The Effect of Vitamin D2 and Vitamin D3 on Intestinal

ORIGINAL E n d o c r i n e

ARTICLE C a r e

The Effect of Vitamin D2 and Vitamin D3 on Intestinal Calcium Absorption in Nigerian Children with Rickets Tom D. Thacher, Michael O. Obadofin, Kimberly O. O’Brien, and Steven A. Abrams Department of Family Medicine (T.D.T.), Mayo Clinic, Rochester, Minnesota 55905; Department of Family Medicine (M.O.O.), Jos University Teaching Hospital, Jos 930001, Nigeria; Division of Nutritional Sciences (K.O.O.), Cornell University, Ithaca, New York 14853; and U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center (S.A.A.), Department of Pediatrics, Baylor College of Medicine Houston, Texas 77030 Context: Children with calcium-deficiency rickets have high 1,25-dihydroxyvitamin D values. Objective: The objective of the study was to determine whether vitamin D increased calcium absorption. Design: This was an experimental study. Setting: The study was conducted at a teaching hospital. Participants: Participants included 17 children with nutritional rickets. Intervention: The participants were randomized to 1.25 mg oral vitamin D3 (n ⫽ 8) or vitamin D2 (n ⫽ 9). Main Outcome Measure: Fractional calcium absorption 3 da after vitamin D administration was measured. Results: Mean baseline 25-hydroxyvitamin D concentrations were 20 ng/ml (range 5–31 ng/ml). The increase in 25-hydroxyvitamin D was equivalent after vitamin D3 (29 ⫾ 10 ng/ml) or vitamin D2 (29 ⫾ 17 ng/ml). Mean 1,25-dihydroxyvitamin D values increased from 143 ⫾ 76 pg/ml to 243 ⫾ 102 pg/ml (P ⫽ 0.001), and the increase in 1,25-dihydroxyvitamin D did not differ between vitamin D2 and vitamin D3 (107 ⫾ 110 and 91 ⫾ 102 ng/ml, respectively). The increment in 1,25-dihydroxyvitamin D was explained almost entirely by the baseline 25-hydroxyvitamin D concentration (r2 ⫽ 0.72; P ⬍ 0.001). Mean fractional calcium absorption did not differ before (52.6 ⫾ 21.4%) or after (53.2 ⫾ 23.5%) vitamin D, and effects of vitamin D2 and vitamin D3 on calcium absorption were not significantly different. Fractional calcium absorption was not closely related to concentrations of 25-hydroxyvitamin D (r ⫽ 0.01, P ⫽ 0.93) or 1,25-dihydroxyvitamin D (r ⫽ 0.21, P ⫽ 0.24). The effect of vitamin D on calcium absorption did not vary with baseline 25-hydroxyvitamin D values or with the absolute increase in 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D values. Conclusions: Despite similar increases in 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D with vitamin D2 or vitamin D3, fractional calcium absorption did not increase, indicating that rickets in Nigerian children is not primarily due to vitamin D-deficient calcium malabsorption. (J Clin Endocrinol Metab 94: 3314 –3321, 2009)

V

itamin D fortification of foods is largely responsible for the near disappearance of nutritional rickets in North America and Europe in the last century. However, nutritional rickets continues to occur in many tropical

countries despite abundant sunlight exposure, and a resurgence of the disease has been noted in developed countries, where the disease was thought to have been eradicated.

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/jc.2009-0018 Received January 5, 2009. Accepted June 18, 2009. First Published Online June 30, 2009

Abbreviations: TRPV6, Transient receptor potential vanilloid type 6.

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J Clin Endocrinol Metab. September 2009, 94(9):3314 –3321

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J Clin Endocrinol Metab, September 2009, 94(9):3314 –3321

