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European Journal of Pediatrics ISSN 0340-6199Volume 171Number 12 Eur J Pediatr (2012) 171:1793-1798DOI 10.1007/s00431-012-1817-2

Status of vitamin D in children with sicklecell disease living in Madrid, Spain

Carmen Garrido, Elena Cela, CristinaBeléndez, Cristina Mata & Jorge Huerta

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ORIGINAL ARTICLE

Status of vitamin D in children with sickle cell disease livingin Madrid, Spain

Carmen Garrido & Elena Cela & Cristina Beléndez & Cristina Mata & Jorge Huerta

Received: 17 March 2012 /Revised: 10 August 2012 /Accepted: 15 August 2012 /Published online: 5 September 2012# Springer-Verlag 2012

Abstract Patients with sickle cell disease have vitaminD deficiency and poor bone health which makes themprone to have an increased risk of fractures and osteo-porosis in adulthood. We performed a prospective,cross-sectional study in children diagnosed with sicklecell disease living in Madrid, Spain. The purpose of thisstudy was to evaluate the status of vitamin D of thesechildren. Patients 0–16 years old were enrolled between2008 and 2011. We studied demographics, calcium me-tabolism, and bone health, especially by measuring lev-els of 25-hydroxyvitamin D (25(OH)D), during differentseasons of the year, and bone densitometry (beyond4 years of age). Seventy-eight children were includedin the study. Mean age was 4.8±4.3 years, and meanserum 25(OH)D level was 21.50±13.14 ng/ml, with nodifferences in 25(OH)D levels within different seasons.Fifty-six percent of children had levels of 25(OH) vita-min D of <20 ng/ml, whereas 79 and 18 % of them hadlevels of <30 and <11 ng/ml, respectively. Secondaryhyperparathyroidism was observed in 25 % of children.Densitometry was performed in 33 children, and anabnormal z-score was seen in 15.2 % of them with nocorrelation with levels of 25(OH)D. Conclusions: Vita-min D deficiency is highly prevalent in children withsickle cell disease, who are residing in Madrid, Spain,and it is detected at a young age. We propose that earlyintervention may increase the possibility of an adequatebone density later in life.

Keywords Sickle cell disease . Vitamin D deficiency .

25-Hydroxyvitamin D

Introduction

Sickle cell disease (SCD) comprises a group of hemoglobindisorders characterized by chronic hemolysis and intermit-tent episodes of vascular occlusion inducing tissue ischemiaas well as acute and chronic organ dysfunction. Thus, anexpert and comprehensive care of patients with this diseasemay decrease their morbidity and increase their lifeexpectancy.

SCD is the most prevalent genetic disease in severalcountries including Spain [8, 12]. At present, the majorityof patients with SCD live at least until mid-adulthood.Although life expectancy in Spain is unknown, it is likelyto be around 40 to 50 years, which is similar to that de-scribed in other countries [37]. In addition, many advancesachieved in recent years in the management of these patientshave increased significantly their quality of life [4, 19, 36].Thus, different lines of research in this disease are opentoday to move forward in providing the best care for patientsand decreasing their morbidity and mortality.

No studies have been done on vitamin D deficiency inchildren and adults with SCD in our country. It is unknownwhether the fact of living in a country with year roundsunlight (latitude 43 N) is associated with lower rates ofvitamin D deficiency in this population. Studies in childrenwith SCD in others countries, including those with a similarlatitude [13, 42], described insufficient levels (<30 ng/ml) of25-hydroxyvitamin D (25(OH)D) in the majority (>90 %) ofthese children.

It has been long assumed that a varied diet and living nearthe equator were requirements for the maintenance of ade-quate levels of 25(OH)D. However, there are reports of

C. Garrido (*) : E. Cela : C. Beléndez :C. Mata : J. HuertaDivision of Pediatric Hematology and Oncology,Department of Pediatrics Medicine,Hospital General Universitario Gregorio Marañón,Maiquez 9,28007 Madrid, Spaine-mail: cgccolino@gmail.com

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vitamin D deficiency in all geographical latitudes, whichseems to question the importance of these factors [30, 41,42].

