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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 6 2156-2166
Copyright © 1998 by The Endocrine Society

Original Studies

Unique 24-Hydroxylated Metabolites Represent a Significant Pathway of Metabolism of Vitamin D2 in Humans: 24-Hydroxyvitamin D2 and 1,24-Dihydroxyvitamin D2 Detectable in Human Serum1

E. Barbara Mawer, Glenville Jones, Michael Davies, Pamela E. Still, Valarie Byford, Neil J. Schroeder, Hugh L. J. Makin, Charles W. Bishop and Joyce C. Knutson

University Department of Medicine, Manchester Royal Infirmary, (E.B.M., M.D., P.E.S.), Manchester; and the Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry (H.L.J.M., N.J.S.), London, United Kingdom; the Department of Biochemistry, Queen’s University (G.J., V.B.), Kingston, Ontario, Canada; and Bone Care International (C.W.B., J.C.K.), Madison, Wisconsin 53713

Address all correspondence and requests for reprints to: Prof. E. B. Mawer, Department of Medicine, Manchester Royal Infirmary, Manchester, United Kingdom M13 9WL.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have produced evidence for a new metabolic pathway for vitamin D2 in humans involving the production of 24-hydroxyvitamin D2 (24OHD2) and 1,24-dihydroxyvitamin D2 [1,24-(OH)2D2]. These metabolites were produced after either a single large dose (106 IU) of vitamin D2 or repeated daily doses between 103 and 5 x 104 IU. We developed assay systems for the metabolites in human serum and showed that in some chronically treated patients, the concentration of 1,24-(OH)2D2 equalled that of 1,25-(OH)2D2 at about 100 pmol/L. The metabolites were identified by high performance liquid chromatography with diode array spectrophotometry for 24OHD2 and by high resolution gas chromatography-mass spectrometry for 1,24-(OH)2D2. We show that 1,24-(OH)2D2 synthesis can be stimulated by PTH, indicating a renal origin for this metabolite and postulate that it is formed from 24OHD2, which may be synthesized in liver. We conclude from this study that vitamin D2 gives rise to two biologically active products, 1,24-(OH)2D2 and 1,25-(OH)2D2, and that 1,24-(OH)2D2 could be an attractive naturally occurring analog of 1,25-(OH)2D3 for clinical use.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IT HAS BEEN generally assumed that vitamin D2 and vitamin D3 are metabolized in an equivalent manner in humans (1), although this equivalency has been shown not to be the case in various other mammals (2) or in avian species (3, 4). At least the major mono- and dihydroxylated forms of vitamin D2 are the same as those of vitamin D3, with identification of 25-hydroxyvitamin D2 (25OHD2), 1{alpha},25-dihydroxyvitamin D2 [1{alpha},25-(OH)2D2], and 24,25-(OH)2D2 as metabolites in human sera (5).

As functional vitamin D status is usually assessed by assaying these common metabolites, particularly 1{alpha},25-(OH)2D, care has been taken in the past to select an assay method that will not discriminate between the D2 and D3 forms during both the purification and detection steps of the procedure. Where a RIA has been used as the detection technique, antibodies have been chosen that will not react preferentially with either 1{alpha},25-(OH)2D2 or 1{alpha},25-(OH)2D3 (6, 7), because in many methods the two forms are not resolved before assay. Although special care has been taken to accurately measure 1{alpha},25-(OH)2D2, the current assay technology is based upon the assumption that the metabolism of the two forms of vitamin D is exactly parallel. By focussing on the conventional metabolites of vitamin D2, particularly 1{alpha},25-(OH)2D2, the analysis has precluded observing any further active forms of vitamin D2 that may be present in plasma.

Studies with animals have recently provided evidence that the subtle differences in the chemistry of the side-chain between vitamins D2 and D3 result in differences in the site of hydroxylation and, hence, differences in hydroxylated products, particularly when large doses of vitamin D are administered (8). Of particular relevance is the direct 24-hydroxylation of vitamin D2 to produce 24OHD2. This metabolite has been observed in the blood of at least three species, including rat (9), bovine (10), and human (11) and is presumed to be of hepatic origin in vivo (12). Several other vitamin D2 metabolites have been reported, either as unique products of the further metabolism of 24OHD2, such as 1{alpha},24-(OH)2D2 (10) or 24,26-(OH)2D2 (13), or the catabolites of 1{alpha},25-(OH)2D2 (e.g. 1{alpha},24,25-trihydroxyvitamin D2, 1{alpha},24,25,26-tetrahydroxyvitamin D2, and 1{alpha},24,25,28-tetrahydroxyvitamin D2) (14) or of 1{alpha},24-(OH)2D2 (e.g. 1{alpha},24,26-trihydroxyvitamin D2) (15).

Of these vitamin D2 metabolites, 1{alpha},24-(OH)2D2 is the most interesting, partly because of its potent antiproliferative properties combined with low calcemic activity (10, 15, 16) and partly because it can be formed either from vitamin D2 by successive 24- and 1{alpha}-hydroxylations (10) or from 1{alpha}OHD2 by hepatic 24-hydroxylation (17). This latter 24-hydroxylation step is unusual, in that it appears to be carried out by the liver cytochrome P450, CYP27, which is currently believed to be the hepatic vitamin D3-25-hydroxylase (18). Although 1{alpha},24-(OH)2D2 has been observed in animals given large doses of vitamin D2 and in human hepatoma cell incubations in vitro, it has not been shown to be formed in humans given vitamin D2.

