Journal of the American College of Nutrition, Vol. 18, No. 5, 470-474 (1999)
Published by the American College of Nutrition
Plasma 25-Hydroxyvitamin D Responses of Younger and Older Men to Three Weeks of Supplementation with 1800 IU/day of Vitamin D
Susan S. Harris, DSc,
Bess Dawson-Hughes, MD and
Gayle A. Perrone, MBA
Jean Mayer United States Department of Agriculture, Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
Address reprint requests to: Susan Harris, D.Sc., Jean Mayer USDA HNRCA at Tufts University, 711 Washington St., Boston, MA 02111
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ABSTRACT
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Objective: The objective of this study was to compare changes in plasma 25-hydroxyvitamin D (25(OH)D) levels of younger and older men after three weeks of oral vitamin D supplementation.
Methods: Nine younger men (22 to 28 years) and nine older men (65 to 73 years) with self-reported vitamin D intakes below 200 IU/d were enrolled in February and randomized to 1800 IU/d of ergocalciferol (vitamin D2, n=11) or to a control group (n=7) and followed for three weeks. Blood was collected at baseline, and after one, two and three weeks for measurement of plasma concentrations of total 25(OH)D, 25(OH)D2 and 25(OH)D3.
Results: In both the younger and older supplemented men, 25(OH)D2 and total 25(OH)D concentrations increased significantly during the study, whereas values of these metabolites did not change in younger or older control subjects. No group showed significant changes in 25-hydroxyvitamin D3. There was a significant interaction between age group and supplement group, suggesting that the effect of vitamin D2 supplementation on changes in 25(OH)D2 changes with age. The mean increase in 25(OH)D2 was greater in the younger supplemented men than in the older supplemented men (37±9 nmol/L vs. 19.5 nmol/L, p=0.027), and this accounted for their significantly greater increase in total 25(OH)D.
Conclusion: These data are consistent with an age-related decline in the absorption, transport or liver hydroxylation of orally-consumed vitamin D.
Key words: vitamin D supplementation, 25-hydroxyvitamin D, vitamin D absorption, aging
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INTRODUCTION
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Vitamin D levels are reduced in older compared with younger adults living in North America [1–3] and Europe [4,5]. This results, in part, from age-related decreases in sun exposure and reduced skin synthesis of vitamin D precursors [6,7]. The consumption of dietary or supplemental vitamin D is an important adjunct to sun exposure for many people, but little is known about the effects of aging on the production of 25-hydroxyvitamin D (25(OH)D), the storage form of the vitamin, from orally ingested vitamin D in non-pharmacologic doses. Vitamin D is absorbed in the small intestine, a process that requires the presence of fat, bile and pancreatic enzymes, and is transported via lymph to the liver where it is hydroxylated to 25(OH)D [8]. Aging has been associated with changes in function of the pancreas [9], liver [10,11] and small intestine [12,13], but it is not known to what extent these or other age-related changes may influence the effectiveness of vitamin D supplementation in healthy elderly men.
The purpose of this study was to determine whether younger and older men with wintertime 25(OH)D levels typical of New England residents with low vitamin D intakes (under 200 IU/d) had similar increases in plasma concentrations of 25(OH)D2 and total 25(OH)D3 after three weeks of consuming 1800 IU/d of supplemental vitamin D2. This amount of supplement, combined with anticipated dietary intake, was close to the maximum amount considered safe for regular consumption (2000 IU/d) [14].
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SUBJECTS AND METHODS
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Ten younger men (ages 20 through 35) and ten older men (ages 60 through 75) with low vitamin D intakes (under 200 IU/d) were recruited in February and randomized to vitamin D supplementation or to no intervention. The study was begun in late winter (February) when 25(OH)D stores are lowest [15] and excluded men who had traveled to southern locations in the previous month, who had used a vitamin D supplement in the previous six months or who worked in an outdoor occupation. Additional exclusion criteria included usual calcium intakes of 600 mg/d or higher, use of a calcium supplement in the past six months, usual consumption of more than three alcoholic beverages a day, past-year use of medications known to affect vitamin D absorption or metabolism, any history of liver disease, kidney disease, gastrointestinal disease resulting in malabsorption syndrome, gastrointestinal surgery, a kidney stone in the past five years or any current medical condition likely to affect vitamin D absorption or metabolism. Eligibility was determined by interviews conducted over the telephone. No biochemical measurements were made prior to enrollment. Two of the randomized subjects, one younger and one older, had unexpectedly high initial wintertime 25(OH)D concentrations (80 and 65 nmol/L respectively) due most likely, in one case, to a trip to Acapulco seven weeks before enrollment and, in the other case, to the use of a fish oil supplement that should have resulted in exclusion from the study. Data for these two men were set aside.