Vitamin D deficiency is related to an increased rate ofrespiratory infections, muscle weakness, and poor dentaleruption in healthy children [24]. More remarkable, childrenwith SCD have 5.3-fold increased risk of developing vita-min D deficiency than healthy African-American controls[42]. This could be related to an increased skin pigmentation[15, 38], reduction of exposure to sunlight, and lower intakeof calcium, vitamin D, vitamin E, folates, and fiber. Thislack of nutrients intake is more remarkable beyond the ageof 4 years [29, 33]. The high prevalence of lactose intoler-ance in this population may also contribute to their nutri-tional deficiencies [11]. Furthermore, bones of children withSCD are affected by infarcts, osteoporosis, osteomyelitis, orosteonecrosis, all associated with an increased risk of frac-tures and bone deformities [7, 32, 44]. Thus, low levels ofvitamin D could further impair the bone comorbidities fre-quently associated to children with SCD.

Purpose

1. To assess the status of vitamin D in children with SCDmostly diagnosed by a neonatal screening program inthe Community of Madrid.

2. To determine potential risk factors involved in the var-iability of 25(OH)D levels in this population.

Methods

Informed consent was signed by parents or guardians of thechildren and by the subject when appropriate.

Patients

Children between 0 and 16 years of age with SCD wereprospectively recruited at the Hemoglobinopathy Clinic ofthe General University Hospital Gregorio Marañón, inMadrid (Spain), between January 2008 and November2011. Most of these children (92 %) had been diagnosedwith SCD through a neonatal screening program based inour hospital to detect structural hemoglobin abnormalitiesthat was launched in May 2003 by the Community ofMadrid. A few children (8 %) were diagnosed in theircountries or by electrophoresis after clinical suspicion.

Data on ethnicity, age, sex, Tanner stage, weight, height,disease severity (measured as days of hospitalization peryear), history of bone fractures and their location, type of

anemia, vitamin D prophylaxis during the first year of life,and duration of breastfeeding were collected. Laboratorytests were performed coinciding with clinical follow-upvisits every 3 or 6 months. Levels of hemoglobin (Hb),reticulocyte count, total calcium, ionized calcium, magne-sium, phosphorus, and C-reactive protein (CRP) were mea-sured. Hormone determinations were performed in thehormone laboratory by the following techniques:

1. Vitamin D was measured by the quantitative determina-tion of 25(OH)D by Liaison 25 OH Vitamin D totalassay (310–600), a direct competitive chemiluminescentimmunoassay.

2. Parathyroid hormone (PTH) was measured in the morn-ing to avoid interference with the physiological noctur-nal rise of this hormone. It was analyzed using theImmulite 2000 analyzer, a sequential immunometricchemiluminescent assay in solid phase. This test meas-ures intact PTH and discriminates between normalpatients and those with primary hyperparathyroidism.

3. Serum osteocalcin by an immunoradiometric assay andbone alkaline phosphatase (BAP) were determined byAccess Ostase assay, an in vitro immunoassay in asingle phase.

4. Bone densitometry (DXA) was performed in children withSCD older than 4 years of age since we considered this agethe limit for cooperation without the need for sedation.Official recommendations from a panel of experts of theInternational Society for Clinical Densitometry (ISCD),regarding the normal values of DXA in children andadolescents published in 2008 and, subsequently, in2010, were followed [5, 6]. The results were interpretedaccording to age, sex, and ethnicity of children.

Reference values

The reference ranges used in this study are:

– Deficit of vitamin D: 25(OH)D of <20 ng/ml (50 nmol/l).

– Borderline values: 25(OH)D between 21 and 29 ng/ml(51–74 nmol/l).

– Normal values: 25(OH)D of >30 ng/ml (75 nmol/l).

These definitions, although based on adults studies, areincreasingly applicable to children [20, 26]. Low bone min-eral density (BMD) is defined as a Z-score≤−2 standarddeviations (SD) adjusted for age, sex, ethnicity, and height[40]. The ISCD defines osteoporosis in children as theassociation of low BMD and a history of bone fractures,such as a long bone fracture in the lower limbs, vertebralcollapse, or two or more fractures in long bones of the upperlimbs [5].