Nutritional rickets in Nigerian children is associated with low dietary calcium intake, and the disease responds well to treatment with calcium with or without vitamin D (1). Nigerian children with nutritional rickets have similar values for calcium absorption compared with those of control children, with mean fractional absorption values of 60% or greater (2– 4). The role of vitamin D in mediating the efficiency of calcium absorption in Nigerian children with rickets has not been clarified. Vitamin D is hydroxylated in the liver to 25-hydroxyvitamin D, and serum concentrations of this metabolite are considered the optimal indicator of vitamin D status. Circulating 25-hydroxyvitamin D is subsequently metabolized in the kidney to 1,25-dihydroxyvitamin D, which acts on vitamin D receptors in the intestine to increase active calcium absorption. Surprisingly, very few human data demonstrate the effects of supplemental vitamin D and its analogs on intestinal calcium absorption, particularly in humans with rickets or osteomalacia. Children with nutritional rickets due to calcium deficiency have high serum 1,25-dihydroxyvitamin D values, suggesting a compensatory response to maximize calcium absorption. Even so, serum values of 1,25-dihydroxyvitamin D increase nearly 2-fold in response to vitamin D, with peak values occurring 3 d after a single oral dose (5). The increase in 1,25-dihydroxyvitamin D in response to vitamin D implies that there is also an element of coexisting vitamin D deficiency, such that when substrate 25hydroxyvitamin D is available, the result is a marked increase in 1,25-dihydroxyvitamin D. Dietary calcium deficiency likely increases the requirement for vitamin D, even when 25-hydroxyvitamin D concentrations are considered adequate. Whether the increase in 1,25-dihydroxyvitamin D that can be measured with vitamin D supplementation is associated with a measurable increase in fractional calcium absorption is unknown. Data from certain studies in adults indicate that calcium absorption increases with increasing 25-hydroxyvitamin D values (6 – 8), but this relationship was not demonstrated in other studies in adolescents (9) and adults (10, 11). Instead there were positive correlations between calcium absorption and 1,25-dihydroxyvitamin D values (9, 11). In Nigerian children with rickets and healthy control children, fractional calcium absorption was unrelated to either 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D concentrations (2– 4). Although vitamin D supplementation increases the serum concentrations of 25-hydroxyvitamin D and 1,25dihydroxyvitamin D, the effect of supplemental vitamin D may depend on the form of vitamin D given. Vitamin D3 is the endogenous form of vitamin D produced by kera-


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tinocytes in the skin in response to UVB radiation from sunlight. Vitamin D2 is produced by irradiation of plant ergosterol and is commonly used for supplementation and food fortification. Both forms of vitamin D are hydroxylated in the liver to 25-hydroxyvitamin D. Because this step is not tightly regulated, serum 25-hydroxyvitamin D is considered the primary indicator of vitamin D status. It has been contended that vitamin D3 is superior to vitamin D2 in sustaining adequate 25-hydroxyvitamin D values in adults (12, 13) because 25-hydroxyvitamin D2 may bind less avidly to vitamin D binding protein and be cleared more rapidly than 25-hydroxyvitamin D3. However, a recent report indicated that both forms of vitamin D are equally effective in maintaining 25-hydroxyvitamin D levels (14). There are no data comparing the effect of vitamin D2 and vitamin D3 on intestinal calcium absorption. The relative roles of calcium and vitamin D nutrition in the etiology of rickets in Nigerian children are uncertain. The primary objective of this study was to test the hypothesis that vitamin D supplementation in calcium-deficiency rickets augments calcium absorption. An additional objective was to determine whether vitamin D3 resulted in a greater increase in calcium absorption than vitamin D2. We conducted a randomized controlled trial to determine the acute effects of oral supplementation with vitamin D on intestinal calcium absorption in Nigerian children with rickets and whether the response differed between vitamin D2 and vitamin D3.

Subjects and Methods Prepubertal children with clinical signs of rickets were recruited from the outpatient department of the Jos University Teaching Hospital (Jos, Nigeria). Subjects required a radiological score of at least 1.5 on a previously validated 10-point scale for assessing the severity of childhood rickets (15). The score is based on the degree of growth plate widening, indicated by cupping and lucency of the long bone metaphyses in radiographs of the wrists and the knees. Children were also required to have achieved bladder control sufficient to allow for a 24-h urine collection. The Ethical Review Committee of Jos University Teaching Hospital and the Institutional Review Board of Baylor College of Medicine and Affiliated Hospitals approved the protocol. The study protocol was explained to potential subjects and their parents in Hausa or English (as they understood), and informed written consent was obtained in all cases. Using stable isotope methods, fractional calcium absorption was determined at baseline and 3 d after vitamin D administration. We randomly assigned eligible subjects by lottery method to either vitamin D3 (cholecalciferol; Bio-Tech, Fayetteville, AR) or vitamin D2 (ergocalciferol; Pliva, Inc., East Hanover, NJ) as a single oral dose of 1.25 mg (50,000 IU). For the baseline study, subjects were given a typical Nigerian meal of 150 ml (233 g wet weight) of maize porridge and 50 ml


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Thacher et al.

