascorbic acid

and subsequent reduction back to ascorbic acid upon cell entry.46 Thus, threonate and other metabolites could theoretically stabi-lize further extracellular oxidation and me-tabolism of dehydroascorbic acid, and/or may enhance the facilitated or active trans-port signaling mechanisms required by vitamin C for intracellular access. Indeed, certain cells, including leukocytes, and di-verse tissues of the human body are capable of vitamin C concentrations many times greater compared with plasma or what can be predicted from saturated plasma curves.41 A complete mechanistic interpre-tation of the reasons for these differences remains open to debate and further basic science research. Still, we recognize the potential shortcomings of these novel mechanistic interpretations. We also have to recognize that these results could have been due to chance. However, the general lack of overlap from the standard error bars (Figures 1-4), the sheer number of blood samples drawn, and the equivalent amount of vitamin C administered to subjects in each study arm, as measured by iodometric titration of the active, nonplacebo tablets, does not suggest a chance observation. For this reason, our preliminary experiment has led to the design and implementa-tion of two randomized trials measuring specific and general immune cell vitamin C function under conditions of unique health-related immunologic stressors.

The strengths of our study have al-ready been highlighted, and include the randomized, double-blind, and four-way crossover design. The total number of se-rial blood values in a study of this size and time period (24 hours) is also unusual,

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and hopefully will set a standard for fu-ture comparison studies. In addition, to our knowledge, this study prescreened and controlled for one of the largest numbers of potential confounding variables of any combined vitamin C plasma and leuko-cyte research dietary supplement study, but again this should establish a paradigm. Our group was successful in controlling for multiple past and recently published vari-ables due to an extensive review of the vita-min C literature, and our desire to try and eliminate any possible issue of confound-ing at the initiation, rather than after the trial, which is one of the benefits of a ran-domized design, well-matched in terms of multiple baseline characteristics. However, we humbly acknowledge that we were also fortunate to have initiated this study after multiple other nutrient investigations had provided the insight into what should and should not be controlled for at baseline in terms of vitamin C concentrations.32

The limitations of our study must also be emphasized, including the small sample size. Regardless, it is still one of the most extensive human studies to have ever ad-dressed this specific issue, and the largest to our knowledge in terms of total serologic measurements over a 24-hour period uti-lizing both plasma and leukocyte concen-trations. Recruiting numerous subjects, for multiple separate blood draws, over several 1-day periods was taxing. This was somewhat expected based on the initial low response rate despite aggressive re-cruitment, and simply due to the repeated concern expressed by multiple potential subjects, regardless of participation status, over the numerous serologic collecting pe-

riods. Thus, our choice to utilize catheter-based measurements in this situation not only may have improved compliance, but we believe strengthens the findings from this investigation. Despite this, over the recruitment period the initial goal was to only include those who had never smoked, but after several chronic smokers inquired about the study it made sense to amend the protocol and strengthen the study and its numbers and interpretations by also using this group of individuals, who are arguably the most sensitive to vitamin C deficiency.6 Another limitation was the acute dosing schedule measured over this short time period. It is unclear from these results whether the differences in con-centration observed in leukocytes mirror the long-term chronic dosing that usually occurs in the case of dietary supplement consumption. Multiple blood draws in our opinion would need to be reduced or even eliminated in this situation. Thus, for this preliminary trial, the choice of study design seemed the most logical. Vitamin C concentrations were measured in total leukocyte counts collected according to a standard method, but there was no differ-entiation between neutrophil, eosinophil, basophil, monocyte/macrophage, or even lymphocyte subsets, which is another lim-iting factor. However, in this case the pre-liminary nature of the study provided the impetus for future studies to assess these potential differences. Finally, a larger trial that includes female participants is needed, and is being recruited in our next study to provide some resolution as to how gender status truly impacts on the reproducibility of these results.


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In conclusion, our data supports the be-lief that short-term vitamin C supplemen-tation significantly improves ascorbic acid concentrations in plasma and leukocytes in those who have never smoked and in chronic smokers over a 24-hour period. A similar but greater concentration of vitamin C in leuko-cytes was also found for both groups receiv-ing vitamin C metabolites compared with ascorbic acid alone or placebo. This suggests a potential positive feedback mechanism for vitamin C when certain cells are exposed to metabolites, or a possible sentinel signaling event for tissues that require greater levels of this nutrient, despite strict control in plasma levels. This phenomenon may be somewhat similar to what has recently been clinically described in the localized vascular net-work surrounding human adrenal tissue.41 Regardless, our data provided the impe-tus for two new clinical trials to further elucidate the mechanisms behind these preliminary, albeit significant, clinical observations.

ACKNOWLEDGMENTS

This research was presented September 27, 2007 at the 48th annual meeting of the American College of Nutrition, Orlando, Florida. NBTY, Inc. owns The Ester C Company and Ester-C® Calcium Ascor-bate, and provided funding for this study and the page charges for publication. An-gelica Vrablic, PhD and Maile Combs are employees, and Mark Moyad, MD, MPH is a paid Research Consultant, of NBTY, Inc. None of the other authors have any financial interest in NBTY, Inc. or The Ester C Company.

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