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Statistical analysis

Descriptive statistics were used for demographic data andbaseline characteristics of the patients. Qualitative variableswere described by frequency and percentage and analyzedby Fisher’s exact, chi-square, or McNemar’s tests. Quanti-tative variables were described by mean and SD of the meanfor parametric analysis and by median and 25th to 75thquartiles for nonparametric analysis. These variables werecompared using Student’s t test or ANOVA for parametricsamples and Wilcoxon rank-sum, Kruskal–Wallis, or Mann–Whitney U tests for nonparametric samples. Correlationswere performed using Pearson or Spearman tests accordingto the distribution of the samples. Standard linear or multi-ple regression analyses were used, including only thosevariables considered important. P<0.05 was considered sta-tistically significant. Data were recorded and analyzed usingSPSS statistics 18.0 (SPSS Science, Chicago, IL).

Results

The study enrolled 78 children, 44.9 % female. Sixty-sevenpercent of them were African (mostly from sub-SaharanAfrica); 29.3 %, African American; and 4 %, Asian. Thetype of anemia that these children had was mostly Hb SS(76.9 %), followed by Hb SC (14.1 %) and Sβo (threepatients) or Sβ + (four patients), (together comprising theremaining 9 % of cases).

The mean age at the time of enrolment was 4.8±4.3 years(range 0–16), with a weight of 20.5±12.2 kg and a height of107.5±29.6 cm. The Tanner stage was prepubertal in 89.3 %of patients. Furthermore, 28.2 % of the children were beingtreated with hydroxyurea, and only six patients (7.7 %) wereon a hypertransfusion regimen. The average length of hos-pitalization/year was 7 days with 85.1 % of children admit-ted to the hospital of <30 days/year.

Ninety-nine percent of the patients had been breastfed foran average of 4 months, and 47.4 % of them had receivedvitamin D prophylaxis for a mean of 8.78±4.7 monthsduring the first year of life. Four patients (5.3 %) had ahistory of fractures: two of them in long bones of the lowerlimbs, and two had vertebral fractures with associated flat-tening. The two latter patients had an abnormal Z-score (≤2SD).

Mean serum 25(OH)D in children from this study was21.50±13.14 ng/ml, with lower levels observed in summer,although it was not statistically significant (p00.58).

When considering vitamin D deficiency 25(OH)D valuesbelow 20 ng/ml, 56.4 % of the children had this deficiency.This percentage increased to 79.5 % if levels of 25(OH)Dbelow 30 ng/ml were considered insufficient. Importantly,17.9 % of the subjects had 25(OH)D levels corresponding

with rickets (<11 ng/ml). When children less than 5 yearsold (n046) were analyzed separately, 41.3 and 67.4 % ofthem had levels of 25(OH)D less than 20 and 30 ng/ml,respectively.

Other remarkable parameters found to be abnormal inthese patients were low mean Hb (8.89±1.48 gr/dl) andan increased reticulocyte count as an indicator of bonemarrow erythropoiesis in response to hemolysis. Al-though PTH levels were normal, secondary hyperpara-thyroidism (PTH>50 ng/l) was found in 25 % of thechildren studied. Furthermore, children with 25(OH)Dof <20 ng/ml had secondary hyperparathyroidism signif-icantly more frequently than children with higher levels(78.9 and 11.8 %; p00.02). This association was notseen when children with levels of <30 ng/ml wereanalyzed.

There was a significant inverse correlation between PTHand phosphorus levels with 25(OH)D (r0−0.40, p00.01;and r0−0.25, p00.05, respectively). There was also a sig-nificant correlation between 25(OH)D and age (r0−0.53)and Z-weight (r0−0.52) and Z-height (r0−0.56) (all p00.01). The remaining quantitative variables, including osteo-calcin, total alkaline phosphatase, BAP, Hb, reticulocytecount, iron, ferritin, CRP, and BMD Z-score, had no asso-ciation with levels of 25(OH)D in this study. The linearregression study (age/level of 25(OH)D) was significant(r200.35; p<0.01). There were no differences in the levelsof 25(OH)D in our patients according to sex, type of SCD,breastfeeding, or ethnicity, although African children had, ingeneral, lower levels of this vitamin. However, higher levelsof 25(OH)D were observed in children receiving vitamin Dprophylaxis at 400 IU/day during the first year of life asrecommended by our health authorities [1] regardless of theduration of prophylaxis (25.70±15.58 vs 18.03±9.73; p00.03).