Vitamin D and Intestinal Calcium Absorption

of orange juice to which 120 mg calcium (as calcium glubionate) and 20 ␮g 46Ca (as calcium chloride) were added. The orange juice containing the isotopes was given after half the porridge had been consumed, and the cup was rinsed with an additional 20 ml of orange juice that the child drank. The remaining porridge was consumed after the orange juice. An iv butterfly needle was inserted to draw a blood sample. After withdrawing blood, 0.5 mg 48 Ca was infused slowly, followed by flushing of the line with 5 ml of saline. A complete urine collection was started immediately before the administration of the isotopes and continued for 24 h. The children remained supervised in the hospital until the urine collection was completed. Four days after the baseline study, a single oral dose of vitamin D2 or D3 was given under direct observation. Three days after the oral dose of vitamin D (1 wk after the baseline study), calcium absorption was determined again. We chose to measure calcium absorption 3 d after vitamin D administration because this is the time when 1,25-dihydroxyvitamin D is maximal and would be most likely to have an effect on calcium absorption. In the second absorption study, 46Ca (12 ␮g) was given iv, and 42Ca (1.8 mg) was given orally, following the same procedure and identical diet as in the first study. In previous studies (Abrams, S.A., unpublished data) with the doses used in this study, there is no measurable residual concentration of oral 46Ca 7 d after dosing. Thus, we considered it unnecessary to correct for residual isotope enrichment in the second absorption study. Serum samples were stored at ⫺70 C until they were transported on ice for biochemical analysis to the Mayo Clinic (Rochester, MN). Serum alkaline phosphatase, phosphorus, and calcium were measured on a Roche/Hitachi MODULAR System (Roche Diagnostics, Basel, Switzerland). Serum albumin was measured with a Roche/Hitachi 912 automatic analyzer (Roche Diagnostics). Measurements of serum cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), 25-hydroxyvitamin D3, and 25-hydroxyvitamin D2 were made by isotope-dilution liquid chromatography tandem mass spectrometry on an API 4000 instrument (Applied Biosystems, Forest City, CA), with sample introduction performed by a cohesive four-channel multiplexed system (Thermo-Fisher, Waltham, MA). Total serum 25-hydroxyvitamin D was the sum of measured 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 values. Measurements of serum 1,25-dihydroxyvitamin D were performed using a 125I RIA (DiaSorin, Stillwater, MN). The lower limits of detection were 1 ng/ml (2.5 nmol/liter) for serum vitamin D2 and vitamin D3 and 5 ng/ml (12.5 nmol/liter) for serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Based on our previous studies, plasma PTH was not measured because it does not vary with changes in calcium absorption or with vitamin D administration (2–5). Urine samples were transported frozen to the Children’s Nutrition Research Center at the Texas Medical Center. Calcium isotope ratios were initially measured as previously described using magnetic sector thermal ionization mass spectrometry (MAT 261; Thermo Scientific, Bremen, Germany) after purification by oxalate precipitation (4, 16). However, because of the extremely low 24-h urine calcium excretion (⬍1 mg) in these children, calcium isotope ratios in the urine samples were subsequently measured in calcium extracted from larger volumes of urine (20 ml) using magnetic sector thermal ionization mass spectrometry (Thermoquest; Triton TI, Bremen, Germany) at the Division of Nutritional Sciences of Cornell University (17). The methodology used for the collection of isotope ratio data are


the same for both mass spectrometers, and the relative SDs of the isotope ratios measures are typically 0.1– 0.2%. This corresponds to a relative SD of approximately 1% for the actual measured absorption at usual doses and absorption values. Data entry and statistical analysis were performed with Epi Info 3.2 (Centers for Disease Control and Prevention, Atlanta, GA) and Excel 2003 (Microsoft Corp., Redmond, WA). Mean values of normally distributed continuous variables were compared between the two absorption studies with a paired t test. Medians and ranges are reported for nonnormally distributed variables, which were compared using the Mann-Whitney test. Proportions were compared with the ␹2 or Fischer exact test, as appropriate. P ⬍0.05 was considered significant.

Results A total of 17 Nigerian children with rickets, ages 2–10 yr, were enrolled, and eight were randomly assigned to receive vitamin D3 and nine to receive vitamin D2. The characteristics of the two groups were similar (Table 1). Among all children enrolled, the mean daily dietary calcium intakes were 182 ⫾ 73 mg, and mean baseline serum 25-hydroxyvitamin D concentrations were 20 ng/ml (range 5–31 ng/ml) [50 nmol/liter (range 12– 80 nmol/ liter)]. Two subjects (12%) had 25-hydroxyvitamin D values in the vitamin D deficient range, less than 12 ng/ml (30 nmol/liter). Baseline alkaline phosphatase concentrations were negatively related to 25-hydroxyvitamin D values (r ⫽ ⫺0.66, P ⫽ 0.005). Serum 25-hydroxyvitamin D values were unrelated to reported daily sun exposure. Baseline 24-h urinary calcium excretion values were very low, with a median of 0.67 mg (range 0.02–7.6 mg). At baseline, all subjects had undetectable values of serum vitamin D2, and one had a detectable 25-hydroxyvitamin D2 value of 12 ng/ml (30 nmol/liter), presumably due to recent ingestion of a vitamin D2-fortified food or supplement that was not recalled by the parent. Three had detectable serum vitamin D3 levels [1.6, 1.7, and 15.3 ng/ml (4.2, 4.4, and 40 nmol/liter)] at baseline. Three days after oral vitamin D, the increase in serum vitamin D3 [52 ⫾ 22 ng/ml (135 ⫾ 57 nmol/liter)] in the vitamin D3 group and the increase in vitamin D2 [48 ⫾ 18 ng/ml (125 ⫾ 47 nmol/liter)] in the vitamin D2 group were equivalent, indicating that the absorption of both drugs was similar. The increase in total serum 25-hydroxyvitamin D was equivalent after administration of vitamin D3 [29 ⫾ 10 ng/ml (72 ⫾ 25 nmol/liter)] or vitamin D2 [29 ⫾ 17 ng/ml (72 ⫾ 42 nmol/liter)]. Mean 1,25-dihydroxyvitamin D values increased from 143 ⫾ 76 pg/ml (343 ⫾ 182 pmol/liter) to 243 ⫾ 102 pg/ml (583 ⫾ 245 pmol/liter) (P ⫽ 0.001), and the increase in 1,25-dihydroxyvitamin D did not differ between vitamin D2 and vitamin D3 [107 ⫾




