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American Journal of Clinical Nutrition, Vol. 87, No. 6, 1738-1742, June 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

25-Hydroxylation of vitamin D3: relation to circulating vitamin D3 under various input conditions1,2,3

Robert P Heaney1, Laura AG Armas1, Judith R Shary1, Norman H Bell1, Neil Binkley1 and Bruce W Hollis1

1 From the Osteoporosis Research Center, Creighton University, Omaha, NE (RPH and LAGA); the Departments of Pediatrics (BWH) and Medicine (JRS and NHB), Medical University of South Carolina, Charleston, SC; and the Department of Medicine, University of Wisconsin, Madison, WI (NB)

2 Supported by US Army grants no. DAMD 17-01-1-0816 (to NHB) and DAMD 17-01-1-0818 (to RPH), grant no M01 RR01070 from the National Institutes of Health for the General Clinical Research Center of the Medical University of South Carolina, and a grant from Dialysis Clinics, Inc, and by the Health Future Foundation.

3 Reprints not available. Address correspondence to RP Heaney, Creighton University, 601 North 30th Street, Suite 4841, Omaha, NE 68131. E-mail: rheaney{at}creighton.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Neither the efficiency of the 25-hydroxylation of vitamin D nor the steady state relation between vitamin D3 and 25-hydroxyvitamin D [25(OH)D] has been studied in humans.

Objective: We aimed to examine the relation between serum vitamin D3 and 25(OH)D in normal subjects after either oral administration of vitamin D3 or ultraviolet-B radiation across a broad range of inputs.

Design: Values for serum vitamin D3 and (OH)D3 were aggregated from 6 studies—1 acute and 5 near-steady state—at various vitamin D3 inputs. In 3 of the steady state studies, vitamin D3 had been administered for 18–26 wk in doses of 0 to 11000 IU/d; in 2 studies, subjects had received solar or ultraviolet-B irradiation.

Results: In the acute study, subjects receiving a single 100000-IU dose of vitamin D3 had a rise in serum cholecalciferol to a mean of 521 nmol/L at 1 d and then a fall to near-baseline values by 7–14 d. Serum 25(OH)D peaked at 103 nmol/L on day 7 and fell slowly to baseline by day 112. In the 5 steady state studies, the relation of serum 25(OH)D to serum vitamin D3 was biphasic and was well described by a combined exponential and linear function: Y = 0.433X + 87.81[1–exp (–0.468X)], with R2 = 0.448.

Conclusions: At physiologic inputs, there is rapid conversion of precursor to product at low vitamin D3 concentrations and a much slower rate of conversion at higher concentrations. These data suggest that, at typical vitamin D3 inputs and serum concentrations, there is very little native cholecalciferol in the body, and 25(OH)D constitutes the bulk of vitamin D reserves. However, at supraphysiologic inputs, large quantities of vitamin D3 are stored as the native compound, presumably in body fat, and are slowly released to be converted to 25(OH)D.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin D deficiency is widespread throughout the United States (1) and the world (2). Deficiency of the vitamin is associated with greater secretion of parathyroid hormone and skeletal remodeling and greater risks of osteoporosis, fractures, and rickets or osteomalacia (1, 3, 4). Vitamin D deficiency has also been implicated in the pathogenesis of certain cancers including breast, ovarian, prostate, and colon cancer and of other diseases, including multiple sclerosis, type 1 diabetes mellitus, lupus erythematosus, and tuberculosis (4–7). In most persons, cutaneous production of vitamin D3 from sunlight is the primary source of the vitamin (8, 9), and the remainder is obtained from dietary sources and supplements. The elderly are at greatest risk of vitamin D deficiency because of their limited exposure to sunlight and lesser cutaneous synthesis of 7-dehydrocholesterol and, hence, their lower production of vitamin D3 (10).

It is conventional wisdom that vitamin D3, a fat soluble molecule, is stored in body fat and that, to become metabolically active, it is first hydroxylated in the 25-position of the sterol molecule. The latter conversion takes place in the liver and is mediated by both microsomal and mitochondrial enzymes (ie, CYP2R1, CYP3A4, and CYP27A) (11–18). However, the partition in the body between the native compound and its 25-hydroxy derivative [25(OH)D3] at various inputs in humans is largely unknown, as are the kinetics of the conversion in vivo. Better understanding of these issues is important both for designing public health strategies to optimize vitamin D3 nutritional status and for avoiding potential toxicity.

