DOES VITAMIN D METABOLITE MEASUREMENT HELP PREDICT 25(OH)D CHANGE FOLLOWING VITAMIN D SUPPLEMENTATION?

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1 ENDOCRINE PRACTICE Rapid Electronic Article in Press Rapid Electronic Articles in Press are preprinted manuscripts that have been reviewed and accepted for publication, but have yet to be edited, typeset and finalized. This version of the manuscript will be replaced with the final, published version after it has been published in the print edition of the journal. The final, published version may differ from this proof. Original Article EP OR DOES VITAMIN D METABOLITE MEASUREMENT HELP PREDICT 25(OH)D CHANGE FOLLOWING VITAMIN D SUPPLEMENTATION? Neil Binkley, MD; Gretta Borchardt, BS; Ellen Siglinsky, BS; Diane Krueger, BS From: University of Wisconsin Osteoporosis Clinical Research Program, Institute on Aging, 2870 University Avenue, Madison, WI Running Title: 25(OH)D Response to Supplementation Correspondence address: Neil Binkley, MD University of Wisconsin Osteoporosis Clinical Research Program 2870 University Avenue, Suite 100 Madison, WI United States nbinkley@wisc.edu

2 All authors have no conflicts of interest. Keywords: Vitamin D; 25(OH)D; 24,25(OH) 2 D; Cholecalciferol; Supplementation Abstract Objective: Variability in 25(OH)D change following vitamin D supplementation exists. Vitamin D metabolite measurement might assist in predicting 25(OH)D response and also contribute to defining vitamin D adequacy. This study assessed utility of vitamin D metabolite measurements to predict 25(OH)D response and explored the relationship of a vitamin D composite index (comprised of the sum of serum 25(OH) D, vitamin D 3 and 24,25(OH) 2 D) with PTH. Methods: Sixty-two postmenopausal women were randomized to daily vitamin D 3 1,800 IU or placebo for 4 months. Blood was drawn at baseline and after 1 and 4 months. Serum 25(OH)D, cholecalciferol (vitamin D 3 ), and 24,25(OH) 2 D were measured by liquid chromatography tandem mass spectroscopy. Free 25(OH)D and PTH were measured by ELISA. Repeated measures ANOVA and regression analyses were performed. Results: Baseline 25(OH)D was positively correlated (p < 0.05) with vitamin D 3, 24,25(OH) 2 D and free 25(OH)D. Daily vitamin D supplementation increased all metabolites (p < 0.001). Substantial individual variability in 25(OH)D change at 4 months was observed but was unrelated to baseline vitamin D 3, 25(OH)D or 24,25(OH) 2 D. Only BMI, body weight and body fat mass was associated with 25(OH)D

3 change at 4 months. The vitamin D composite score was associated with serum PTH but this association was similar to that observed with 25(OH)D alone. Conclusion: This study does not support measurement of vitamin D metabolites in a composite index to assist in prediction of 25(OH)D response to supplementation. Overweight individuals have less robust 25(OH)D response to supplementation, but variability precludes prediction of the result following daily supplementation. Abbreviations: 25(OH)D = 25-hydroxyvitamin D; 24,25(OH) 2 D = 24, 25 dihydroxyvitamin D; PTH = Parathyroid hormone; IU = International units; Vitamin D 3 = cholecalciferol; ELISA = Enzyme-linked immunosorbent assay; ANOVA = Analysis of variance; BMI = Body mass index; LC-MS/MS = liquid chromatography tandem mass spectroscopy; DXA = Dual energy x-ray absorptiometry.

