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Departments of Laboratory Medicine and Pathology (R.J.S., R.L.T., S.K.G.G.) and Medicine (S.K.G.G.), Mayo Clinic, Rochester, Minnesota 55905; and Epimer, LLC (G.S.R.), Providence, Rhode Island 02906
Address all correspondence and requests for reprints to: Stefan K. G. Grebe, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905. E-mail: grebe.stefan{at}mayo.edu.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD2 or 25OHD3 in human serum, and 2) identify the patient populations that might be affected.
Study Design: We modified our LC-MS/MS method to allow detection of C-3 epimers. We retested specimens from four patient groups with the new method and an extracted RIA: 1) children less than 1 yr old, 2) children 1–18 yr old, 3) adults aged 20–87 yr with liver disease, and 4) adults aged 19–91 yr without liver disease.
Results: In 172 children from group 1 with detectable 25OHD2 or 25OHD3, we identified C-3 epimers in 39 (22.7%). The epimers contributed 8.7–61.1% of the total 25-OHD. There was an inverse relationship between patient age and epimer percentage (r = 0.48; P < 0.002). The RIA gave accurate 25-OHD results that correlated with the modified LC-MS/MS method. No C-3 epimers were detected in any of the other groups.
Conclusions: Significant concentrations of C-3 epimers of 25OHD2 or 25OHD3 are commonly found in infants. This can lead to overestimation of 25-OHD levels. Measurements in children less than 1 yr should therefore be performed with an assay that allows accurate detection of 25-OHD in the presence of its C-3 epimers.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Unfortunately, there are substantial discrepancies between test results obtained with different 25-OHD assays. Most 25-OHD assays are competitive immunoassays or competitive assays based on vitamin D binding proteins (11, 12). For such assays, 25-OHD is a difficult analyte because of its hydrophobicity and relatively low serum concentrations (10, 12). This often necessitates sample extraction and concentration before analysis, potentially increasing assay variability. Furthermore, equal detection of 25OHD2 and 25OHD3 represents a challenge, in particular for assays based on vitamin D binding protein, because binding proteins from many species show higher affinity for 25OHD3 than for 25OHD2 (13). As a consequence of all these factors, only 50–60% of the approximately 100 laboratories that participate in the international quality assessment scheme for vitamin D metabolites (DEQAS), meet performance criteria consistently, and the results obtained for the same sample can differ up to 2- to 4-fold, sometimes even for the same assay, when performed in different laboratories (11).
In an attempt to overcome these problems, we replaced our previous 25-OHD extracted RIA with a candidate reference method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) (14, 15). The method uses deuterated 25OHD3 as an internal standard in each sample, thereby enabling correction of extraction variability and allowing accurate, separate measurement of 25OHD2 and 25OHD3. In our assay validation, it agreed well with our previous RIA, which some authors have considered a gold standard (Fig. 1
) (12, 16).
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) (17, 18, 19, 20, 21). Furthermore, 3-epi-1,25-OHD has some unique properties of potential clinical significance. It is nearly as potent as 1,25-OHD in suppressing PTH secretion (22, 23) but has significantly reduced calcemic effects (24, 25). Distinguishing between 25-OHD and 3-epi-25-OHD could therefore be of relevance. The C-3 epimers can be converted to their respective 3-epi-1,25-OHD metabolites, and their presence might also be an indicator of elevated C-3 epimerization at the level of 1,25-OHD synthesis.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Our study aims were to 1) determine the prevalence of detectable 3-epi-25OHD2 or 3-epi-25OHD3 in human serum, and 2) determine the patient populations that might be affected.
The samples for this study were consecutive waste specimens from routine testing received in our laboratory, selected based only on age and, for adult samples, the likely presence or absence of hepatic dysfunction. They included samples from four groups of subjects: group 1, children less than 1 yr old (n = 183; 116 males and 67 females); group 2, children 1–18 yr old (n = 47; 19 males and 28 females); group 3, adults aged 20–87 yr with liver disease (hepatoma surveillance patients with elevated serum 
-fetoprotein concentrations; n = 53; 25 males and 28 females); and group 4, adults 19–91 yr of age without biochemical evidence of liver disease (n = 147; 35 males and 112 females).
