Background:

Serum 25-hydroxyvitamin D (25(OH)D) is low in obese adults.

Objective:

To examine serum 25(OH)D in obese (BMI >95th percentile for age) vs. non-obese (BMI = 5th–75th percentile for age) 6–10-year-old African American children and compare their differences in therapeutic response to vitamin D supplementation.

Methods and Procedures:

In an open label non-randomized pre-post comparison 21 obese (OB) and 20 non-obese (non-OB) subjects matched for age, sex, skin color, and pubertal maturation were treated with 400 IU of vitamin D₃ daily for 1 month. Serum 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)₂D), parathyroid hormone (PTH), leptin, and markers of bone turnover (serum bone-specific alkaline phosphatase (BSAP), osteocalcin (OC), and urine n -telopeptide cross-links of type 1 collagen (urine NTX)) were measured. Vitamin D deficiency was defined as serum 25(OH)D 20 ng/ml and insufficiency as 21–29 ng/ml respectively.

Results:

Vitamin D deficiency occurred in 12/21 (57%) OB vs. 8/20 (40%) non-OB at baseline (P = 0.35) and persisted in 5/21 (24%) OB vs. 2/18 (11%) non-OB (P = 0.42) after treatment. When the cohort was stratified by the baseline levels of 25(OH)D, there were differences in the response to treatment in the obese and non-obese cohorts.

Discussion:

Vitamin D deficiency was common among OB and non-OB preadolescent African American children, and 400 IU of vitamin D₃ (2 the recommended adequate intake) daily for 1 month was inadequate to raise their blood levels of 25(OH)D to 30 ng/ml.

Introduction

Serum 25-hydroxyvitamin D (25(OH)D), an indicator of vitamin D status, has been found to be low in obese adults (1,2,3,4). The explanation for this association has been the sequestration of vitamin D in the subcutaneous body fat and its consequent reduced bioavailability (3). Sedentary lifestyles of obese individuals could be associated with less outdoor activities and reduced sunlight exposure, perhaps also contributing to reduced endogenous vitamin D production and compromised vitamin D status. Vitamin D insufficiency associated with obesity is likely to be functionally significant, as compensatory hyperparathyroidism has been observed in obese adults with low 25(OH)D levels (1,2,3,4).

Bone metabolism, bone turnover, and bone mineral content are altered in severe obesity. Higher levels of serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) were reported in small cohorts of obese adults and children, which is understandable since the secondary hyperparathyroidism increases the renal production of 1,25(OH)₂D (1,2,5). However, recently reported large cross-sectional cohort studies of obese adults did not find elevated levels of 1,25(OH)₂D (4,6). Increased bone mineral content and lower levels of serum osteocalcin (OC) were noted in stable severely obese adults compared to non-obese adults (7).

The relationship between obesity and vitamin D status has not been well characterized during childhood. Therefore, we designed a study to extend the existing knowledge regarding vitamin D status and obesity in adults to a vulnerable childhood population. Our study objective was to examine the vitamin D status of obese vs. non-obese healthy preadolescent African American children and explore differences in their therapeutic response to 400 IU of vitamin D₃ daily, twice the recommended daily adequate intake for vitamin D, for 1 month (8).

We have previously reported the prevalence of vitamin D insufficiency in the study cohort (inclusive of both obese and non-obese children) (9). In this report, we focused on the obese versus non-obese differences in vitamin D status and their response to 400 IU of vitamin D₃ daily for 1 month.

Methods and Procedures

Subjects

Forty-two healthy preadolescent African American children aged 6–10 years were recruited from the Primary Care Center of the Children's Hospital of Pittsburgh and its satellite clinic at Wilkinsburg, PA. Exclusion criteria included ingestion of vitamin D or multi-vitamin supplements, anticonvulsants or systemic glucocorticoids, presence of hepatic or renal disease, metabolic rickets, malabsorptive disorders (Crohn's disease, cystic fibrosis, and celiac disease), and cancer. The study was conducted during winter and early spring over 2 years (December 2001 through April 2002 and December 2002 through April 2003). The research protocol was approved by the Human Rights Committee (Institutional Review Board) of the Children's Hospital of Pittsburgh. All parents and the children who participated in the study gave their informed consent and assent.