4 Introduction Vitamin D is essential for bone health (1-3) and potentially many other conditions (4-9). Unfortunately, what constitutes vitamin D inadequacy is contentious and clinical guidelines are incongruent (10, 11). Measurement of serum 25(OH)D is the accepted approach to evaluate an individual s vitamin D status (11, 12). However, there is major between-individual variability in surrogate markers of vitamin D status (e.g., serum PTH and bone osteoid volume) at a given 25(OH)D level (8, 13, 14). Similarly, there is substantial, and currently inadequately understood, variability of 25(OH)D response to supplementation and with high-level UV exposure (15-18). Indeed, in our clinical experience, patients of similar body weight and age may require substantially different daily vitamin D supplement doses (e.g., 400 IU to 4,000 IU) to attain a 25(OH)D of 30 ng/ml. Therefore, identifying factors that predict the 25(OH)D result following supplementation would be beneficial. Ideally, clinicians could estimate a daily dose that would assure attainment of the desired 25(OH)D concentration. To this end, it has been reported that low initial 25(OH)D level is associated with a greater increment and conversely that high body weight blunts the 25(OH)D response.(19) Additionally, it is logical that variation in gut absorption and/or subsequent utilization of vitamin D contributes to these between-individual differences. Consistent with this, we, and others, have reported that vitamin D metabolite measurement may allow estimation of response (20, 21). As an over-simplification, it could be expected that elevated 24,25(OH) 2 D levels could be viewed as a surrogate for vitamin D degradation/utilization; therefore, elevated values might predict a less robust 25(OH)D

5 response to supplementation. Similarly, serum cholecalciferol (vitamin D 3 ) concentration might be viewed as a surrogate of vitamin D absorption with low levels thus predicting poorer 25(OH)D increase. This work explores this possibility. Additionally, it is plausible that the singular measurement of serum total 25(OH)D is a less than ideal approach to defining vitamin D status. Indeed, circulating 25(OH)D does not have direct physiologic consequence; it only serves as a marker of vitamin D intake and utilization. As other vitamin D metabolites e.g., vitamin D 3 and 24, 25(OH) 2 D have vitamin D biologic activity (22-28), it is plausible that consideration of other metabolites, in combination with 25(OH)D, might enhance vitamin D inadequacy identification. To our knowledge, the possibility of developing a vitamin D composite score consisting of 25(OH)D plus other vitamin D metabolites has not been attempted. As a first step towards this, we explored the approach of simply adding vitamin D metabolites that have previously been reported to have a vitamin D physiologic effect and that also circulate at comparable, i.e., ng/ml concentrations. Finally, as the biologic vitamin D activity may be related to free 25(OH)D there is much interest in the potential utility of this measurement in considering what constitutes vitamin D inadequacy. Thus, the purposes of this study were to evaluate the possibility that vitamin D metabolite measurements might enhance the ability to predict resultant 25(OH)D increase following supplementation and to begin exploring the possibility that a vitamin D composite score might have clinical utility in assessment of vitamin D status.

6 Methods Subjects/Study Design A cohort of 62 generally healthy, community-dwelling postmenopausal women with no clinically significant laboratory abnormalities was recruited for this study. Briefly, study inclusion criteria required being postmenopausal and having a baseline serum 25(OH)D concentration of < 40 ng/ml as measured by liquid chromatography tandem mass spectroscopy (LC-MS/MS). All volunteers were required to be willing to use sunscreen (SPF 15) when sun exposure of more than 15 minutes was expected. Exclusion criteria included hypercalcemia at baseline, the presence of any measurable circulating 25(OH)D 2 at screening and treatment with drugs known to interfere with vitamin D metabolism. Study volunteers were randomly assigned to receive 2,000 IU of commercially available vitamin D 3 daily (NOW Foods, Bloomington, IL) or matching placebo (Tishcon, Salisbury, MD) daily for 4 months. The study preparation (the labeling for which stated 2,000 IU) was validated to contain a mean of 1800 (SD 160) IU of vitamin D 3 in the University of Wisconsin laboratory of Professor H. DeLuca. Blood was drawn at baseline and after one and four months of daily supplementation. This study was reviewed and approved by the University of Wisconsin Health Sciences IRB. Informed consent was obtained and documented prior to the conduct of any studyrelated procedure. Laboratory At screening, a serum chemistry panel was run in routine clinical manner and serum 25(OH)D 3 and 25(OH)D 2 were measured using HPLC. At baseline, serum vitamin D 3,