Standards/calibrators, internal standard, and controls
25OHD2 and 25OHD3 standards/calibrators and 25OHD3-d6 internal standard were purchased from Sigma Chemical Co. (St. Louis, MO) and As Vitas (Oslo, Norway), respectively. Manufacturer-stated concentrations were confirmed by UV spectrophotometry. The 3-epi-25OHD3 standards were prepared and verified by one of the authors (G.S.R.) as described previously (21). Stock solutions of all standards were stored at –20 C.
Three pools of control samples each were prepared for 25OHD2 and 25OHD3. Control target values of 25OHD2 were established at 4.2, 17, 42, and 110 ng/ml (10.2, 41.3, 101.9, and 267 nmol/liter). Target values of 25OHD3 were established at 1.7, 24, 55, and 132 ng/ml (4.25, 60, 137.5, and 330 nmol/liter).
Sample preparation
Twenty-five microliters of working internal standard at a concentration of 200 ng/ml (492.6 nmol/liter) were added to each 200 µl of patient samples, controls, or calibrators. The mixtures were incubated for 15 min at room temperature to allow the internal standard to equilibrate with binding proteins. Proteins were precipitated by addition of 200 µl of acetonitrile and centrifugation. The supernatant was transferred to 96-well plates for analysis.
LC-MS/MS
For the standard 25-OHD method, online extraction and HPLC chromatography of the supernatants were performed using a TX4 Turbo Flow system (Cohesive Technologies, Franklin, MA) with 1.0 x 50 mm Cyclone extraction columns and 3.3 cm x 4.6 mm, 3-µm LC-18 (Supelco, St. Louis, MO) analytical columns. After online extraction, the analytes were eluted onto the analytical column for 90 sec with a mobile phase of 39.5% vol/vol methanol, 0.005% vol/vol formic acid. There was a step gradient to 87% vol/vol methanol, 0.005% vol/vol formic acid for the analytical column. The analytes then entered an API 4000 triple-quadrupole mass spectrometer (ABI-Sciex, Toronto, Canada) and were ionized in an atmospheric-pressure chemical-ionization source and detected by multiple reaction monitoring of the following ion pairs: m/z 413.0/395.3 for 25OHD2, m/z 401.4/383.3 for 25OHD3, and m/z 407.4/389.5 for 25OHD3-d6. The raw signals of 25OHD2 and 25OHD3 in the calibrators, controls, and samples were normalized to their respective internal standard 25OHD3-d6 signals, and concentrations in the samples and controls were calculated off the normalized six-point calibration curves [0–200 ng/ml (0–500 nmol/liter)]. Samples with concentrations that exceeded the highest calibrator were diluted and run again. The total 25-OHD concentrations of each control and sample were calculated by summing the measured values of 25OHD2 and 25OHD3.
For separation of epimers, the standard LC-18 column was replaced with a longer 5-dinitrobenzoyl-(R)-phenylglycine column (Chirex-PGLY and DNB 250 x 4.6 mm; Phenomenex, Torrance, CA) and 100 µl of the supernatant was injected. The step gradient extends only up to 67% vol/vol methanol, 0.005% vol/vol formic acid at an analytical column flow rate of 0.9 ml/min. The mass spectrometer settings remained unchanged. The concentrations of 25OHD2, 25OHD3, and 25-OHD were calculated as above. The concentrations of any detected C-3 epimers of 25OHD2 or 25OHD3 were also calculated off the normalized 25OHD2 and 25OHD3 calibration curves, and the total 3-epi-25-OHD concentration is the sum of 3-epi-25OHD2 and 3-epi-25OHD3 concentrations.