Study design

Study design was an open label non-randomized pre-post comparison of the effect of vitamin D supplementation in obese vs. non-obese preadolescent African American children. Categorization of the subjects as obese or non-obese was based on age and sex specific BMI. The obese subject had a BMI >95th percentile for age and the non-obese had a BMI between 5th and 75th percentile for age. Subjects were Tanner stage I or II for pubertal maturation and type 4 or 5 for skin color. Subjects assessed their pubertal status utilizing a self-assessment tool based on Tanner and Marshall's drawings of stages of secondary sexual characteristics (10,11,12,13). Skin color typing was based on the classification of Pathak et al. and Jimbow et al. (14,15). All the subjects were supplemented with vitamin D₃ 400 IU daily for 1 month (Delta D₃ 400 IU vitamin D tablets, Freeda Vitamins, Long Island City, NY). High-performance liquid chromatography analysis of five vitamin D tablets showed a mean vitamin D₃ content of 406 IU/tablet (16). Serum 25(OH)D, 1,25(OH)₂D, calcium, phosphorus, albumin, leptin and parathyroid hormone (PTH) levels, and markers of bone turnover (markers of bone formation: serum OC and bone-specific alkaline phosphatase (BSAP) and marker of bone resorption: urine n -telopeptide cross-links of type 1 collagen (urine NTX)), and a food frequency questionnaire were assessed at study entry and completion. Blood samples for biochemical measurements were obtained by venipuncture in a vacutainer tube with no anticoagulant in a non-fasting state during the day. Subjects were asked to return the container used for dispensing the vitamin D to assess compliance.

Assessment of vitamin D and calcium intake

Dietary intakes of vitamin D and calcium were estimated using a youth and adolescent food frequency questionnaire. Youth and adolescent food frequency questionnaire is a validated and reproducible self-administered food frequency questionnaire to assess the dietary intake of older children and adolescents (17,18). Although the subject and/or the parent were asked to complete the youth and adolescent food frequency questionnaire, most often the parent completed the questionnaire.

Biochemical measurements

Serum 25(OH)D was measured using Nichols Advantage 25(OH)D chemiluminescent assay (Nichols Institute, San Clemente, CA). The intraassay and interassay coefficients of variation were: 4.5 and 14.5% at 11 ng/ml and 3.6 and 7.8% at 57.8 ng/ml. Serum 1,25(OH)₂D was determined using radio immunoassay (Diasorin, Stillwater, MN). The intraassay and interassay of coefficients of variation were 12.5 and 18.7% at 23.2 4.3 pg/ml and 10.5 and 13.1% at 70.7 9.3 pg/ml. PTH was measured using IMMULITE Intact PTH chemiluminescent assay (Diagnostic Products, Los Angeles, CA). The intraassay coefficient of variation was 5.4% at the level of 26 pg/ml (n = 20) and interassay coefficient of variation was 5.0% at the level of 28 pg/ml (n = 20). BSAP was measured by enzyme immunoassay (Metra BAP; Quidel, San Diego, CA). The intraassay and interassay coefficients of variation were 4.7 and 4.8% at 14.3 0.7 U/l and 3.3 and 4.8% at 69.6 3.3 U/l. OC was measured by an enzyme immunoassay (Quidel, San Diego, CA). The intraassay and interassay coefficients of variation were 4.9 and 3.4% at 4.9 0.16 ng/ml and 5.0 and 4.6% at 20.5 0.94 ng/ml. Urinary NTX was measured using a competitive-inhibition enzyme-linked immunosorbent assay (Ostex international, Seattle, WA). The intraassay and interassay coefficients of variation were 11.3 and 10.7% at 378 40.5 nmol/l bone collagen equivalent (BCE) and 9.1 and 7.2% at 1,390 101.2 nmol/l BCE. Leptin was measured in a single radioimmunoassay (HL-81K; Linco Research, St. Charles, MO); the intraassay coefficient of variation was 5%.