In this study, we set out to use some of the recent advances in assay technology [cartridges, high performance liquid chromatography (HPLC), and RIA] to search for some of these unique vitamin D2 metabolites, found previously in the blood of animals and generated in vitro, in the blood of patients treated with large doses of vitamin D2 for various clinical conditions. We were able to measure in such human serum samples the usual 25-hydroxylated metabolites represented by the mono- and dihydroxylated forms: 25OHD2, 1{alpha},25-(OH)2D2, and 24,25-(OH)2D2. We also detected the metabolites 24OHD2 and 1{alpha},24-(OH)2D2, suggesting the existence of a unique 24-hydroxylation pathway of vitamin D2. As might be expected from such a sequence of metabolism involving first 24-hydroxylation and then renal 1{alpha}-hydroxylation, we also found that the synthesis of 1{alpha},24-(OH)2D2 was stimulated by the infusion of a potent 1{alpha}-hydroxylase inducer, PTH, and correlated, although not quite significantly, to the concentration of the substrate, 24OHD2.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Details of subjects are given in Table 1Go.


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  		Table 1. Details of subjects treated with vitamin D2

 
Consent for the study protocols was given by the Manchester Royal Infirmary ethical committee, and samples were taken with the informed consent of the patients after they had received a full explanation of the study.

Vitamin D-deficient subjects given a single large dose of vitamin D2 (subjects 1–7)

Seven vitamin D-deficient patients (initial serum concentration of 25-hydroxyvitamin D, 11.25 ± 1.64 nmol/L) were each given 65 µmol vitamin D2, orally (106 IU). Blood samples were taken before treatment and at intervals for up to 90 days after treatment, and serum was analyzed for vitamin D metabolites. In one of these patients, a larger volume of blood (40 mL) was extracted for definitive identification of certain metabolites.

Vitamin D-deficient subjects treated with small daily doses of vitamin D2 (subjects 8–10)

Two vitamin D-deficient subjects were treated with 400 IU (26 nmol)/day vitamin D2 for 14 days, followed by 4000 IU (260 nmol) vitamin D2/day. In addition, archival samples were reanalyzed from a patient given 1000 IU (65 nmol)/day each of vitamins D2 and D3.

Subjects treated with large daily doses of vitamin D2 (subjects 11 and 13–21)

Archival serum samples were reanalyzed from five patients with X-linked hypophosphatemic osteomalacia (XLH) who had been treated for several years with vitamin D2 [50,000 IU (3.25 µmol)/day] and from one additional XLH patient treated with 50,000 IU each of vitamins D2 and D3. Samples were also examined from two patients with idiopathic hypoparathyroidism receiving 50,000 IU vitamin D2/day and from one patient with Crohn’s disease given 50,000 IU each of vitamins D2 and D3. Archival samples were analyzed for vitamin D metabolites from two of the XLH patients who had received a PTH infusion. The patients had received two infusions of 200 U human PTH-(1–34) (Parathar, Rhone Poulenc-Rorer, Paris, France), iv, over 10 min at 1000 and 1600 h on the day of study. Blood samples were taken immediately before the first infusion and 12, 24, 48, and 72 h after the first infusion.

Vitamin D sterols

Standard vitamin D compounds were donated as follows; 25OHD3, 24,25-(OH)2D3, and 1,25-(OH)2D2, Dr. Milan Uskokvic; 24OHD2, 1,24-(OH)2D2, and 1,25-(OH)2D3, Bone Care International (Madison, WI); 25OHD2, Dr. M. A. Maestro, University of Coruna (Coruna, Spain); 24,25-(OH)D2, Dr. T Kobayashi, Kobe Pharmaceutical University (Kobe, Japan); and 25,26-(OH)D2, Dr. Y. Mazur, Weizman Institute (Tel Aviv, Israel). [26,27-Methyl-3H]-25OHD3 (6.5 tetrabecquerels/mmol) and [26,27-methyl-3H]1,25-(OH)2D3 (6.7 tetrabecquerels/mmol) were obtained from Amersham International (Aylesbury, UK).

Extraction, chromatography, and assay of metabolites

Sequential serum samples were extracted and analyzed for vitamin D metabolites as described in Fig. 1AGo. The scheme used was a development of a previously published method (19). Elution positions of standard compounds were determined in the preparative HPLC system used, namely a Zorbax-SIL 3-µm particle size column eluted with a mobile phase of n-hexane:propan-2-ol:methanol (30:1:1, vol/vol/vol) at a flow rate of 2 mL/min (Fig. 1BGo).



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  		Figure 1. Assay system used to measure the metabolites of vitamin D2 in human serum. A, Scheme of extraction, purification, and HPLC of samples before assay by UV detection or RIA. B, Typical HPLC profile of standard vitamin D2 and D3 metabolites showing baseline resolution. C, Collection of samples and typical RIA data for specific fractions across the HPLC profile showing three major peaks of cross-reactivity corresponding to 1{alpha},24-(OH)2D2, 1{alpha},25-(OH)2D2, and 1{alpha},25-(OH)2D3, respectively. Note that in C the fraction containing the 1{alpha}-dihydroxylated metabolites had been prepared by immunoextraction.