Assay performance parameters
The performance of the two LC-MS/MS methods was similar for 25OHD2 and 25OHD3. Interassay coefficients of variation (CV) for the 25OHD2 controls were 14, 5, 7, and 6%, respectively. Corresponding interassay CV for 25OHD3 controls were 13, 8, 8, and 6%. The recovery of analyte spiked into patient samples was 82–115% (mean, 102%) of predicted for 25OHD2 and 88–115% (mean, 103%) for 25OHD3. The 25-OHD concentrations obtained by the two LC-MS/MS methods matched each other closely (Fig. 3).
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In the 25-OHD RIA (Diasorin, Stillwater, MN), samples were extracted with acetonitrile, followed by a competitive RIA. According to the Diasorin packet insert, the RIA uses 125I-labeled 25OHD3 and a goat polyclonal antibody specific to the 25-OH-containing side chain; in the original published assay description, a rabbit-derived antibody was used (26, 27). A secondary antibody against the primary antibody was used as a precipitating reagent.
Interassay CV for this assay range from 8.5–14.4% across the reportable range. The assay compares closely with the LC-MS/MS standard method (Fig. 1
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Testing of study samples
All study samples were assayed by the standard and the modified LC-MS/MS method. In addition, all samples with total 25-OHD concentrations more than 100 ng/ml (250 nmol/liter) underwent confirmatory retesting. Chromatograms obtained with the modified method were inspected for the presence of C-3 epimer peaks. All detectable 25OHD2, 25OHD3, 3-epi-25OHD2, and 3-epi-25OHD3 peaks were quantitated, as described above, and the total 25-OHD and 3-epi-25-OHD concentrations were calculated. For every sample with detectable C-3 epimer peaks, we calculated the percent contribution of total 3-epi-25-OHD to the corresponding total 25-OHD concentration.
We also assayed all samples with epimer peaks and sufficient residual sample volume with the Diasorin RIA. The results were compared with the total 25-OHD values obtained by the modified LC-MS/MS assay.
Data analysis
The frequencies of occurrences of C-3 epimers were tabulated for the different study groups and compared with each other by 
2-analysis, using appropriate degrees of freedom. In each group, 2 testing was also used to compare the likelihood for the presence of C-3 epimer peaks depending on whether patients had detectable 25OHD2 or 25OHD3 as well as to determine whether there was a relationship between patient gender and the presence of detectable C-3 epimers in any of the groups. Yates correction was used for all single degree of freedom 2 tests.
Regression analysis and ANOVA, respectively, were applied to all cases with detectable C-3 epimers to determine whether there was any relationship between the percent contribution of the C-3 epimers to the total 25-OHD values and the patient’s age or gender. Statistical comparisons between different analytical methods were performed using Passing-Bablock linear regression.
For all statistical tests, a P value of <0.05 was considered significant, corrected if necessary for degrees of freedom or multiple comparisons.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We found 3-epi-25OHD2 (n = 2) or 3-epi-25OHD3 (n = 38) in 39 of the 172 children (22.7%) with detectable 25OHD2 or 25OHD3 (Fig. 4). All subjects with detectable C-3 epimers also had detectable 25OHD2 or 25OHD3. Among the 39 epimer-positive children, 31 had only detectable 25OHD3, one had only detectable 25OHD2, and seven had detectable 25OHD2 and 25OHD3. Of the eight children in this subgroup with detectable 25OHD2, two, including the one who only had 25OHD2, had 3-epi-25OHD2 peaks. 3-Epi-25OHD3 was detected in all 38 children with detectable 25OHD3 peaks.
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There was sufficient sample volume for RIA testing in 34 of the 39 children with detectable 3-epi-25OHD2 or 3-epi-25OHD3. The standard LC-MS/MS method and the Diasorin RIA were in good agreement in this group with a Passing-Bablock regression slope of 1.059, an intercept of –5.8, and a correlation coefficient of 0.8.