Vitamin D status

Vitamin D status was classified as deficiency (serum 25(OH)D 20 ng/ml), insufficiency (serum 25(OH)D >20 to <30 ng/ml), and sufficiency (serum 25(OH)D 30 ng/ml) (19–21).

Statistical analysis

Results are expressed as mean s.d. Comparisons of means between two groups were done using an independent t -test for continuous outcomes and Fisher's exact test for categorical outcomes. Comparisons within each group over time were done using repeated measures ANOVA. Pearson or Spearman correlations were used to assess associations. All the analyses were done as intent to treat and all statistical tests were non-directional, and all statistical assumptions were met. Significance level was set at P 0.05.

Pearson correlation coefficients were calculated to measure the relationship between weight-based vitamin D intake from the supplement and diet and the corresponding changes in serum 25(OH)D. The P values from the z -test for each of the correlations were tested using a Fisher's transformation, thus detecting non-zero correlations (P < 0.05).

Results

Clinical characteristics

Forty-one of the forty-two enrolled children (obese: 21, non-obese: 20) were analyzed. The excluded subject was an outlier, with a normal basal serum 25(OH)D (35 ng/ml), normal serum calcium and a significantly elevated corresponding serum PTH (93 pg/ml, normal 65 pg/ml). There was no left over serum from the outlier for repeating the serum 25(OH)D and PTH assays. The obese and the non-obese cohorts were matched for age, sex, skin color, and pubertal maturation (Table 1). As expected, the mean weight (49.1 14.3 kg vs. 29.9 5.8 kg) and BMI (25.5 4.8 kg/m² vs. 16.3 0.9 kg/m²) was significantly different between the obese and the non-obese cohorts (P < 0.0001, Table 1). The study cohort comprised 27 males and 14 females, with a mean age of 8.9 1.2 years (Table 1). The majority of the children were Tanner stage I (n = 33) and had type 4-skin color (n = 33). The mean dietary intake of vitamin D was 277 146 IU/day (n = 41). The obese cohort had a significantly lower dietary intake of vitamin D than the non-obese (218.1 112 IU/day vs. 339 153 IU/day, P = 0.007). Vitamin D intake was below the dietary reference intake for age (<200 IU/day) in 12/21 (57%) obese vs. 5/20 (25%) non-obese, P = 0.058. Two of the subjects (non-obese) did not complete the study due to personal reasons.

Table 1 - Baseline characteristics.

Full table

Vitamin D status data at baseline

At baseline, there was no difference in the proportion of vitamin D deficiency and insufficiency between obese and non-obese cohorts. The proportion of vitamin D deficiency was 12/21 (57%) obese vs. 8/20 (40%) non-obese (P = 0.35) and vitamin D insufficiency was 5/21 (24%) obese vs. 5/20 (25%) non-obese (P = 0.99). There were no significant differences between the obese and the non-obese cohorts for serum calcium, phosphorus, albumin, PTH, 25(OH)D, 1,25(OH)₂D, and markers of bone turnover (OC, BSAP, and urinary NTX), at baseline (Table 2).

Table 2 - Baseline laboratory data.

Full table

Vitamin D status post-intervention

Following oral supplementation with 400 IU of vitamin D₃ daily for 1 month the proportion of vitamin D deficiency decreased from 12/21 (57%) to 5/21 (24%) in the obese group and from 8/20 (40%) to 2/18 (11%) in the non-obese group and the proportion of vitamin D insufficiency decreased from 5/21 (24%) to 1/21 (5%) in the obese group and from 5/20 (25%) to 2/18 (11%) in the non-obese group. There were no significant differences between baseline and post-supplementation values in the bone turnover markers for the obese group (OC: 24.8 6.2 ng/ml vs. 23.7 6.0 ng/ml, P = 0.40; BSAP: 158 55.4 U/l vs. 158.4 58.2 U/l, P = 0.92; urine NTX: 322.0 169.2 nmol/l BCE/mmol/l creatinine vs. 332.7 127.2 nmol/l BCE/mmol/l creatinine, P = 0.82) and non-obese group (OC: 29.2 9.7 ng/ml vs. 32.1 7.9 ng/ml, P = 0.14; BSAP: 172.1 35.4 U/l vs. 164.9 42.2 U/l, P = 0.74; urine NTX: 354.6 160.2 nmol/l BCE/mmol/l creatinine vs. 455.3 168 nmol/l BCE/mmol/l creatinine, P = 0.22).