 
Monohydroxylated metabolites that eluted in the positions corresponding to standard 24OHD2, 25OHD2, and 25OHD3 were measured by quantitative HPLC (19), and dihydroxylated metabolites corresponding to 1,24-(OH)2D2, 1,25-(OH)2D2, and 1,25-(OH)2D3 were measured by RIA after HPLC separation (20). In a modification of the method, immunoextraction was used (see Results) to prepare the 1{alpha}-hydroxylated metabolites and establish their elution positions (Fig. 1CGo). Competitive protein binding assay using human plasma as a source of vitamin D-binding protein (21) was used to assay a metabolite eluting in the position of 24,25-(OH)2D2. A novel RIA using monoclonal antibody (mAb) 5F2 (8) was devised for 1,24-(OH)2D2. As no radiolabeled vitamin D2 metabolites were available, the assumption was made that recovery from serum was similar to that of comparable vitamin D3 compounds, which was assessed by the use of isotopically labeled metabolites.

The validity of using archival samples that had been stored at -20 C for up to 10 yr was checked by comparing the values obtained on reanalysis with those originally obtained. In most cases the methodology was identical, but in some early samples 1,25-(OH)2D2 and 1,25-(OH)2D3 had been measured by RIA using polyclonal antibodies (19) instead of mAb 5F2 (20) as used in in-house assays since 1989. However, the reference ranges and assay characteristics for the two methods are extremely similar and do not introduce any systematic bias.

Cartridges incorporating mAb 1G7 (7) (IDS, Tyne and Wear, UK), were used for immunoextraction of 1{alpha}-hydroxylated metabolites (22) as part of the validation procedure for the assay of 1,24-(OH)2D2.

Assay of PTH was performed using a Nichols Institute (Saffron Walden, UK), Allegro immunoradiometric assay kit for PTH-(1–84).

Extraction and chromatography for identification of metabolites

A 40-mL volume of serum from one subject was extracted with acetonitrile (23) and used to confirm the characterization of 24OHD2, 25OHD2, 24,25-(OH)2D2, and 1,24-(OH)2D2 by comparison with standards. Retention times of HPLC-purified vitamin D metabolites prepared from serum by the above method were compared to those of authentic standards in a further HPLC system consisting of a Zorbax-SIL 3-µm particle size column eluted with n-hexane:propan-2-ol:methanol (91:7:2 or 94:5:1, vol/vol/vol) at a flow rate of 1 mL/min (24). Eluting peaks were monitored using a photodiode array spectrophotometric detector (model 990, Waters Associates, Milford, MA) set to scan in the UV (200–400 nm) range.

In additional experiments, 200 mL serum were extracted to obtain metabolite peaks for analysis by gas chromatography-mass spectrometry (GC-MS).

Mass spectrometry

Two serum samples were extracted and purified by straight phase HPLC as described above, but with an additional step in that the fraction corresponding to 1,24-(OH)2D2 was rechromatographed in the same system, and the 1,24-(OH)2D2 fraction was collected. One sample came from subject 17 (Table 1Go) who was taking large daily doses of vitamin D2 and had an assayed concentration of 1,24-(OH)2D2 of 34 pg/mL (79 pmol/L). The other sample came from a control subject with no detectable vitamin D2 metabolites in serum, to which standard 1,24-(OH)2D2 (20 pg/mL; 47 pmol/L) had been added. To the final extract from each sample, 500 pg (1.2 pmol) 1,25-dihydrotachysterol [1,25-(OH)2DHT3] were added in a small volume of ethanol; this was used as an internal standard during GC. These extracts were subjected to an additional straight phase HPLC separation (Lichrospher Si 60, Merck, Darmstadt, Germany) using a solvent system hexane:isopropanol:methanol (92:4:4) at a flow rate of 1.2 mL/min. Solvent eluting between 11–14 min was collected [this fraction should contain 1,24-(OH)2D2 (11.56 min) and the internal standard 1,25-(OH)2DHT3 (12.83 min)]; in this system 1,25-(OH)2D3 has a retention time of 14.85 min. Pertrimethylsilyl ethers were made using trimethylsilylimidazole and were separated on Lipidex 5000 columns as previously described (25). The samples were dissolved in a small volume of N-methyl-N-trimethylsilyl-trifluoroacetamide (20 µL) before injection into the GC-MS system. Chromatography, using an HP6890 GC linked to an Autospec high resolution mass spectrometer (Micromass, Manchester, UK), was carried out on a WCOT capillary column (Hewlett Packard, Bracknell, United Kingdom) (HP1, cross-linked methyl silicone gum, 12-m x 0.2-mm x 0.33-µm film thickness). The end of the GC column was inserted directly into the ion source of an Autospec high resolution mass spectrometer (Micromass, Manchester, UK). Ions previously identified by analysis of standard 1,24-(OH)2D2 at a resolution of 10,000 [m/z 513.3584, m/z 554.3975, and m/z 601.3929, representing the ions at (M-132)+, (M-90)+ and (M-43)+] were monitored.