7 24,25(OH) 2 D and total 25(OH)D were measured by LC-MS/MS. Briefly, these measurements were performed as follows: 100 ul of serum was combined with 300 ul 1% formic acid in acetonitrile and internal standard at 250 ng/ml d6-25(oh)d 3, d6-25(oh)d 2 and 75 ng/ml of d6-24,25(oh) 2 D 3 for protein precipitation. Samples were run through solid phase extraction (HybridSPE) and then derivitized with 4-Phenyl-1,2,4- triazole-3,5-dione (PTAD). Liquid chromatography separated the vitamin D metabolites on a Phenomenex Kinetex 2.6 um C18, 100A (100 mm x 2.1 mm; gradient: A=0.1%formic acid in ultrapure water, B=methanol with methylamine 5 mm ; program %B at 0 minutes, 50% up to 98% over 18 minutes, held until 20 minutes and equilibrated at 50% for 5 minutes on a Shimadzu Prominence (Addison, Il, USA) integrated PLC interfaced with an AB SCIEX (Foster City, CA) QTRAP 5500 Quadrupole Linear Ion Trap Mass Spectrometer equipped with an ESI source. The 25(OH)D results obtained using this methodology are traceable to the NIST standards. Coefficient of variation ranged from %. Serum free 25(OH)D was measured by ELISA using kits from Diasource (Louvain-La-Neuve, BE) with Inter/Intra assay CVs of 10.3 and 4.3. Serum Intact PTH was measured by ELISA kit (Alpco, Salem, NH) with Intra- and Inter assay CVs of 9.1 and 15.2%. LC-MS/MS methodology has capability to measure metabolites of both vitamin D 3 and vitamin D 2. In this cohort, 25(OH)D 2 levels were low, near the limit of quantification, and 24,25(OH) 2 D 2 levels were virtually always unable to be measured; as such, D 2 metabolites were not reported here. Total Body DXA Body composition was measured at baseline by total body DXA using a GE

8 Healthcare Lunar idxa densitometer. Total body bone, fat and lean mass data were obtained from these scans. All DXA scans were performed and analyzed by International Society for Clinical Densitometry (ISCD) certified technologists with encore software v13.31 or 13.6 following ISCD guidelines. Statistical Analyses Relationship of total 25(OH)D with vitamin D 3 and 24,25(OH) 2 D at baseline was evaluated using linear regression analyses. Change in serum concentration of vitamin D 3, total/free 25(OH)D, 24,25(OH) 2 D and PTH over time was assessed with a group by time interaction using repeated measures ANOVA. An exploratory composite vitamin D score was developed by addition of the serum concentrations of 25(OH)D, vitamin D 3 and 24, 25(OH) 2 D and the relationship of this composite score with PTH at baseline and after 4 months of vitamin D supplementation was assessed by linear regression analyses. Finally, the relationship of change in total 25(OH)D after 4 months in the supplemented volunteers with baseline vitamin D metabolite levels and with clinical parameters, e.g., body weight and total body fat mass was assessed by linear regression. All tests were performed using Statview software (Cary, NC). Results Subjects Study participant mean (SD) age was 69.7 (9.7) years; range 47 to 87 years. Their mean BMI (SD) was 27.9 ± 5.7 kg/m 2 ; range 20.0 to 48.0 kg/m 2. No between treatment group differences were present in age, BMI, serum 25(OH)D or serum chemistry results (Table 1). Overall supplement compliance was 99.1% as determined by pill count. In