There were no significant differences between the obese and non-obese cohorts for serum calcium, phosphorus, albumin, PTH, 25(OH)D, 1,25(OH)₂D, and BSAP at post-intervention (Table 3). However, the post-intervention OC and urinary NTX were both significantly lower in the obese cohort compared to the non-obese (OC (ng/ml): 23.7 6 vs. 32.1 7.8, P = 0.001; NTX (nmol/l BCE/mmol/l creatinine) 332.7 127.2 vs. 455.3 168.5, P = 0.015, Table 3).

Table 3 - Post-supplementation laboratory data.

Full table

Treatment response effects were different between obese and non-obese cohorts when they were stratified into tertiles based on basal serum 25(OH)D (group 1: 20 ng/ml, group 2: >20 to <30 ng/ml, group 3: 30 ng/ml, Table 4). In the non-obese cohort, post-supplementation serum 25(OH)D was significantly different between any two groups (Table 4). However in the obese cohort, the post-supplementation serum 25(OH)D was significantly different only between group 1 (20 ng/ml) and 2 (>20 to <30 ng/ml), and group 1 (20 ng/ml) and 3 (30 ng/ml), and not between group 2 (>20 to <30 ng/ml) and 3 (30 ng/ml) (Table 4). It was apparent that the threshold level of serum 25(OH)D associated with response to therapy was different between obese and non-obese cohorts, and was 30 ng/ml in the non-obese cohort compared to 20 ng/ml in the obese cohort.

Table 4 - Difference in post-supplementation 25-hydroxyvitamin D (25(OH)D) between groups.

Full table

Leptin status

As anticipated, the obese cohort had significantly higher levels of serum leptin compared to the non-obese cohort at baseline (26.3 16.5 ng/ml vs. 3.6 1.6 ng/ml, P < 0.001, Table 2) and post-supplementation (25.6 18.2 ng/ml vs. 3.4 1.8 ng/ml, P < 0.001, Table 3).

Significant correlations

Basal 25(OH)D had a significant negative correlation with PTH only in the non-obese (r = -0.507, P = 0.022). Leptin had a positive correlation with OC in the obese at baseline (r = 0.513, P = 0.05). There were no significant correlations between baseline PTH and NTX or OC for the obese and non-obese groups. However, a significant negative correlation was observed between baseline PTH and BSAP only in the non-obese group (r = -0.515, P = 0.04).

Compliance and response

Empty medication containers were returned by 75% of the subjects (13/21 (62%) obese vs. 16/18 (88%) non-obese, P = 0.074). The remainder of the subjects reported taking all of the prescribed medication; however they were not compliant in returning the empty medication containers. Response to therapy was defined as an increase of 25(OH)D by 5 ng/ml following 1 month of vitamin D supplementation. No association between response and compliance was noted within each of the subgroups and for the entire cohort.