There was a greater likelihood of detectable 3-epi-25OHD3 than 3-epi-25OHD2, with 38 of 163 25OHD3-positive individuals having 3-epi-25OHD3 peaks, whereas only two of 39 patients with detectable 25OHD2 showed 3-epi-25OHD2 peaks [odds ratio = 5.62; confidence interval (CI) = 1.24–35.40; 2 = 5.46; P < 0.02]. In the patients with detectable C-3 epimer peaks, regression analysis revealed an inverse relationship between patient age and percentage of total 3-epi-25-OHD detected, with a linear fit resulting in a correlation coefficient 0.48 (P < 0.002), whereas an exponential decay function fit resulted in a marginally higher correlation coefficient of 0.49 (P < 0.007) (Fig. 5).
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In group 2 (children 1–18 yr of age), total 25-OHD concentrations were 0–58 ng/ml [0–145 nmol/liter; median, 34 ng/ml (85 nmol/liter); mean, 33 ng/ml (82.5 nmol/liter)]. Thirty-nine children had only detectable 25OHD3, six had detectable 25OHD2 and 25OHD3, one had only 25OHD2, and one had neither 25OHD2 nor 25OHD3. We did not detect any C-3 epimer peaks in any of these 47 older children.
Total 25-OHD concentrations in group 3 (adults with compromised liver function, 20–87 yr old) were 0–57 ng/ml [0–142.5 nmol/liter; median, 17 ng/ml (42.5 nmol/liter); mean, 19 ng/ml (47.5 nmol/liter)]. Three patients had no detectable circulating 25-OHD, 39 had detectable 25OHD3, 10 had detectable 25OHD2 and 25OHD3, and one had only 25OHD2. No patient had any detectable C-3 epimer peaks.
In group 4, the 147 adults aged 19–91 yr, without known liver disease, total 25-OHD levels were 4–330 ng/ml [10–825 nmol/liter; median, 28.5 ng/ml (71.2 nmol/liter); mean, 31.3 ng/ml (78.2 nmol/liter)]. All patients had detectable 25OHD3, and 47 also had detectable 25OHD2. No C-3 epimer peaks were detected.
The differences in C-3 epimer detection rates between the four groups were highly significant with an overall 2 (three degrees of freedom) of 60.81 (P < 0.00001), whereas individual paired comparisons of group 1 against groups 2, 3, and 4 yielded 2 values of 11.21 (P < 0.00082; multicomparison corrected P < 0.00246), 12.23 (P < 0.00047; multicomparison corrected P < 0.00141), and 35.89 (P < 0.00001; multicomparison corrected P < 0.00003), respectively. There were no significant differences between groups 2, 3, and 4.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Within the limitations of our study, the phenomenon seems to be confined to children under the age of 1 yr. Furthermore, within this group, it is inversely correlated with age (Fig. 5). This suggests that high rates of C-3 epimerization might be a function of immaturity of vitamin D metabolism. It also suggests that C-3 epimerization could be a major metabolic pathway for 25-OHD under certain circumstances. In fact, recent in vitro studies have hinted at the possibility that C-3 epimerization of 25-OHD could play an equal or more important role than epimerization of 1,25-OHD. Microsomal enzyme systems from a variety of cell lines show greater specificity and substrate conversion rates for the C-3 epimerization of 25-OHD than for the corresponding conversion of 1,25-OHD (28). However, the exact nature of the conditions, which might favor C-3 epimerization of 25-OHD, remains to be determined. Our studies seem to exclude deranged hepatic metabolism, despite the fact that hepatic microsomal cytochrome enzymes are known to play a major role in vitamin D metabolism and the associated clinical observation that liver patients often have biochemical evidence of disturbed vitamin D metabolism. This is consistent with in vitro experiments that have failed to identify the enzyme involved in C-3 epimerization of vitamin D metabolites among a group of known enzymes in the vitamin D pathway, including CYP24, CYP27A1, CYP27B1, and 3(-ß)-hydroxysteroid epimerase (28). It therefore appears that although microsomal enzyme systems seem to play a role in C-3 epimerization of vitamin D metabolites, the actual enzymes involved are distinct from the classical hepatic enzyme systems of vitamin D metabolism.