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TABLE 1. Baseline characteristics of study subjectsa

Characteristic Age (months) Sex (male/female) Familial rickets Daily sun exposure (h) Height-for-age z-score Weight-for-height z-score Weight-for-age z-score Daily calcium intake (mg) Radiographic score Serum biochemistriesb Calcium (mg/dl), reference range 9.6 –10.6 Phosphorus (mg/dl), reference range 3.7–5.4 Albumin (g/dl), reference range 3.5–5.0 Alkaline phosphatase (U/liter), reference range 149 – 476 25-Hydroxyvitamin D (ng/ml), optimal range 25– 80 1,25-Dihydroxyvitamin D (pg/ml), reference range24 – 86

All subjects (n ⴝ 17) 44.5 (28 –118) 6/11 8 (47%) 3.25 (0.5– 8) ⫺3.6 (⫺7.7 to ⫺0.1) 0.3 (⫺1.0 to 1.6) ⫺1.9 (⫺4.6 to ⫺0.3) 172 (56 –309) 2.75 (1.5–10) 8.8 ⫾ 1.0 4.0 ⫾ 1.1 4.2 ⫾ 0.4 686 (182–2476) 20 (5–32) 143 ⫾ 76

Vitamin D2 group (n ⴝ 9) 44.5 (28 – 66) 2/7 4 (44%) 2.75 (1– 8) ⫺3.6 (⫺7.7 to ⫺1.8) 0.3 (⫺1.0 to 1.6) ⫺2.0 (⫺4.6 to ⫺0.3) 172 (56 –261) 3.5 (1.5–10) 8.6 ⫾ 1.1 3.6 ⫾ 0.8 4.2 ⫾ 0.3 751 (182–2476) 18 (10 –32) 156 ⫾ 74

Vitamin D3 group (n ⴝ 8) 45.6 (32.5–118) 4/4 4 (50%) 3.5 (0.5–5) ⫺3.5 (⫺4.5 to ⫺0.1) 0.2 (⫺0.9 to 0.8) ⫺1.6 (⫺3.1 to ⫺0.3) 186 (82–309) 2.25 (1.5– 8) 9.0 ⫾ 1.0 4.5 ⫾ 1.3 4.1 ⫾ 0.5 447 (205–1055) 23 (5–32) 130 ⫾ 82

a

Values for nonnormally distributed continuous variables are shown as medians with range in parentheses. Normally distributed variables are shown as means ⫾ SD. b To convert values of serum calcium to millimoles per liter, multiply by 0.25; to convert values of serum phosphorus to millimoles per liter, multiply by 0.32; to convert values of albumin to grams per liter, multiply by 10; to convert values of 25-hydroxyvitamin D to nanomoles per liter, multiply by 2.5; to convert values of 1,25-dihydroxyvitamin D to picomoles per liter, multiply by 2.4. Pediatric reference ranges are provided for the age range of children in the study.

110 and 91 ⫾ 102 ng/ml (257 ⫾ 264 and 218 ⫾ 245 nmol/liter), respectively]. The degree of rise in 1,25-dihydroxyvitamin D values was explained almost entirely by the baseline 25-hydroxyvitamin D concentration (Fig. 1; r2 ⫽ 0.72; P ⬍ 0.001). The intersection of the regression lines with the x-axis suggests that with 25-hydroxyvitamin D values of 25–30 ng/ml (63–75 nmol/liter), there appears to be little increase in 1,25-dihydroxyvitamin D in response to additional vitamin D. However, there is some variability among subjects with some exhibiting no increase with baseline 25-hydroxyvitamin D values less than 20 ng/ml (50 nmol/liter) and others with a significant rise in 1,25Increase in 1,25(OH)2D after oral vitamin D (pg/mL)

350 300 Vitamin D2 Vitamin D3 Vitamin D3 Vitamin D2

250 200 150 100

R2 = 0.72 P