Impact of weight-based vitamin D intake from supplement and diet on serum 25(OH)D

We analyzed the change in serum 25(OH)D in response to weight-based dosage of vitamin D₃ supplement (Table 5). Pearson correlation coefficients for measuring the relationship of weight based dosing of vitamin D₃ supplement and changes in serum 25(OH)D were calculated for the obese, non-obese and the overall group by Fischer's transformations to detect non-zero correlations (P < 0.05). The correlations of weight-based vitamin D₃ supplement dose and change in serum 25(OH)D were not significant for the obese (r = -0.112, P = 0.63), non-obese (r = -0.187, P = 0.43) and overall groups (r = -0.170, P = 0.29). We also calculated the change in serum 25(OH)D based on weight-based intake of vitamin D from the supplement and diet (Table 5) and noted a significant negative correlation between weight-based vitamin D intake from the supplement and diet and change in serum 25(OH)D for the non-obese (r = -0.478, P = 0.03), suggesting that higher intakes of vitamin D from diet and supplement (25.4 6.4 IU/kg/day vs. 13.9 5.6 IU/kg/day, P < 0.001) induced relatively small changes in serum 25(OH)D in the non-obese cohort (2.4 7 ng/ml vs. 4 9.4 ng/ml, P = 0.54).

Table 5 - Relationship between weight-based vitamin D intake and changes in serum 25-hydroxyvitamin D (25(OH)D).

Full table

Discussion

We have examined the therapeutic response to 400 IU of vitamin D₃ daily, for 1 month during winter, in vitamin D deficient and insufficient, obese and non-obese 6–10 year old African American children. There was no significant difference in the proportion of vitamin D deficiency or insufficiency between obese and non-obese cohorts, even though vitamin D deficiency (50%) and insufficiency (24%) was common among the study population.

Treatment response effects were different between obese and non-obese cohorts. Treatment was effective in the non-obese cohort for those with basal 25(OH)D <30 ng/ml and in the obese cohort for those with basal 25(OH)D 20 ng/ml. We acknowledge that this is a tentative conclusion as the group size was small. Therefore, the threshold levels of serum 25(OH)D associated with response to vitamin D therapy in obese and non-obese children need to be further characterized with a larger group size. Conceivably, a greater degree of sequestration of vitamin D in the body fat stores of obese children and potential differences in the metabolism of 25(OH)D between obese and non-obese children could explain the differential treatment response effect between obese and non-obese children, if our findings are confirmed with a larger group size (1,2,3,22).

It's also important to note that the vitamin D inputs from dietary sources and the prescribed intervention (vitamin D₃ 400 IU daily) were inadequate to optimize the serum 25(OH)D (30 ng/ml) in both obese and non-obese preadolescent African American children. However, the mean dietary intake of vitamin D was significantly lower among obese children compared to non-obese children (218.1 112 vs. 338.6 153, P = 0.007).

The correlations of changes in serum 25(OH)D in response to weight-based dosage of vitamin D₃ supplement were not significant for the obese (r = -0.112, P = 0.63), non-obese (r = -0.187, P = 0.43) and overall groups (r = -0.170, P = 0.29). However there was a significant negative correlation between weight-based vitamin D inputs from the tablet and diet and change in serum 25(OH)D for the non-obese (r = -0.478, P = 0.03), suggesting that higher intakes of vitamin D from diet and supplement induced relatively small changes in serum 25(OH)D in the non-obese cohort. It was apparent that the non-obese, despite receiving higher inputs of vitamin D based on body weight (25.4 6.4 IU/kg/day vs. 13.9 5.6 IU/kg/day, P < 0.001) did have less of an absolute change in serum 25(OH)D (2.4 7 ng/ml vs. 4 9.4 ng/ml, P = 0.54). This observation suggests that body weight is not an important determinant for maintenance of serum 25(OH)D levels.

There were significant differences, in changes to markers of bone turnover with vitamin D supplementation, between obese and non-obese children. The anticipated compensatory increase in PTH in response to vitamin D deficiency and insufficiency was observed only among the non-obese (r = -0.507, P = 0.022), suggesting that the innate PTH response to vitamin D deficiency/insufficiency may be blunted in the obese state. In addition, the post-supplementation OC (marker of bone formation) and urinary NTX (marker of bone resorption) were significantly lower in obese children compared to non-obese children (Table 3), possibly suggesting lower rates of bone turnover in obese children. We are unable to explain the lack of increase in PTH with vitamin D insufficiency/deficiency in obese children. Blunted PTH response in the presence of vitamin D insufficiency (that has been reported in a subset of elderly women with osteoporosis), and parathyroid glandular dysfunction (i.e., abnormal calcium sensing receptors or abnormal 1,25(OH)₂D receptors) or intracellular magnesium deficiency resulting in impaired PTH synthesis, are the proposed explanations for this phenomenon (23,24).