The immediate clinical consequence of our findings lies in the potential for inaccurate measurement of 25-OHD in young children. In our laboratory, we have changed our practice based on this study. We now use the alternative LC-MS/MS method for all 25-OHD measurements in children under the age of 1 yr. It is interesting to speculate how other LC-MS/MS or HPLC-UV detection methods in other laboratories might be performing with regard to C-3 epimer separation. Similarly, for the most part, it remains to be determined whether the various assays based on vitamin D binding protein and immunoassays are able to distinguish 25-OHD from its C-3 epimers. Cross-reactivity of 25-OHD assays with one or several of the over 40 known natural vitamin D metabolites is common (10, 29). For example, the Diasorin RIA cross-reacts with 24,25-(OH)2D3, 25,26-(OH)2D3, and 25OHD3-26,23-lactone (26, 27). Traditionally, these and similar cross-reactivities in other 25-OHD assays have been regarded as clinically irrelevant because the serum concentrations of the cross-reactants are between one and two orders of magnitude lower than those of 25-OHD. However, as we have shown, 3-epi-25OHD2 and 3-epi-25OHD3 concentrations are much higher and could represent a relevant interference. Reassuringly, the Diasorin extracted RIA does not appear to cross-react with 3-epi-25OHD2 or 3-epi-25OHD3, giving accurate total 25-OHD results that correlate well with our modified LC-MS/MS method. However, we did not study any of the other 25-OHD assays that are currently in clinical use in the United States or Europe, and some of these might display cross-reactivity with 3-epi-25OHD2 or 3-epi-25OHD3.
Another issue that needs to be discussed is whether distinguishing between 25-OHD and its C-3 epimers is of clinical importance. Although the phenomenon is fairly prevalent among young infants and significant concentrations of 3-epi-25-OHD are found in affected children, the biological consequences depend on whether 25-OHD and its C-3 epimers differ in their physiological effects. This question can be separated into two parts. Because 25-OHD is a prohormone that needs to be converted into 1,25-OHD, we need to determine first whether 3-epi-25-OHD is converted to 3-epi-1,25-OHD and then to dissect the differential biological effects of 1,25-OHD vs. 3-epi-1,25-OHD.
With regard to 3-epi-25-OHD conversion to downstream metabolites, the literature indicates that it is a substrate for 1-hydroxylase and is converted into 3-epi-1,25-OHD (21). In addition, the conditions that favor C-3 epimerization of 25-OHD probably also favor the same metabolic pathway for 1,25-OHD, possibly involving extrarenal tissues (18, 19, 20, 21, 30, 31) (Fig. 2). It therefore seems highly probable that children with detectable 3-epi-25-OHD might also have 3-epi-1,25-OHD. Final proof, however, will have to await the development of a method for measurement of serum 3-epi-1,25-OHD.
The issue of the bioactivity of 1,25-OHD vs. that of 3-epi-1,25-OHD is more complex. 3-Epi-1,25-OHD can stimulate gene transcription through the vitamin D receptor (VDR) despite the fact that it appears to have weaker binding affinity to the VDR than 1,25-OHD (32, 33). The lower receptor binding affinity does not translate into universally reduced biological effects of 3-epi-1,25-OHD in all vitamin-D-responsive tissues. The transcriptional, as well as the ultimate physiological, response to 3-epi-1,25-OHD has been found to be highly variable for different VDR-regulated genes in different tissues (20, 22, 23, 25, 30, 31, 34, 35). These differences may in part relate to the longer half-life of 3-epi-1,25-OHD (30) but could also reflect selective partial agonistic-antagonist effects, such as have been described for a number of synthetic vitamin D analogs (36). Potential examples of apparently contradictory biological effects include the C-3 epimers’ reduced calcemic properties and less potent gene-regulatory effects on some VDR-responsive genes involved in bone metabolism, such as osteocalcin (18, 24, 25, 34), whereas, on the other hand, suppression of gene transcription of PTH, antiproliferative effects in epithelial cells, and induction of surfactant gene transcription in pulmonary type II alveolar cells are comparable to 1,25-OHD (22, 23, 30, 35).