The principal reason for contemporary ignorance of these matters is that few human studies of controlled vitamin D3 inputs have been performed or published, and essentially none of the studies to date, either cross-sectional or prospective, have provided data for both serum vitamin D3 and serum 25(OH)D3 in the same subjects.

In this report, we attempt to address this deficiency, assembling data from 6 different studies in which values for both vitamin D3 and 25(OH)D3 were measured. The studies fall into 2 groups: 1 acute dosing study permitting semiquantitative description of the partition and conversion and 5 near-steady state studies in which the equilibrium relation between vitamin D3 and 25(OH)D3 can be estimated. The vitamin D3 values and their relation to 25(OH)D3 concentration are published here for the first time.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Design
The studies providing data for this analysis are listed in Table 1Go, and pertinent descriptive information from each study is provided. Further details on the 5 published studies (studies A and C-F), including identification of the principal investigators and institutions, are contained in their respective reports (19–23). The other study (study B), whose data have not been completely published, was conducted at the Medical University of South Carolina, with Normal Bell as the principal investigator.


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  		TABLE 1. Studies providing serum data for both vitamin D3 and 25-hydroxyvitamin D3 concentrations

 
Treatment duration in studies B, C, and D ranged from 18 to 26 wk, and the serum concentrations of vitamin D3 and 25(OH)D were measured throughout the treatment period. Study E was performed in Hawaiian sports participants going about their customary outdoor activities, and study F, conducted in participants who previously had little solar exposure, involved 3 sessions/wk, for 4 wk, of controlled ultraviolet-B (UV-B) exposure in a dermatology light box (HOUVA-A II; National Biological Corp, Twinsburg, OH). For studies of the relation between vitamin D3 inputs or concentrations and serum 25(OH)D3 concentrations, values were taken from the end of the period of treatment, so as to approximate the true equilibrium status.

For all studies, participants gave written informed consent. All of the study designs had been approved by the respective institutional review boards.

Analytic methods
The analytic methods were described in detail in the primary reports of the studies concerned [(19–23) also: N Bell, et al, unpubished observations, 2005]. Serum vitamin D3 in study D was measured by Tai Chen (Boston University). For all of the other studies, the measurements of serum vitamin D3 were performed in the laboratory of one of us (BWH) by using methods described elsewhere (24–26). Briefly, serum vitamin D3 and 25(OH)D3 were determined by reverse-phase HPLC and radioimmunoassay, respectively, as previously described (19–26). For 25(OH)D3, the methods used employed an external vitamin D quality assurance survey [eg, DEQAS (27)].

Statistical analysis
The area under the curve (AUC) for the data of study A was calculated by using the trapezoidal method. Data from studies B through F were analyzed by fitting to a combination of exponential and linear functions with the use of the curve-fitting routine of SIGMAPLOT software (version 10; Systat Inc, Richmond, CA). Because the methods for vitamin D3 assay in study D differed from those in studies B, C, E, and F, the data from study D were not pooled with the others. Routine descriptive statistics and linear regressions were computed by using the statistical functions of EXCEL software (version 2003; Microsoft Inc, Redmond, WA). Results are expressed as means ± SDs or SEMs, as appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conversion of vitamin D3 to 25-hydroxyvitamin D3
The time course for serum vitamin D3 and 25(OH)D3 after a single oral dose of 100000 IU cholecalciferol (study A) is plotted in Figure 1Go. Serum vitamin D3 peaked on day 1 at a mean concentration of 521 nmol/L, whereas serum 25(OH)D3 rose more slowly, peaking on day 7. The increment above baseline at the maximal concentration (Cmax) for vitamin D3 was 515 nmol/L, and that for 25(OH)D3 was 34 nmol/L. Whereas the vitamin D3 concentration fell rapidly, being close to baseline by 7 d, the concentration of 25(OH)D3 fell slowly, reaching baseline by 112 d. The AUC for the increment above baseline for vitamin D3 calculated to day 14 was 1493 nmol·d/L, and that for 25(OH)D3 was 1911 nmol·d/L. The lower AUC for vitamin D3 would not have been predicted, and it probably reflects a failure to capture the actual Cmax with the 0-, 1-, and 3-d sampling frequencies used in the present study. Nevertheless, the similar magnitudes of the values for vitamin D3 and for 25(OH)D3 suggests complete conversion of the dosed vitamin D3 to 25(OH)D3 by day 112 at, therefore, an average rate approximating 1000 IU/d.