Statistical analysis

Statistical analysis was undertaken using Instat instant statistics software (GraphPad, San Diego, CA). Results were expressed as the mean ± SE. Associations between variables were examined using Pearson’s correlation for normally distributed data and Spearman’s rank correlation for nonparametric data.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Identification of vitamin D2 metabolites

Vitamin D metabolites, isolated from serum using an acetonitrile extraction, were rechromatographed using a Zorbax-SIL column, with various strengths of the ternery solvent system n-hexane:propan-2-ol:methanol from 91:7:2 to 94:5:1 (vol/vol/vol) required to achieve optimal resolution of peaks. In the results shown in Fig. 2Go, each quadrant represents the comparison of purified vitamin D2 metabolites from human serum with its chemically synthesized standard. In each case the putative metabolite comigrates exactly with its standard, providing strong evidence of correct identification.



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  		Figure 2. Comigration with synthetic standards and UV spectra of metabolites of vitamin D2 extracted from human serum: A, 24OHD2; B, 25OHD2; C, 24,25-(OH)2D2; and D, 1{alpha},25-(OH)2D2.

 
Insets within each quadrant of Fig. 2Go contain the background-subtracted UV spectrum from the apex of the peak for each biologically derived metabolite. Although the quality of these spectra improves as the concentration of metabolite increases, in each case the spectrum is consistent with a vitamin D compound possessing the classical vitamin D chromophore ({lambda}max = 265 nm; {lambda}min = 228 nm). This provides further confidence that the identification is correct and indicates that each metabolite is not the previtamin D ({lambda}max = 260 nm; {lambda}min = 235 nm) for the corresponding vitamin D3 metabolite, which routinely runs close to the vitamin D2 metabolite (e.g. 24OHD2 runs close to 25-hydroxyprevitamin D3, both of which run ahead of 25OHD3 on straight phase HPLC). Particular attention should be paid to Fig. 2DGo, because this HPLC peak, representing 1{alpha},25-(OH)2D2, was calculated to contain only 4.7 ng (10.8 pmol) solute. This corresponds to a concentration of around 200 pg/mL (500 pmol/L) in the original serum, which agrees quite well with the RIA result in this case. For the sample processed here for identification purposes, the fraction containing 1{alpha},24-(OH)2D2 did not contain sufficient material for detection by UV methodology. However, the application of high resolution MS to the analysis of extracts from a patient taking high doses of vitamin D2 was sufficiently sensitive to detect the presence of 1{alpha},24-(OH)2D2 in this extract (see Fig. 3Go). The identification of this metabolite as 1{alpha},24-(OH)2D2 relies upon two pieces of evidence. The metabolite had a relative retention time corresponding to that of standard 1{alpha},24-(OH)2D2 that had either been injected directly onto the mass spectrometer or had been added to a serum sample which was then extracted in the same way as the sample from a patient taking pharmacological doses of vitamin D2. The identification also relies upon the fact that the ratio of the three ions monitored during GC-MS was the same as that obtained from both the standard and the extract of supplemented control serum; these data are summarized in Table 2Go. Such criteria of specificity are widely accepted as satisfactory evidence for the presence of a particular compound in a biological fluid (26).



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  		Figure 3. High resolution mass fragmentography of an extract of serum from a patient taking vitamin D2. When injected into the heated zone of the gas chromatograph, vitamin D molecules are cyclized to form pyro- and isopyro-isomers. The mass spectral interpretation inserted in this figure has been applied to the pyro-isomer. Ion chromatograms of pertrimethylsilylated 1{alpha},24-(OH)2D2 monitoring three separate ions, m/z 513.3584 (A), m/z 554.3975 (B), and m/z 601.3929 (C), showing the trace between 9 and 14 min. The peaks from the pyro-isomer of 1{alpha},24-(OH)2D2-TMSi are shaded.

 

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  		Table 2. High resolution mass spectrometry of 1,24-(OH)2D2 peak from serum extracts

 
Assays of vitamin D metabolites levels in serum

Assay systems were developed to measure those vitamin D2 metabolites not included in our routine assay procedure (20). 24OHD2 was measured by UV absorbance in an extension of our system for 25OHD2 and 25OHD3 (20). Standard 24OHD2 was provided as an unresolved mixture of the 24R- and 24S-diastereoisomers that eluted as a single peak at 10.06 min on HPLC using a Zorbax-SIL column and a mobile phase consisting of n-hexane:propan-2-ol:methanol (24:1:1, vol/vol/vol; data not shown). On examining the serum extracts from the vitamin D2-treated patients, a peak was identified in all samples that corresponded to the standard peak for 24OHD2. Quantitation of this peak in the patient samples was achieved by measuring the UV absorbance.