Volunteers in the supplemented group received 1800 IU/d of ergocalciferol (vitamin D2) as Drisdol (Sanofi Winthrop Pharmaceuticals, New York, NY) in liquid form, taken in the morning with food. Volunteers were instructed by the nursing staff in how to measure out drops of the supplement and, in the presence of the nursing staff, took their first dose and recorded the dose in a supplement diary that they then continued to keep until the end of the study. The control group received no intervention or placebo. The supplemented and control group volunteers returned to the Center weekly over the subsequent three weeks for a total of four study visits. The study was approved by the Tufts University Human Investigation Review Committee and written informed consent was obtained from each participant.
Information about dietary vitamin D intake was obtained with an interviewer-administered food frequency questionnaire at the baseline and final visits. Medical history, medication and dietary-supplement use and travel history were obtained by interview at the same visits, as were measurements of height and weight. Supplement diaries were reviewed with volunteers at each follow-up visit, and blood for 25(OH)D measurements was drawn at each visit after an overnight fast.
Plasma 25(OH)D2 and 25(OH)D3 were batch-analyzed at the end of the study. One mL aliquots were spiked with 25hydroxy[26,27-methyl-3H] cholecalciferol (Amersham Pharmacia Biotech, Arlington Heights, IL) and HPLC purified just before use as an internal standard. Samples were then extracted and cleaned up by batch chromatography on C18 and silica cartridges following an adaptation of a method from Dr. Bruce Hollis (personal communication). Tracer recoveries were monitored for each step of the process and used to correct sample results. Final HPLC chromatography was performed on a Waters, Inc. system using the method of Shepard et al. [16], with a variable wavelength UV detector at the peak maximum of 265nm. The 25(OH)D2 standard was provided by Dr. Ron Horst of USDA, Ames IA; the 25(OH)D3 standard was provided by Dr. M. Uskokovic of Hoffmann-LaRoche, Nutley, NJ. The sensitivity of the method was three ng; when translated to a one mL sample with total loss of about 50% through all steps of the assay, this is equivalent to a detection limit of six ng/mL or 15 nmol/L. Undetectable concentrations were set to zero for the analyses. Total 25(OH)D, by this method, is calculated as the sum of 25(OH)D2 and 25(OH)D3. The inter- and intra-assay coefficients of variation of 25(OH)D by this method are 10% and 8% respectively [16].
Baseline total 25(OH)D was also measured by the competitive protein binding (CPB) method of Chen et al. [17] without the optional chromatography step. The sensitivity of this method is 0.025ng, corresponding to a detection limit of five ng/mL or 12.5 nmol/L. As described by Mayer and Schmidt-Gayk [18], comparable CPB methods result in measurements that are higher than those obtained by the HPLC method because the former does not remove compounds that result in nonspecific interference at the DBP binding site. Total 25(OH)D, 25(OH)D2 and 25(OH)D3 are presented in nmol/L, equivalent to 2.496 ng/mL.
Characteristics and laboratory values of young and old men within each supplement group were compared with Student’s t test for two independent samples conducted at the two-tailed 0.05 level. Three-week changes in 25(OH)D values were compared to zero with Student’s one-sample t test conducted at the 0.05 level. The age difference in effectiveness of the supplement was tested statistically with an analysis of variance model in which the three-week change in 25(OH)D2 was included as the dependent variable and age group, supplement group and the interaction of the two were included as factors. Within the supplemented group, mean changes in 25(OH)D were adjusted for age differences in selected baseline characteristics by analysis of covariance.
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RESULTS
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Selected characteristics of the eighteen subjects are shown by age and supplement group in Table 1. Within the supplemented group, the younger men had a significantly lower body mass index (BMI) than the older men, modestly lower mean vitamin D intake and a mean baseline 25(OH)D concentration that, though not significantly different, was 7.5 nmol/L lower than that of the older men (Table 2). As expected, baseline measurements of 25(OH)D measured by the HPLC method were lower than those measured by the binding protein method (57.8±19 nmol/L in the younger supplemented men and 61.4±10.4 in the older supplemented men) and constituted about 60% of the latter measurements in both groups.
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Table 2. Mean±sd Baseline Plasma 25(OH)D Concentrations, nmol/L and Changes after Three Weeks of Vitamin D Supplementation (Supplemented Group) or No Intervention (Control Group)
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Among the supplemented subjects, concentrations of 25(OH)D2 increased significantly over the three-week period in both the young and old subjects (Fig. 1a and Table 2). Among the control group subjects, there were no statistically significant changes in either younger or older men (Fig. 1b and Table 2). Concentrations of 25(OH)D3 did not change significantly in any group.
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Fig. 1. Changes in plasma 25-hydroxyvitamin D2, D3, and total in younger men (solid line) and older men (dashed line) during the three-week study.