As expected, leptin levels were significantly higher in obese children. There was a significant positive correlation between basal leptin and OC only in obese children, and this association may suggest that leptin may have a positive influence on bone formation in obese children. However, previous studies in children and post-menopausal women found no correlation between serum leptin and OC (25,26,27).Compensatory hyperparathyroidism associated with the hypovitaminosis D has the potential to lead to increased rates of bone turnover (28,29). Compston et al. demonstrated the increased risk for histological evidence of metabolic bone disease among vitamin D deficient obese adults (30).

We did not find significant differences between obese and non-obese preadolescent African American children for the proportion of vitamin D deficiency or insufficiency. The expected changes with vitamin D supplementation during vitamin D deficiency or insufficiency, namely a reduction in PTH and/or increase in serum 1,25(OH)₂D were not significant in either obese or non-obese children. It's possible that if our trial was of longer duration or if we had used a higher dosage of vitamin D we might have seen the expected response. Limitations of our study include short duration of intervention, inadequate dosage of vitamin D, non-randomized open label design, and lack of sunlight exposure data, although it was controlled since the study was done in the winter.

There are potential limitations to our serum 25(OH)D assay (Nichols Advantage assay). Recent reports have shown significant variability in serum 25(OH)D measurements between different assays and laboratories (31,32). Most commercial assays overestimate serum 25(OH)D measurements when compared to the gold standard high-performance liquid chromatography method (20,32). Underestimation of serum 25(OH)D₂ is a recognized limitation of our serum 25(OH)D assay (Nichols Advantage assay) (20,32). As our vitamin D supplement (tablet) contained only vitamin D₃ and the typical main dietary source of vitamin D is vitamin D₃–fortified milk (20), it's unlikely that underestimation serum 25(OH)D₂ would have impacted our reported serum 25(OH)D measurements. High-performance liquid chromatography or liquid chromatography–mass spectrometry method would have yielded a more accurate measurement of serum 25(OH)D (20,31,32). However, we are reassured by the recent report by Holick et al. (20), showing a strong correlation (r = 0.698) between the liquid chromatography–mass spectrometry method and Nichols Advantage assay in estimation of serum 25(OH)D. Rates of diagnosis of vitamin D inadequacy in this cohort of postmenopausal women (n = 296) based on liquid chromatography–mass spectrometry were nearly similar to Nichols Advantage assay (serum 25(OH)D <30 ng/ml: 52.7% vs. 52%, and <20 ng/ml: 18.9% vs. 18.2%).

We conclude that vitamin D deficiency and insufficiency are common among obese and non-obese preadolescent African American children and 400 IU of vitamin D₃ daily for 1 month is inadequate for achieving vitamin D sufficiency status. There were differences in bone turnover and treatment effect in response to vitamin D supplementation between the obese and non-obese preadolescent African American children. Further characterization of response to vitamin D therapy and differences in bone metabolism between obese and non-obese children should be studied utilizing a more obese cohort, increased dosage (vitamin D₃ 1,000 IU daily), and longer duration of intervention (33).

DISCLOSURE

The authors declared no conflict of interest.

References

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Acknowledgments

The Research Advisory Committee and the National Institutes of Health General Clinical Research Center of Children's Hospital of Pittsburgh (M01 RR00084) funded this study. We acknowledge the Endocrine Immunoassay Core Laboratory of the University of Pittsburgh School of Medicine for performing the assays and Ms Deborah Cleary of the Endocrine Immunoassay Core Laboratory for her technical assistance with the performance of the assays. We also would like to thank Mr Paul Hoffmann, the research pharmacist at the Children's Hospital of Pittsburgh, for his help with this project.