A peak corresponding to 1,24-(OH)2D2 was measured by RIA after separation from 1,25-(OH)2D2 and 1,25-(OH)2D3 by HPLC. Efficient displacement of [3H]1,25-(OH)2D3 was obtained with 50% binding of 13 pmol/L (Fig. 4AGo), enabling a standard curve to be established for the assay, which was validated as follows. A serum extract was prepared from patient 12 (who had previously received vitamin D2) using immunoextraction cartridges based on mAb 1G7 (22), which binds 1,25-(OH)2D2 and 1,25-(OH)2D3. These are retained on the column and separated from monohydroxylated metabolites and dihydroxylated metabolites that lack a 1{alpha}-hydroxyl group and are readily removed from the column. The cartridges, however, retain 1,24-(OH)2D2, so a combined 1,24- and 1,25-dihydroxylated fraction can be prepared. This was done, and the fraction was then applied to a Zorbax-SIL column (4.6 mm x 25 cm) eluted with n-hexane:propan-2-ol:methanol (30:1:1, vol/vol/vol). One-minute fractions were collected across the whole volume corresponding to the HPLC elution positions of standards for the three metabolites. Clear baseline values were obtained in the fractions collected between the positions of the three peaks, showing that the values corresponding to 1,24-(OH)2D2 were a discrete peak and not a shoulder of the 1,25-(OH)2D2 peak (Fig. 1CGo).



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  		Figure 4. Assay of 1{alpha},24-(OH)2D2. A, Standard curve, using mAb 5F2, showing displacement of [3H]1{alpha},25-(OH)2D3 by 1{alpha},24-(OH)2D2. B, Validation of assay by measuring recovery of 1{alpha},24-(OH)2D2 added to human serum. C, Assay of 1{alpha},24-(OH)2D2 peak from human serum after purification on straight phase HPLC (as described in Materials and Methods) followed by reverse phase HPLC (on Zorbax-ODS, eluted with methanol:water, 78:22). The upper trace shows the retention times of standards in the reversed phase system; immunoassayable activity in 1-min fractions from the HPLC (below) shows a clear peak in the position of 1{alpha},24-(OH)2D2.

 
This was further tested by supplementing plasma containing no vitamin D2 metabolites with 46 pmol/L (19.6 pg/mL) 1,24S-(OH)2D2 and 1840 pmol/L (762 pg/mL) 1,25-(OH)2D2 and running the acetonitrile extract on a Zorbax-SIL column eluted with hexane:propan-2-ol:methanol (24:1:1, vol/vol/vol). Even under these conditions a clear separation of the peaks was achieved. The material from serum corresponded in elution position to 1,24S-(OH)2D2 (17). Recovery of added 1,24S-(OH)2D2 to two pools of 1,25-(OH)2D-deficient serum obtained from renal failure patients showed linearity over the range 23–115 pmol/L. Accuracy could not be determined with certainty because recovery was estimated from [3H]1,25-(OH)2D3 added to the two pools of serum, as [3H]1,24-(OH)2D2 was not available (Fig. 4BGo).

Further evidence for the identity of the material as 1,24S-(OH)2D2 came from a study in which serum from a patient chronically treated with vitamin D2 was extracted and the 1,24S-(OH)2D2 fraction prepared as described in Fig. 1BGo on straight phase HPLC. This fraction was then rechromatographed in a reverse phase system consisting of a Zorbax-ODS column eluted with methanol:water (78:22), and 1-min fractions were collected and assayed as described in Fig. 4AGo. A clear peak of immunoassayable activity was observed in the position corresponding to 1,24S-(OH)2D2 in the reverse phase system (see Fig. 4CGo).

Time course of vitamin D2 metabolism in vitamin D-deficient subjects given a single large dose of vitamin D2

The 30-day time course for the mean concentration of the monohydroxylated metabolites and 24,25-(OH)2D2 from seven vitamin D-deficient subjects (Table 1Go, no. 1–7) is shown in Fig. 5AGo, and the time course for the three dihydroxylated metabolites with a 1{alpha}-hydroxy function is shown in Fig. 5BGo. Similar patterns of metabolism were observed in all seven subjects, although the extent of the response varied. The rapid rise in 25OHD2 became a plateau between days 3–17, with a slow decline thereafter (Fig. 5AGo); the level was still high (170 nmol/L) in one subject sampled on day 90. In contrast, levels of 25OHD3 remained low and constant at about 10 nmol/L. The metabolite that cochromatographed with standard 24OHD2 achieved its maximum level on day 1 with a value of 34 ± 8.4 nmol/L (mean ± SE) and declined slowly over 30 days, although low values could still be detected up to 90 days. 24,25-(OH)2D2 was initially undetectable, but rose to about 10 nmol/L by day 17. Quantitatively, 1,25-(OH)2D2 was the major 1{alpha}-hydroxylated metabolite (Fig. 5BGo), with a peak by day 2 of 584 ± 205 pmol/L; 1,24S-(OH)2D2 was present at about 1/10th the concentration, again peaking on day 2 at 57.5 ± 19.9 pmol/L. The endogenous 1,25-(OH)2D3 declined throughout the course of the study from an initial value of 72.7 ± 14.8 pmol/L to 14.5 ± 4.2 pmol/L by day 30. To investigate whether the metabolic response to vitamin D2 depended on the degree of vitamin D deficiency, correlations were examined between levels of the dihydroxylated metabolites and indices of vitamin D deficiency. The peak level of 1,25-(OH)2D2, but not that of 1,24-(OH)2D2, correlated significantly and inversely with the initial serum calcium level (P = 0.048; Spearman’s rank correlation, rs, -0.786), but neither metabolite correlated with initial serum 25OHD or PTH levels.