When performing multiple regression studies in thesechildren, the best predictive model for 25(OH)D levelswas achieved with Z-height, Z-weight, and length of hospi-talization (r200.53; p<0.01). However, age was not signif-icantly associated with 25(OH)D when these studies wereperformed. There was not a relationship between differentlaboratory parameters and the levels of 25(OH)D. Regard-ing the BMD, a pathological Z-score (≤2 SD) was found in17.5 % of children studied (n040) without a significantcorrelation with 25(OH)D levels.

Discussion

There are numerous studies in the medical literature world-wide demonstrating a relationship between vitamin D defi-ciency and rickets both in healthy children and in childrenwith chronic diseases [16, 18, 21–23, 31, 41]. The Institute

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of Medicine, along with many other experts, agrees aboutthe importance of vitamin D in bone health [45].

The definition of normal or desirable levels of 25(OH)Dhas been a subject of debate in recent years. Althoughwithout an absolute consensus, most experts define thefollowing vitamin D rankings based on levels of 25(OH)D[25]. These definitions, although based on adults studies, areincreasingly applicable to children [20, 26].

The current study, unlike previous studies, evaluatesyounger children, including children less than 12 monthsold (11 cases), with a mean age of 4.8±4.3 years, which ismuch lower than those previously evaluated. Vitamin Ddeficiency (defined as 25(OH)D of <20 ng/ml) was foundin 56.4 % of cases, and vitamin D insufficiency (25(OH)Dof <30 ng/ml), in 79.5 % of cases in this study. This highprevalence of low levels of 25(OH)D in these children is inagreement with other previous studies [9, 14, 42]. Further-more, we observed that 17.9 % of these children had levelsbelow 11 ng/ml, which is consistent with rickets. Remark-ably, these children did not demonstrate any clinical signs orsymptoms. Although abnormal levels of 25(OH)D werefound since the first year of life, the group of childrenbetween 0 and 5 years of age had the most optimum levelsfor appropriate bone health, and beyond that age, none ofour children had optimum levels of 25(OH)D. Other studieshave also seen higher levels of 25(OH)D in younger chil-dren [29]. In the current study, breastfeeding or the age atwhich whole milk was introduced did not explain this dif-ference in 25(OH)D levels between the two age groups.Vitamin D status of mothers during pregnancy may havean impact on the levels of 25(OH)D of their children, butthis parameter was not evaluated in this study. A significantdifference was found between children who received anyprophylaxis with vitamin D in the first year of life, com-pared with those children who did not receive it (26 vs 18;p00.03). It is possible that this extra intake of vitamin Dmay have helped maintain higher levels of 25(OH)D, espe-cially during the first year of life. Other factors, such asseverity of illness, intake of calcium and vitamin D fromfood, exercise, and growth rate, are likely to play an impor-tant role in explaining this difference, but these parameterswere not evaluated in this study.

In the study, there was 25 % of secondary hyperparathy-roidism which was significantly more frequent in childrenwith 25(OH)D levels <20 ng/ml and inversely correlatedwith levels of 25(OH)D and phosphorus. The present studydid not find any other correlation between this and othermarkers of bone resorption, such as osteocalcin or BAP, inagreement with other publications [13, 14].

We did not detect any seasonal variation in 25(OH)Dlevels in our study, confirming prior reports [39, 46] butcontradicting the high rates of vitamin D deficiency seenduring spring by Buison et al. [10]. Rovner et al. [42]

studied 61 African children of 9–18 years of age living inPhiladelphia with SCD and compared them with healthychildren with the same characteristics in terms of race,geographical location, and age. Severe vitamin D deficiency(levels of <11 ng/ml) was found in 33 % of children withSCD, compared to 9 % in the control group (p<0.05).Furthermore, 93 % of children with SCD and SS hemoglo-bin had insufficient levels (<30 ng/ml). They also observeda lower BMD (16.1±2.8 vs 19.5±4.1) and increased PTHlevels (25 vs 17 %) in children with SCD. Two other studies,one in California [30] and one in Philadelphia [9], describevitamin D deficiency in 65 % of these children despite theirdifferent latitudes. However, the cutoff level of 25(OH)Dused in these studies was different. The latter study analyzeddata from 65 children aged 5 to 18 years and found vitaminD deficit (<11 ng/ml) in 65 % of them. They also observedan increase in PTH levels in these patients not clearly relatedwith 25(OH)D concentrations. They concluded that thisdeficit may be related to skin color and a lower intake ofvitamin D of this population along with the season studied(spring was associated with the lowest levels of 25(OH)D).