Figure 1
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  		FIGURE 1.. Time course of the rise from baseline of serum vitamin D3 and 25-hydroxyvitamin D3 for study A (23) after a single oral dose of 100000 IU cholecalciferol to 30 healthy adults of both sexes. Baseline serum vitamin D3 was 5.1 ± 1.3 nmol/L, and baseline 25-hydroxyvitamin D3 was 67.6 ± 3.5 nmol/L.

 
Serum vitamin D and orally dosed vitamin D
The time course of the relation of serum vitamin D3 concentration as a function of continuing intake (4000 IU/d from study B and 5500 and 11000 IU/d from study D) is shown in Figure 2Go. As is evident from inspection, vitamin D3 concentration plateaued after {approx}3 wk of daily dosing. The regression of vitamin D3 dose on the equilibrium concentration of vitamin D3 in the 78 subjects of studies B and D is shown in Figure 3Go. (The usual x- and y-axes have been reversed to facilitate estimation of effective dose from measured steady state concentration.) Basal, unsupplemented serum vitamin D3 concentration in studies A–D and F (the 5 studies for which such data were available) averaged 10.6 ± 9.3 nmol/L (4.1 ± 3.6 ng/mL). Using the regression equation of Figure 3Go, that value is what would be produced by an input from all sources of {approx}39 µg/d, or just shy of 1600 IU/d.


Figure 2
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  		FIGURE 2.. Time course of serum cholecalciferol concentration after oral vitamin D3 doses of 4000, 5500, and 11000 IU/d in healthy human subjects. Serum vitamin D3 increased rapidly during the first week, and the rate of increase then declined, plateauing by {approx}3 wk.

 

Figure 3
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  		FIGURE 3.. Relation of equilibrium serum vitamin D3 concentration to oral vitamin D3 doses of 4000, 5500, and 11000 IU/d in the subjects from studies B and D.

 
Serum vitamin D3 and 25-hydroxyvitamin D3
The relation between steady state concentrations of vitamin D3 and 25(OH)D3 is shown in Figure 4Go and Figure 5Go. Figure 4Go plots the data of studies B, C, E, and F, and Figure 5Go is based on the data of study D. Both graphs show the replicability of the pattern of the relation. In both, the mean concentration of 25(OH)D rises very steeply from values close to zero to values of {approx}100 nmol/L (40 ng/mL) and even higher. As is suggested visually, the relation is biphasic, with serum 25(OH)D rising very rapidly at low serum vitamin D3 concentrations and then more slowly, but with no apparent tapering off at progressively higher serum vitamin D3 concentrations. This slow phase begins at serum vitamin D3 values of {approx}15 nmol/L (5.8 ng/mL) and at 25(OH)D concentrations of {approx}80–100 nmol/L. Consistent with standard enzyme kinetics, involving a first-order reaction at low substrate concentrations and then a zero-order reaction at higher concentrations, the data were fitted to a combination exponential and linear function, according to the following equation:

Formula 1(1)
where Y = serum 25(OH)D (nmol/L) and X = serum vitamin D3 (nmol/L). The R2 value (0.443) indicated a good fit (P < 0.001) to the selected function. In Figure 5Go, the fit was even better (R2 = 0.718, P < 0.001). The data in study D (Figure 5Go), although identical in pattern, produced slightly different equation variables. Because of likely systematic differences in the assay results of one or both of the 2 measured variables in study D, we did not attempt to use study D to define the values of those variables, only to confirm that the pattern of the relation was the same as for the aggregate of studies B, C, E, and F.


Figure 4
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  		FIGURE 4.. Plot of the relation between serum concentrations of vitamin D3 and 25-hydroxyvitamin D after 18–20 wk of treatment with various doses of vitamin D3. {Delta}, subjects from study B; {circ}, subjects from study C; {square}, subjects from study F. The regression line is a least-squares fit of the data to a combination exponential and linear function.