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  		Figure 5. Time course of serum vitamin D metabolite concentrations (mean ± SE) in seven human subjects (Table 1Go, no. 1–7) given a single dose of vitamin D2 (106 IU). A, Monohydroxylated metabolites, 25OHD2 (•), 24OHD2 ({blacksquare}), 25OHD3 ({blacktriangleup}), and 24,25-(OH)2D2 ({circ}). B, Dihydroxylated metabolites, 1{alpha},25-(OH)2D2 (•), 1{alpha},24-(OH)2D2 ({blacksquare}), and 1{alpha},25-(OH)2D3 ({blacktriangleup}).

 
Relationships between vitamin D metabolites

Linear correlations were examined between the principal metabolites over the 30 days after treatment. No significant relationships were found, however, for three sets of data; these were reported as being "almost significant." The Pearson correlation coefficient for 1,24S-(OH)2D2 and 24OHD2 was 0.699 (P = 0.053), that for 1,25-(OH)2D2 and 25OHD2 was 0.71 (P = 0.051), and that for 1,24S-(OH)2D2 and 25OHD2 was 0.66 (P = 0.073). The stronger relationships between the 1{alpha}-hydroxylated forms and their corresponding respective 24- or 25-hydroxylated compounds suggest a precursor-product relationship.

Production of 1,24-(OH)2D2 in patients treated daily with large or small doses of vitamin D2

Data are presented in Table 3Go for vitamin D2 and D3 metabolite concentrations in patients treated with large (50,000 IU) daily doses, usually of vitamin D2 (subjects 13–17, XLH patients; subjects 20–21, hypoparathyroid patients), but in two cases (subject 11, vitamin D deficient; subject 18, XLH) they were given equimolar doses of each form of the vitamin. Metabolites corresponding to 1,24S-(OH)2D2 and 24OHD2 were seen in most patients. A decline in both of these metabolites was seen in patients 15 and 16, in whom therapy was discontinued in 1993 and 1987, respectively. Patient 18 was vitamin D deficient when she started taking equimolar doses of vitamin D2 and D3, but with vitamin D repletion, 1,24-(OH)2D2 became measurable in serum. Patient 11 was a vitamin D-deficient patient with Crohn’s disease and was given the same treatment; he responded in a similar fashion. Table 4Go shows results for vitamin D-deficient patients treated with near-physiological doses of vitamin D2. Both 1,25-(OH)2D2 and 1,24-(OH)2D2 were detectable in the serum of patient 9. Low concentrations of 1,24-(OH)2D2 were detectable even when this patient was taking only 400 IU/day vitamin D2. In contrast, only trivial amounts of 1,24-(OH)2D2 were assayed in patient 8, whose 1,25-(OH)2D2 did not reach high levels with either 400 or 4,000 IU doses; however, PTH concentrations were much lower than those in patient 9. In the serum of the third patient treated with low doses of vitamin D2 and D3 (1,000 IU/day), assayable amounts of 1,24-(OH)2D2, but not 24OHD2, were detected.


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  		Table 3. Serum vitamin D metabolite concentrations in patients treated with large daily doses of the vitamin

 

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  		Table 4. Serum vitamin D metabolite concentrations in patients treated with small daily doses of the vitamin

 
Production of 1,24-(OH)2D2 in patients infused with PTH

Archival samples were available for two patients with XLH (subjects 15 and 19) who were given an infusion of PTH (see Materials and Methods). Serum samples were collected for 3 days, and the stored samples were analyzed for vitamin D metabolites and PTH as described above. Results for subject 19 (shown in Fig. 6Go) demonstrate that changes in the concentration of 1,24-(OH)2D2 mirror those in 1,25-(OH)2D2 and rise in response to the increase in PTH. In patient 15, levels rose somewhat later in relation to PTH, but the same basic pattern was seen.



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  		Figure 6. Elevation of serum 1{alpha}-hydroxylated vitamin D metabolites in response to PTH infusions (subject 19). {blacksquare}, 1{alpha},24-(OH)2D2; •, 1{alpha},25-(OH)2D2; {blacktriangleup}, 1{alpha},25-(OH)2D3. Arrows indicate the times of PTH infusions.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Evidence is presented in this paper that in humans, vitamin D2 metabolism differs from that of vitamin D3. We show here that in addition to the conventional metabolites formed from vitamin D2, namely 25OHD2, 1{alpha},25-(OH)2D2, and 24,25-(OH)2D2, which are routinely measured in clinical assays for vitamin D3 metabolites, there exists a series of metabolites based upon 24-hydroxylation of the D2 side-chain. These unique derivatives include 24OHD2 (9, 10, 11) and a biologically active form, 1{alpha},24-(OH)2D2, observed previously only in animal studies and in experiments employing cultured cells in vitro (10, 17).

In our studies, we were able to detect 1{alpha},24-(OH)2D2 by a combination of high resolution HPLC and a sensitive RIA based upon an antibody known to detect both 1{alpha},25-(OH)2D2 and 1{alpha},25-(OH)2D3 (7). Levels of 1{alpha},24-(OH)2D2 were measured in the low picomolar range after the administration of large doses of vitamin D2 to a variety of patients with disorders of calcium homeostasis. In some patients receiving long term vitamin D2 therapy, the levels of 1{alpha},24-(OH)2D2 exceeded or were equivalent to the values reached by the alternative 1{alpha}-hydroxylated metabolite, 1{alpha},25-(OH)2D2. The detection of 1{alpha},24-(OH)2D2 was in most cases accompanied by a parallel increase in the level of 24OHD2 and appeared to be PTH dependent, suggesting that an alternative pathway of activation of vitamin D2 through 24OHD2 occurs in humans.