9 those assigned to receive daily vitamin D, two individuals were 72% and 75% compliant; all others in this group were 90+% compliant with supplementation. Vitamin D Metabolites at Baseline At baseline, the mean (SD) serum total 25(OH)D, 24,25(OH) 2 D and vitamin D 3 concentrations were 27.2 (5.6), 2.2 (0.9) and 2.0 (2.54) respectively. Baseline serum vitamin D 3 was below the lower limit of quantitation (< 0.19 ng/ml) in 16 study participants. The mean (SD) free 25(OH)D was 5.1 (1.8) pg/ml; thus free 25(OH)D was present at ~ 0.02% of total serum 25(OH)D. Serum 25(OH)D at baseline was positively correlated with vitamin D 3 (p < 0.05), 24,25(OH) 2 D (p < 0.001) and free 25(OH)D (p < 0.01; Figures 1a-c) and negatively correlated with PTH (p < 0.01; Figure 1d). Baseline 25(OH)D was unrelated to age, body weight, BMI and total body fat mass (data not shown). Change in Vitamin D Metabolites Over Time Daily vitamin D supplementation increased serum 25(OH)D, vitamin D 3, 24,25(OH) 2 D and free 25(OH)D (p < 0.001; Figures 2a-d). In the supplemented group, the mean increase at four months in 25(OH)D, vitamin D 3, and 24,25(OH) 2 D was 10.4, 6.8 and 1.1 ng/ml respectively. Supplementation increased free 25(OH)D at 4 months by 2.4 pg/ml. A downward trend in PTH (p = 0.09) was observed in the vitamin D supplemented group (Figure 2e).

10 Vitamin D Composite Score A vitamin D composite score was calculated by the addition of total 25(OH)D, vitamin D 3 and 24,25,(OH) 2 D and was found to be inversely correlated (p < 0.01) with PTH at both study baseline and at four months (Figure 3a/b). When compared with the association of total 25(OH)D and PTH (Figure 3c/d) the regressions were not materially different. Further exploratory analyses arbitrarily limiting the data to those volunteers whose 25(OH)D was < 30 ng/ml or < 20 ng/ml at baseline did not yield evidence that the composite score was more highly correlated with PTH than was 25(OH)D alone (data not shown). Relationship of Baseline Vitamin D Metabolites and Clinical Parameters With 25(OH)D Change at Four Months Substantial between-individual variation in the 25(OH)D change observed after four months of daily vitamin D supplementation was observed (Figure 4). Baseline vitamin D metabolite levels did not predict 4 month 25(OH)D change; specifically, baseline 25(OH)D, vitamin D 3, 24,25(OH) 2 D and the ratio of 25(OH)D to 24,25(OH) 2 D were unrelated to 25(OH)D change at 4 months (Figures 5a-d). Similarly, age was unrelated to 25(OH)D change (data not shown). In contrast, BMI, total body fat mass (Figures 5ef) and total body weight (data not shown) were negatively correlated with 25(OH)D change (p < 0.05). As noted in figure 5h, total body fat mass was no more highly correlated with change in 25(OH)D than was BMI. Despite BMI being correlated with resultant 25(OH)D change, substantial variability in 25(OH)D response within BMI category was observed (Figure 6).

11 Discussion In this study, only BMI, total body fat mass and body weight were predictive of subsequent response to daily vitamin D supplementation. Disappointingly, and in contrast to some other reports, measurement of serum vitamin D 3, 24,25(OH) 2 D and the ratio of 25(OH)D/24,25(OH) 2 D were not predictive of 25(OH)D concentration following four months of daily supplementation. As such, this work supports the suggestion of the Endocrine Society that obese individuals receive large vitamin D supplementation doses, but does not support utility, at least at this time, of other vitamin D metabolite measurements to assist with clinical decision making regarding vitamin D dose selection. Variability in 25(OH)D increase in response to a given vitamin D dose is recognized (19). It is widely reported that 25(OH)D levels are lower in those with higher BMI (29, 30), which may reflect a larger volume of distribution, i.e., sequestration into larger fat mass. However, not all studies find this relationship (31, 32). Nonetheless, overweight individuals often have low 25(OH)D levels and the Endocrine Society Clinical Practice Guideline suggests that obese individuals receive 2-3 times more vitamin D than nonobese people (12). Consistent with this, a recent equation to predict 25(OH)D response following supplementation does include BMI (33). Moreover, a small prospective study found the 25(OH)D response following supplementation to be less robust in obese compared with normal-weight subjects (34). Our data support a less profound increase in obese individuals and demonstrate that greater amount of total body fat as measured by DXA is associated with less of a 25(OH)D increase. Thus, a reasonable clinical rule