Chapelon et al. studied 53 children with SCD with amean age of 12.8±2.4 years and observed a vitamin Ddeficit (defined as 25(OH)D of <12 ng/ml) in 76 % and anincrease in PTH level in 27 % of them, respectively [14].

It is known that the disease affects bone health. In addi-tion, the frequent dactylitis affecting infants with this con-dition is the result of necrosis of the epiphysis of thephalanges and results in permanent shortening of the carpusand metacarpus. Children and adolescents with this disorderalso have an increased risk of necrosis of the femoral head.On the other hand, there is bone marrow hyperplasia withwidening of the bone marrow spaces in long bones andcortical thinning which leads to increased bone fragility.

Small sample studies in children with SCD have shown adecreased BMD in this population [30]. The present studyevaluated BMD (Z-score) in children older than 3 years ofage and found that 17.5 % of these children had a patholog-ical BMD Z-score (≤−2 SD). We did not find a correlationbetween the BMD Z-score and the levels of 25(OH)D,which could be explained by the fact that the measurementsof 25(OH)D were performed at only certain time points, andthus, it did not analyze a more continuous correlation.Therefore, it is possible that a correlation may have beenfound between these two factors in these children if theevaluation had been performed more frequently and extend-ed sufficiently over time. The current study found a positivecorrelation between BMD Z-score with hemoglobin levelsand an inverse correlation with reticulocyte count (p<0.01),which may indicate that greater hemolysis and, therefore,disease severity could be associated with lower BMD inthese children. In addition, children with more severe he-molysis have an increased erythroid hyperplasia that may

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cause trabecular destruction leading to lower bone density[34]. These results have also been observed by Baldanzi etal. [3].

In fact, there are only few publications analyzing the riskof fractures in children with SCD, mostly single case reportsand a cohort of children with osteomyelitis [2, 17, 28, 43].Further studies are needed to assess the possible associationbetween vitamin D levels and the risk of fractures in thispopulation. In this regard, a prospective study performed innon-SCD African children between 5 and 9 years of age, toinvestigate the relationship between vitamin D status andbone fracture, concluded that a significant number of chil-dren with fractures had, in fact, low 25(OH)D levels (59 %)[43].

This relationship between low BMD and fractures inthese children was not evaluated in this study due to thescarce number of patients with fracture, which was detected(n04; two with a pathological DXA). Other previouslyreported associations, such as vitamin D status and chronicor acute pain [27, 35], were not evaluated.

In this study, children of >5 years of age had the mostsignificant 25(OH)D deficiency in agreement with previousstudies [29]. Nevertheless, even children under 5 years hadfrequently low levels of this vitamin. Of note is the fact thatinfants younger than 12 months of age also had low levels of25(OH)D despite the fact that most of them were undergo-ing vitamin D prophylaxis. These findings suggest thatlevels of 25(OH)D should be determined in these childrenearly in life to prevent a state of chronic vitamin D deficien-cy and, therefore, maintain appropriate bone health.

Conclusion and new directions

In conclusion, the present study found a high prevalence ofeither insufficient levels or deficiency of vitamin D in chil-dren with SCD from the Community of Madrid, whichoccurred early in life. The cause of this deficit is probablymultifactorial and not well known, but it is obvious that thefact of living in a country with enough sunlight is notsufficient to maintain adequate 25(OH)D levels in thispopulation.

The authors do not know at this point which may be themost appropriate treatment and/or prophylaxis for thesechildren, but it seems that early intervention may be impor-tant. Pending resolution of the current questions seemsappropriate to develop strategies to improve the peak bonemass and prevent fractures and osteoporosis in these chil-dren in the future.

Physicians involved in the care of these patients shouldbe aware of the importance of vitamin D deficiency anddetermine its levels for possible interventions and an appro-priate follow-up. They should insist in the importance of

good nutrition and assure adequate calcium and vitamin Dintake for these children.

Acknowledgments The authors would like to thank Dr. CynthiaMcCoig and Dr. Jesús Saavedra-Lozano for reviewing the manuscript.

Conflict of interest None.

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