Although the group of vitamin D-deficient subjects who received a large single dose of vitamin D2 were predominantly of Asian origin (i.e. from the Indian subcontinent), as this immigrant group in the United Kingdom is particularly prone to vitamin D deficiency, there is no reason to believe that there is a genetic component to the response. We were fortunate in having access to stored samples from patients with XLH and hypoparathyroidism dating from the time when treatment of these conditions with large doses of vitamin D2 was standard. These patients were all Caucasian, and they showed qualitatively the same response as the vitamin D-deficient Asians. In the study employing a single large dose of vitamin D2, the level of 1{alpha},25-(OH)2D2 formed correlated with the level of serum calcium, one of the indices of vitamin D deficiency, although not with the initial 25OHD level, possibly because the range of the latter was small. In addition, concentrations of 25OHD tend to be labile, changing quickly with small differences in intake of the vitamin.

The identification techniques used in this paper have rarely been applied to blood derived from clinical studies. For most of the metabolites of vitamin D2, we were able to analyze the compounds not only by chromatographic mobility with standards but also by diode array spectrophotometry, which indicated that the compounds have the correct chromophore for vitamin D. For the peaks containing tens of nanograms of material, such as 25OHD2 and 24OHD2, this approach is relatively straightforward and provides good confirmation of peak identity. For the analysis of the 1{alpha},25-(OH)2D2 peak, which contains many fewer nanograms of material, we were able to obtain a UV spectrum, albeit of lower quality.

Despite this remarkable sensitivity of the HPLC diode array detector, we were unable to analyze 1{alpha},24-(OH)2D2 by this method, and we, therefore, used a combination of high resolution HPLC and a sensitive RIA. In the sample analyzed in this way, however, 1{alpha},24S-(OH)2D2 was present at only about 10% of the concentration of 1{alpha},25-(OH)2D2. It should be noted that no other known metabolite comigrates with chemically synthesized 1{alpha},24S-(OH)2D2 on this chomatographic system, and extreme care was taken not to confuse this metabolite with closely migrating 1{alpha},25-(OH)2-previtamin D2 or 1{alpha},25-(OH)2-previtamin D3 during collection. As previtamin D/vitamin D equilibrium ratios rarely reach 1/10 in any vitamin D samples, and given that 1{alpha},24-(OH)2D2 values in some cases approached those of 1{alpha},25-(OH)2D2 in our study, and 1{alpha},25-(OH)2D3 levels were usually very low, it seems highly unlikely that the measured fraction simply represents poorly resolved 1{alpha},25-(OH)2-previtamin D2 or 1{alpha},25-(OH)2-previtamin D3. To resolve the question of whether 1{alpha},24-(OH)2D2 is present in human serum after the administration of vitamin D2, we used the highly sensitive technique of high resolution GC-MS of the pertrimethylsilyl ether derivative. Although the concentrations of 1{alpha},24-(OH)2D2 were insufficient to obtain a complete mass spectrum, monitoring three ions [one of which is specific for the 1{alpha},24-(OH)2D2 structure] gave a peak with the appropriate retention time with ion ratios that corresponded with those found in standard 1{alpha},24-(OH)2D2. This technique provides satisfactory specificity to allow the conclusion that 1{alpha},24-(OH)2D2 is present in human serum after the administration of vitamin D2.

Based upon experience with vitamin D3, current dogma in the vitamin D field suggests that 24-hydroxylation of the side-chain only occurs after prior 25-hydroxylation (27). Furthermore, this 24-hydroxylation is renal or target cell based and involves the cytochrome P450, CYP24 (28). Although this may well be true for vitamin D3, it certainly does not appear to be the case for vitamin D2, where a wealth of information gained from animal models (8, 9, 10) suggests that direct 24-hydroxylation of the vitamin D2 side-chain can occur, and 24OHD2 is a significant product. From various clues gathered in these studies, investigators have speculated that 24OHD2 is formed in the liver (12, 29). Further support for 24-hydroxylation by the liver has come from direct in vitro work with hepatoma cell lines, HepG2 and Hep3B, and with the liver cytochome P450, CYP27, transfected into COS-1 cells (17, 18). Thus, it seems that unlike vitamin D3, for which 24-hydroxylation is mainly performed in target cells and not the liver, the 24-hydroxylation of vitamin D2 can occur in the liver. The 1{alpha}-hydroxylation step for 24OHD2, however, appears to be carried out by the same renal enzyme that is involved in the formation of 1{alpha},25-(OH)2D2 and 1{alpha},25-(OH)2D3. The observed pattern of PTH stimulation of the biosynthesis of 1{alpha},24-(OH)2D2 is consistent with this hypothesis, as PTH is a well characterized stimulator of the renal enzyme. Work by Horst et al. (10) in the bovine species also found evidence of the same pathway of 24-hydroxylation followed by 1{alpha}-hydroxylation, which has profound implications when contemplating the clinical use of vitamin D2.