12 of thumb, is that obese individuals will generally require larger daily vitamin D supplementation doses. Unfortunately, this does not apply to all patients; other factors explaining variability should be sought. Until the mechanism(s) explaining 25(OH)D variability are clarified, it seems clinically reasonable to recheck 25(OH)D four to six months following vitamin D supplement initiation to validate that the clinician s target 25(OH)D is attained. It is logical that variability of vitamin D absorption and degradation alter the response to vitamin D supplementation. Circulating 24,25(OH) 2 D results from 24 hydroxylation of 25(OH)D; a process mediated by CYP24A1 (35). This is often viewed as the first step in vitamin D degradation (36). In this study, consistent with prior reports, we found serum 24,25(OH) 2 D to be present at ~10% of the 25(OH)D level and highly correlated with total 25(OH)D concentration (37-40), but was not predictive of 25(OH)D response to supplementation. This is contrary to prior work from our group that did suggest utility of vitamin D metabolite measurement in estimation of 25(OH)D response to supplementation (20). It is unclear why the current study does not support/replicate these results; nonetheless, these data do not currently support use of vitamin D metabolite measurements to predict response to vitamin D supplementation. Additional research to clarify if/how vitamin D metabolite measurement might facilitate clinical decision making regarding vitamin D dose selection is reasonable. Measurement of total 25(OH)D is currently accepted as the clinical approach to defining an individual s vitamin D status. However, it is possible that other approaches might be superior. Indeed, it has been suggested that assessment of vitamin D status

13 include consideration of the parent vitamin D compound, cholecalciferol (vitamin D 3 ), as it may have direct physiologic vitamin D effects (22, 23). Similarly, some reports find 24,25(OH) 2 D to possess vitamin D effects on cartilage, bone metabolism and calcium absorption (24-28). Thus approaches that might improve the definition of vitamin D inadequacy, such as our vitamin D composite score are reasonable. Unfortunately, the data reported here do not support such a simplistic approach. Perhaps other approaches in which various vitamin D metabolite concentrations are weighted differently and/or various metabolite ratios are utilized are indicated. In this regard, it has been suggested that a ratio of 24,25(OH) 2 D with 25(OH)D, a so-called vitamin D metabolite ratio, may be used to improve assessment of vitamin D deficiency/insufficiency (38). This is plausible in that vitamin D inadequacy may lead to reduced 24-hydroxyase activity. Indeed, in those with a 25(OH)D of less than 10 ng/ml, markedly lower 24,25(OH) 2 D levels are present (40). Moreover, it could be postulated that individuals with a relatively less active 24-hydroxylase activity, manifested as low 24,25(OH) 2 D, might have a more robust increase in 25(OH)D following supplementation. Consistent with this expectation, the 24,25(OH) 2 D/25(OH)D ratio two weeks after the initiation of supplementation has been reported to be correlated with the increase in 25(OH)D four weeks later (21). In contrast, we observed no relationship between this ratio and 25(OH)D change. Notable differences between the Wagner, et. al., report and the current study include weekly vs. daily dosing and our use of baseline vitamin D metabolite values. In summary, this study does not support clinical utility of vitamin D metabolite measurement at this time. Perhaps larger studies, measurement of additional vitamin D metabolites or considering differential potency of

14 various vitamin D metabolites could be used in a vitamin D composite score to further definition of vitamin D inadequacy. Further study of this possibility is indicated. Study strengths include utilization of a 25(OH)D assay traceable to the National Institutes of Standards and Technology (NIST) standard reference materials and use of LC-MS/MS methodology for 25(OH)D and vitamin D metabolite measurements. Notable limitations include small sample size, a rather limited range of 25(OH)D values at baseline and study only of postmenopausal women. In conclusion, this study does not support measurement of vitamin D metabolites to assist in the clinical prediction of 25(OH)D response to daily vitamin D supplementation. Overweight individuals, in general, have a less robust 25(OH)D response to supplementation, but substantial variability precludes prediction of the result following daily supplementation. Given this variability, it seems clinically prudent for clinicians to validate that whatever serum 25(OH)D they elect to target is achieved following daily supplementation.