In conclusion, significant serum concentrations of 3-epi-25-OHD are commonly found in infants. Although the biological consequences of this phenomenon remain uncertain, in clinical practice, it can lead to overestimation of serum 25-OHD levels. Because the calcemic effects of the active downstream metabolite 3-epi-1,25-OHD are low, this might result in inappropriate reduction or omission of 25-OHD treatment in some children or unjustified anxiety about possible 25-OHD overdosing or toxicity in other children. Serum 25-OHD in children below the age of one should therefore be measured with an assay that either does not cross-react with 3-epi-25-OHD or allows unequivocal separation of 3-epi-25-OHD from 25-OHD. Currently, the only assays that we have verified to fulfill these requirements are our modified LC-MS/MS assay and the extracted Diasorin RIA. Laboratories that use other assays should evaluate whether their assays measure 25-OHD accurately in the presence of 3-epi-25-OHD.
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Acknowledgments |
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Footnotes |
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First Published Online May 23, 2006
Abbreviations: CV, Coefficients of variation; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 25-OHD, 25-hydroxyvitamin D; VDR, vitamin D receptor.
Received April 3, 2006.
Accepted May 17, 2006.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1,25-dihydroxy-3epi-cholecalciferol. Exp Cell Res 241:194–201[CrossRef][Medline],25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed. Bone 24:457–463[Medline],25-Dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1,25-dihydroxyvitamin D3 in rats. FEBS Lett 448:278–282[CrossRef][Medline] or C-24 hydroxylation. J Biol Chem 279:15897–15907,25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem 73:106–113[CrossRef][Medline]-hydroxy-3-epi-vitamin D3, a potent suppressor of parathyroid hormone secretion. J Cell Biochem 96:569–578[CrossRef][Medline],25-(OH)2-Vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys 329:228–234[CrossRef][Medline],25-Dihydroxy-3-epi-vitamin D3 a physiological metabolite of 1,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes. Mol Cell Endocrinol 170:91–101[CrossRef][Medline],25-dihydroxyvitamin D3 and its C-3 epimer 1,25-dihydroxy-3-epi-vitamin D3 in neonatal human keratinocytes. Steroids 66:441–450[CrossRef][Medline],25-dihydroxyvitamin D3 retain biologic activity mediated through the vitamin D receptor. J Cell Biochem 78:112–120[CrossRef][Medline],25 dihydroxyvitamin D3 stimulate transcription through the vitamin D receptor. J Steroid Biochem Mol Biol 72:29–34[CrossRef][Medline],25-dihydroxyvitamin D3: evaluation of actions in bone. Steroids 66:347–355[CrossRef][Medline],25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells. Mol Genet Metab 76:46–56[CrossRef][Medline]This article has been cited by other articles:
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  R. Sakurai, E. Shin, S. Fonseca, T. Sakurai, A. A. Litonjua, S. T. Weiss, J. S. Torday, and V. K. Rehan 1{alpha},25(OH)2D3 and its 3-epimer promote rat lung alveolar epithelial-mesenchymal interactions and inhibit lipofibroblast apoptosis Am J Physiol Lung Cell Mol Physiol, September 1, 2009; 297(3): L496 - L505. [Abstract] [Full Text] [PDF]
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E. Orwoll, C. M. Nielson, L. M. Marshall, L. Lambert, K. F. Holton, A. R. Hoffman, E. Barrett-Connor, J. M. Shikany, T. Dam, J. A. Cauley, et al. Vitamin D Deficiency in Older Men J. Clin. Endocrinol. Metab., April 1, 2009; 94(4): 1214 - 1222. [Abstract] [Full Text] [PDF]
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