 

Figure 5
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  		FIGURE 5.. Plot of the relation between serum vitamin D3 and 25-hydroxyvitamin D in study D only. As in Figure 4Go, the regression line is a least-squares fit of the data to a combination exponential and linear function.

 
That the relation is truly biphasic is supported in 3 ways. First, the 2 datasets depicted in Figures 4 Goand 5Go each showed the identical biphasic pattern; second, separate analysis of the lower end of the 2 continua [ie, serum vitamin D3 values <15 nmol/L (5.8 ng/mL)] showed a significant upward trend with a linear slope that was nearly 10 times as great as the corresponding best fit slope for the slower, linear phase above vitamin D3 concentrations of 15 nmol/L (5.8 ng/mL). Third, the early and late phase slopes were significantly different from each other (P < 0.01).

The slope of the linear portion of the curve in Figure 4Go—ie, 0.433—indicates that serum 25(OH)D rose by 0.433 nmol/L (0.164 ng/mL) for every 1-nmol/L (0.385-ng/mL) rise in serum vitamin D3. This finding may be interpreted to mean that {approx}43% of the daily vitamin D3 input supporting the circulating D3 concentration is being converted to 25(OH)D3 and that the remainder is building up, both in blood and in storage depots (presumably, fat depots). In contrast, the much steeper slope at the low end of the curve indicates near-quantitative conversion of vitamin D3 to 25(OH)D3. That such was the case is strongly suggested by results in several of the subjects in study F who showed a median rise of 21 nmol/L (8.1 ng/mL) in serum 25(OH)D3 with UV-B exposure but no rise whatsoever in serum vitamin D3 (which remained below the detection limit of the assay after 4 wk of controlled UV-B radiation).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present findings clearly establish for the first time that the concentration of serum 25(OH)D in response to input of vitamin D3 in humans is biphasic: a rapid increase occurs at low vitamin D3 concentrations and a slower response occurs at higher concentrations. The ability to elevate serum 25(OH)D to a meaningful extent by additional input of vitamin D3—with little and, in some persons, no appreciable change in serum vitamin D3 itself—suggests essentially complete conversion of the vitamin D3 to 25(OH)D without an appreciable buildup in the concentration of the precursor. But, as Figures 4Go and 5Go show graphically, above a serum vitamin D3 concentration of {approx}15 nmol/L (5.8 ng/mL), the relation shifts dramatically. Serum 25(OH)D concentration continues to rise, but D3 now rises as well, at better than twice the rate of the product molecule; ie, with continuing increases in input, the precursor is accumulating faster than it can be converted. We may calculate from Figure 3Go that a serum vitamin D3 concentration of 15 nmol/L would be produced by continuing daily total inputs (from all sources) of {approx}50 µg (ie, 2000 IU). Whether the same biphasic response of serum 25(OH)D also occurs with vitamin D2 is not known. In lactating women given vitamin D2 at doses of 1600 or 3600 IU/d for 3 mo, increments in serum vitamin D2 and serum 25(OH)D2 were modest, and only a steep response was observed (28).

We suggest that these observations point to 3 conclusions. First, at typical inputs of vitamin D3 (whether cutaneous or oral), there is rapid and near-quantitative conversion of vitamin D3 to 25(OH)D, which then serves not only as the functional status indicator of the nutrient but, more important, as its principal storage form in the body. Second, above typical serum vitamin D3 concentrations (ie, above {approx}15 nmol/L), which are probably equivalent to a daily input of 2000 IU, the hepatic 25-hydroxylases become saturated and the reaction switches from first order to zero order. Third, the constant (maximal) production of 25(OH)D, irrespective of precursor concentration of vitamin D3, must be in excess of metabolic consumption, which is the reason that serum 25(OH)D continues to rise as vitamin D3 concentrations rise.

If correct, this explanation may help to clarify many of the uncertainties surrounding vitamin D physiology, one of which is the determination of the approximate concentration of serum 25(OH)D that may be considered optimal for health. Our data offer a different approach to estimating this value. One could plausibly postulate that the point at which hepatic 25(OH)D production becomes zero-order constitutes the definition of the low end of normal status. This value, as suggested from the equation in Figure 3Go, is at a serum 25(OH)D concentration of {approx}88 nmol/L (35.2 ng/mL) (the y-axis intercept of the linear portion of the equation in Figure 3Go). It is interesting that this estimate is very close to that produced by previous attempts to define the lower end of the normal range from the relations of serum 25(OH)D to calcium absorption (29) and to serum parathyroid hormone concentration (ie, {approx}75–85 nmol/L, or 30–34 ng/mL) (30).