Examination of the pattern of results obtained from patients treated either with one large dose or repeated doses of vitamin D2 suggests that levels of both 24OHD2 and 1{alpha},24-(OH)2D2 are higher in the patients receiving chronic therapy. This would support the theory that the initial 24-hydroxylation of vitamin D2 occurs in the liver, as vitamin absorbed from the diet and entering the circulation in chylomicrons would undergo a type of first pass phenomenon; hence, the concentration of this metabolite would remain high when there was daily oral treatment of the parent vitamin. In contrast, a single large dose would pass through the liver initially on absorption, but thereafter would be largely redistributed into body tissues (30), from where it would be mobilized slowly on the vitamin D-binding protein. The sharp peak of 24OHD2 seen in contrast to the steadily maintained level of 25OHD2 supports this hypothesis. As it is postulated that 1{alpha},24-(OH)2D2 is formed from 24OHD2, then a similar pattern was expected for this metabolite and was seen after the single dose. In contrast, consistently raised levels of 1{alpha},24-(OH)2D2 and 24OHD2 are seen after chronic treatment, e.g. in the XLH patient 17. An additional reason for this is suggested by experiments using the murine model of XLH, which indicate increased renal 24-hydroxylase messenger ribonucleic acid levels and activity (31, 32); it is possible that a hepatic 24-hydroxylase could also be up-regulated. Support for the renal origin of 1{alpha},24-(OH)2D2 is provided by the increase seen in response to PTH infusion, as the renal 1{alpha}-hydroxylase is stimulated by PTH. The data in Table 3Go suggest that serum 1{alpha},24-(OH)2D2 levels may be higher in XLH patients than in those with hypoparathyroidism (who by definition had low PTH levels), all of whom had the same intake of vitamin D2, but unfortunately numbers were too small for a definitive comparison. In general, in chronically treated patients, concentrations of 1{alpha},24-(OH)2D2 seem higher in relation to 1{alpha},25-(OH)2D2 than the ratio of 24OHD2 to 25OHD2 would warrant. This may indicate either that 24OHD2 is more readily 1{alpha}-hydroxylated than 25OHD2 or that the turnover of 1{alpha},24-(OH)2D2 is slower than that of 1{alpha},25-(OH)2D2.

To the best of our knowledge, only one preliminary report has presented evidence for the existence of the alternative monohydroxylated form, 24OHD2, in human plasma samples (11). This lack of corroborative evidence reflects the rigidity of current analytical approaches that focus only on conventional metabolites. Furthermore, the S-isomer of 1{alpha},24-(OH)2D2, which is also the natural isomer formed in hepatomas in vitro (17) and is found in human blood, possesses a biological activity approaching that of 1{alpha},25-(OH)2D3 (15, 17) in vitamin D-dependent transcription assays and cell antiproliferation assays. Clinical vitamin D assay strategies (33), therefore, should be designed to take these new forms of vitamin D2 into account, especially where there is a heavy use of vitamin D2 in the population. A reminder that assay technology should be measuring all of the active forms of the given analog of vitamin D has been sounded of late based on the realization that certain active analogs may give rise to one (or more) biologically active products. Examples of such analogs include F6-1{alpha},25-(OH)3D3 and KH1060, which are not only both active in their own right, but also are converted to other stable and biologically active forms: F6-1{alpha},23,25-(OH)3D3 and 26OHKH1060, respectively (34, 35). Thus, the concept that a vitamin D analog, in this case vitamin D2, gives rise to two biologically active products in the form of 1{alpha},24-(OH)2D2 and 1{alpha},25-(OH)2D2 is not without precedent.

We conclude from these studies that the biological effects of vitamin D2 in humans are different, perhaps only subtly, from those of vitamin D3. Our conclusion builds logically on those of other researchers who have reported different effects of the two vitamins in other mammals (36, 37). It is also consistent with the observed lower toxicity of vitamin D2 (38, 39, 40, 41) and 1{alpha}OHD2 (42, 43, 44, 45, 46). Such a toxicity advantage appears likely for the synthetic analog 1{alpha}OHD2 and for 1{alpha},24-(OH)2D2 over their vitamin D3 equivalents (8, 45, 46) and could make 1{alpha}OHD2 and 1{alpha},24-(OH)2D2 attractive alternatives to the corresponding vitamin D3 analogs for future clinical use. Thus, although vitamin D2 has long been considered as equivalent to its natural homolog, the current studies suggest that it may possess a sufficiently different metabolic pattern so as to offer advantages in its toxicity profile, advantages that may be carried over into the design of future generations of vitamin D analogs, which can be used as clinical therapies.


   Acknowledgments
 
We thank Dr. J. L. Berry for help with the immunoextraction, and Mrs. J. Martin and Mrs. J. Burgess for skilled technical assistance. We are grateful to Nick Ordsmith and Erik Williams of Micromass for allowing us access to an Autospec mass spectrometer.


   Footnotes
 
1 This work was supported by Program Grant 902 6370 from the Medical Research Council, United Kingdom (to E.B.M. and M.D.) and Joint University/Industry Grant UI-11884 from the Medical Research Council of Canada (to G.J.). Back

Received December 12, 1997.

Revised February 3, 1998.

Accepted February 18, 1998.


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 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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