15 Acknowledgement: This study was sponsored by an investigator-initiated research grant from Merck & Co., Inc. The sponsor had no role in study design, conduct, data analysis/interpretation or reporting. Kits to measure free 25(OH)D were supplied by Diasource.

16 Table 1: Study Participant Demographic and Laboratory Data at Baseline Parameter Age BMI 25(OH)D 24,25(OH) 2 D D 3 PTH Free 25(OH)D Creatinine Albumin Calcium Group years kg/m 2 ng/ml ng/ml ng/ml pg/ml pg/ml mg/dl mg/dl mg/dl Overall (n = 62) (9.1) (6.1) (5.6) (0.9) (2.4) (28.0) (1.8) (0.13) (0.2) (0.3) Vitamin D (n = 31) (9.7) (5.7) (5.9) (1.1)* (2.9) (27.8) (1.9) (0.13) (0.2) (0.3) Placebo (n = 31) (8.2) (6.5) (5.1) (0.6) (1.8) (28.4) (1.5) (0.13) (0.2) (0.3) Note: Data as mean (SD); * = different from placebo group, p < BMI = body mass index, 25(OH)D = 25 hydroxyvitamin D, 24,25(OH) 2 D = 24, 25 dihydroxyvitamin D, D 3 = cholecalciferol (vitamin D 3 ), PTH = parathyroid hormone

17 Figure Legends: Figure 1: Baseline relationship of 25(OH)D with other vitamin D metabolites and with PTH. As could be expected, baseline levels of cholecalciferol (vitamin D 3 ) (1a), 24,25(OH) 2 D (1b) and free 25(OH)D (1c) were all positively correlated, while serum PTH (1d) was negatively correlated with 25(OH)D. Figure 2: Effect of Vitamin D Supplementation on Vitamin D Metabolites and PTH. Supplementation increased 25(OH)D, cholecalciferol (vitamin D 3 ), 24,25(OH) 2 D and free 25(OH)D (a p < 0.001). In the supplemented group, PTH trended downward (p = 0.09). Figure 3: Relationship of a Vitamin D Composite Score, Total 25(OH)D and Free 25(OH)D With PTH. A vitamin D composite score was obtained by simple addition of serum 25(OH)D, cholecalciferol (vitamin D 3 ) and 24,25(OH) 2 D. While this composite score was correlated with PTH at both baseline and four months (3a-b), the relationship is not substantially different than that observed when comparing PTH with 25(OH)D (3cd). Free 25(OH)D was not correlated with PTH at baseline (p = 0.22; 3e) or at four months (p = 0.07; 3f). Figure 4: Individual Variability in 25(OH)D Response to Vitamin D Supplementation. In the 31 women receiving ~1,800 IU of vitamin D daily, substantial variation in the 25(OH)D change observed at 4 months is apparent. Suboptimal compliance does not explain these changes; the two volunteers with compliance < 90% (72% and 75%) are noted by the dashed rectangles.

18 Figure 5: Relationship of Various Parameters with Change in 25(OH)D. In the 31 women receiving ~1,800 IU of vitamin D 3 daily for four months, the change in 25(OH)D was unrelated to baseline 25(OH)D (5a), cholecalciferol (vitamin D 3 ) (5b), baseline free 25(OH)D (5c), baseline composite score (5d), 24,25(OH) 2 D (5e) or the ratio of 25(OH)D to 24,25(OH) 2 D (5f). In contrast, 25(OH)D change with supplementation was negatively correlated (p < 0.05) with BMI (5g) and total body fat mass measured by DXA (5h). Figure 6: Effect of BMI Category on 25(OH)D Change. Overall, a greater increase in 25(OH)D was observed among normal weight (BMI < 25 kg/m 2 ) volunteers in the vitamin D supplemented group. However, a substantial range of 25(OH)D increase was seen within all three BMI categories (normal weight < 25, overweight 25- < 30 and obese 30 kg/m 2 ). For example, in the normal weight volunteers, the 25(OH)D increase ranged from 8-21 ng/ml whereas in the obese volunteers it changed by -3 to + 12 ng/ml.

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