In study A, with a supraphysiologic input, slow release from storage depots is indicated by the slow fall in 25(OH)D3 from its Cmax. The half-time of 25(OH)D is typically on the order of 20–30 d, whereas the approximate half-time in study A for the increment above baseline was >50 d. Fat is the most likely storage depot, although muscle storage cannot be ruled out. Fat storage of vitamin D3 is certainly the case in the rat (as well as in humans) when serum vitamin D3 concentrations are high. Analysis of body distribution in rachitic animals given 14C-labeled vitamin D3 every day for 2 wk showed that the largest amount, {approx}10%, appeared in body fat and was slowly released into the circulation over the next several months along with a more polar metabolite—probably 25(OH)D, which had not been identified at that time (31). In obese human subjects, serum 25(OH)D is lower, serum vitamin D may be very low, and rises in serum vitamin D and 25(OH)D after either UV-B irradiation or oral administration of vitamin D2 are significantly lower in obese than in nonobese persons (32, 33).

Deposition in body fat almost certainly occurs in cases of vitamin D intoxication, and persistence of hypercalcemia for months has been attributed to sustained release of vitamin D from such body stores. Fat storage is also the best explanation for the seeming disappearance of vitamin D3 from the serum in the acute dosing experiment (study A). We cannot rule out some excretion of the large dose of vitamin D3, either directly or by various catabolic reactions; however, the fact that the AUC for the increment in serum 25(OH)D was not lower than that for the increment in serum vitamin D3 suggests little or no wastage of the ingested 100000 IU.

Taken together, these results show that, as is typical for enzyme systems, there is a practical limit to the first-order 25-hydroxylation of vitamin D3 and that, when vitamin D3 input exceeds that limit, vitamin D3 itself accumulates within the body, both in serum and probably in body fat. From the data presented in Figure 4Go, it would seem that that threshold occurs at a serum vitamin D3 concentration of {approx}15 nmol/L. In turn, such a concentration, from the data of Figure 3Go, is reached on average at a vitamin D3 input of 2000 IU/d. We suggest that, below this input (whether cutaneous or oral), near-quantitative conversion of vitamin D3 to 25(OH)D3 occurs. Thus, at typical inputs, 25(OH)D3 would constitute the principal storage form of the vitamin.

The strengths of the present study are several. First, it includes both sexes and a broad range of ages and races in the 206 participants of the component studies, which enhances the generalizability of the findings to the adult population. Second, the measurements in most of the component studies were made in a single laboratory by using research-quality assay methods. Third, these studies provide the largest body of simultaneous human vitamin D3 and 25(OH)D3 serum values ever published, which permits for the first time an estimation of the quantitative dynamics of the system. The principal weakness of the study lies in the lack of measurement of tissue (eg, fat) vitamin D metabolite content. For the latter reason, all conclusions about fat (or other tissue) storage must be considered tentative.

In summary, results of studies in which vitamin D3, in doses of 0 to 11000 IU/d, was given to normal subjects and subjects receiving exposure to either sunlight or UV-B irradiation showed that the response of 25(OH)D to increases in serum vitamin D3 is biphasic: ie, an initial steep slope is followed by a more gradual rise. These findings are interpreted to mean that the conversion of substrate to 25(OH)D is nearly quantitative at low vitamin D3 inputs but less than half-quantitative at supraphysiologic intakes.


   ACKNOWLEDGMENTS
 
The authors' responsibilities were as follows: RPH: data generation, data analysis, and manuscript preparation; LAGA: data generation and manuscript review; JRS: data generation and manuscript review; NHB: data generation, data analysis, and manuscript preparation; NB: data generation and manuscript review; and BWH: laboratory analysis, data generation, and manuscript review. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication January 2, 2008. Accepted for publication March 4, 2008.




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A. A. Ginde, J. M. Mansbach, and C. A. Camargo Jr
Association Between Serum 25-Hydroxyvitamin D Level and Upper Respiratory Tract Infection in the Third National Health and Nutrition Examination Survey
Arch Intern Med, February 23, 2009; 169(4): 384 - 390.
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