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An analysis of variance of the three-week change in 25(OH)D2 indicated a significant interaction between age group and supplementation group (p=0.020), consistent with an age-dependent difference in the response to supplementation. The increase in 25(OH)D2 after three weeks of supplementation was 18 nmol/L greater in younger compared with older men (Table 2), and this difference was the main contributor to the 23 nmol/L age group difference in total 25(OH)D increases over the same period. Adjustment for initial total 25(OH)D reduced the age group difference in the three-week change in total 25(OH)D only slightly (adjusted values were 29.2 nmol/L in the younger men compared with 8.9 nmol/L in the older men, p=0.025), whereas adjustment for BMI increased the estimated difference. By the end of the study, the mean total 25(OH)D concentration of the younger men was 62.8±3.7 nmol/L, compared with 47.4±18.3 nmol/L in the older men, p=0.073. No subjects reported side effects of supplementation over the three-week study period.
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DISCUSSION
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This study provides preliminary evidence that daily supplementation with 1800 IU of vitamin D2 for three weeks causes smaller increases in plasma concentrations of 25(OH)D2 and total 25(OH)D in older compared with younger men. We are aware of no previous studies that compared vitamin D or 25(OH)D changes of younger and older men during weeks of continuous supplementation, but several studies have examined short-term responses of older and younger men to one-time administration of an oral or intravenous vitamin D dose. Two of these studies compared the increase in serum vitamin D2 of younger and older adults after a single oral dose of 50,000 IU vitamin D2 [19,20], and neither found any effect of age on vitamin D absorption at that high dose. Two other studies examined the appearance of radiolabelled vitamin D3 in blood after a single small dose of vitamin D3 (<100 IU) with added tracer. One reported a 40% lower appearance of tracer in older compared with younger women [21] after six hours. Starting 25(OH)D concentrations of the older women in that study were substantially lower than those of the younger women. Since starting 25(OH)D levels are inversely associated with supplement-induced changes in 25(OH)D [22], it is possible that an even greater age difference may have been observed if the groups had been more similar initially. The other study, in which initial 25(OH)D concentrations were not reported, did not show evidence of an age difference in vitamin D absorption or metabolism in three younger compared with six older adult men and women [23]. Finally, a small group of elderly subjects had a substantially smaller increase in plasma 25(OH)D than younger subjects after both groups received a subcutaneous injection of 50,000 IU ergocalciferol [24]. This may have been due to differences in the blood uptake and transport of the vitamin or to its hydroxylation in the liver, but may also have been influenced by medication use among the older subjects.
We considered the possibility that the 7.5 nmol/L lower initial 25(OH)D of the younger men in our study explained their greater response to supplementation, but adjustment for initial 25(OH)D reduced the age-related response difference only slightly. Similarly, the difference in body mass index of the younger and older men did not appear to explain the greater response of the younger men to supplementation. We cannot determine from this study whether the difference we observed would have remained over a period longer than three weeks. A limited number of short-term supplementation studies demonstrate widely varying periods needed to reach plateau levels, and age may be one of a number of factors that influence this time course [25,26]. The detection limit for 25(OH)D in our laboratory (15 nmol/L) was higher than that reported by Shepard et al. for the HPLC method on which ours was based (2.5 nmol/L) [16], probably because the volume of our plasma samples was smaller. An inability to detect small baseline concentrations of 25(OH)D2 in one or both age groups could have biased the estimated age difference in response to supplementation. We think a strong bias is unlikely because total baseline 25(OH)D by the HPLC method (computed as a sum of 25(OH)D2 plus 25(OH)D3) represented a similar proportion of total 25(OH)D by an independent method (RPB) in the two age groups. Nevertheless, this potential bias provides another reason that this work needs to be replicated. Finally, we do not know whether similar results would be observed in subjects given vitamin D3 instead of vitamin D2 or in subjects with higher usual calcium intakes than those of men in the present study (under 600 mg/d compared with a national median of 865 mg/d) [14].
This study, although limited by its small size and short duration, provides preliminary evidence that there may be an age-related difference in the circulating 25(OH)D response to orally consumed vitamin D. Such a difference could result from alterations in the absorption, transport or liver hydroxylation of orally-consumed vitamin D. Age-related differences in sun exposure and skin synthesis of vitamin D already support the present higher recommended dietary intakes of vitamin D for older age groups [14]. A confirmation of the present finding would provide further support for increased dietary consumption of vitamin D with aging.
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ACKNOWLEDGMENTS
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The authors thank Ms. Amanda Yeaton in the Calcium and Bone Metabolism Laboratory, Ms. Irene Ellis, Ms. Bella Gindelsky and Dr. Elias Seyoum in the Nutrition Evaluation Laboratory and the staff of the Metabolic Research Unit for their substantial contributions to this study.
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FOOTNOTES
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This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 58-1950-9-001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors, and do not necessarily reflect the view of the U.S. Department of Agriculture.
Received January 1, 1999.
Accepted July 1, 1999.
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ABSTRACT
INTRODUCTION
