MON-386 Determining Vitamin D Status: Analytical Variability Between Available Assays

Abstract Clinical interest to evaluate serum 25-hydroxyvitamin D[25(OH)D] to assess Vitamin D status in health risks continue to increase exponentially over the last decade. Variability of 25(OH)D measurements remains controversial despite the international initiative Vitamin D Standardization Program (VDSP) to standardise the assays. The aim of the present study was to examine the correlation of 25(OH)D concentrations measured by different assays. We measured 25(OH)D using the new Abbott Alinity and DiaSorin LiaisonXL chemiluminescence immunoassays against the National Institute of Standards and Technology (NIST)-traceable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The immunoassay method were compared with the LC-MS/MS using Passing-Bablok regression and Bland-Altman analysis. Common 25(OH)D cut-point for classification of vitamin D deficiency was used to compare the different assays. 125 adult serum samples were randomly selected and measured 25(OH)D levels ranged between <10 nmol/L to 290 nmol/L as determined by LC-MS/MS. Compared to the LC-MS/MS, both immunoassays demonstrated strong positive relationship based on Passing-Bablok regression analysis. The results were as follows: Abbott Alinity = 0.85x + 1.29nmol/L, 95% CI: -2.39 to 5.03 (r=0.94); DiaSorin LiaisonXL= 0.74x + 2.54nmol/L, 95% CI: -2.53 to 6.18 (r=0.91). Despite apparent good correlation, the overall mean bias was -12.6% for Abbott Alinity and -24.4% for DiaSorin LiaisonXL assays. A higher percentage of patients were classified as vitamin D deficient (25(OH)D <50 nmol/L) using DiaSorin LiaisonXL (53%) followed by Abbott Alinity (49%), when compared with LC-MS/MS (34%). Using 25(OH)D ≥ 50nmol/L as “adequate” determined by LC-MS/MS method, 22% (18/82) and 28% (23/82) of patients were classified as “deficient” when analysed on Abbott Alinity and DiaSorin LiaisonXL respectively. Clinician should be aware of the inter-method variability among different Vitamin D assays despite standardization efforts. These differences could be due to cross-reactivity with 25(OH)D2 and vitamin D metabolites, such as 24,25(OH)2D and epimeric forms. 3-epi-25(OH)D is not separated chromatographically by the LC-MS/MS method used in this study. Therefore, the negative bias observed might be due to interference from the 3-epi-25(OH)D, whereby clinical significance is uncertain. It is present in low concentrations in adult human serum, 1 but seen in significant amounts in neonatal serum.2 It is advisable to monitor serum 25(OH)D level following treatment in the same laboratory. 1. Lensmeyer G, et al. The C-3 epimer of 25-hydroxyvitamin D3 is present in adult serum. JCEM 2012; 97: 163–8. 2. Singh RJ, et al. C-3 epimers can account for a significant portion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. JCEM 2006; 91: 3055-61.

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Vitamin D Metabolism and Profiling in Veterinary Species

Metabolites ◽

10.3390/metabo10090371 ◽

2020 ◽

Vol 10 (9) ◽

pp. 371 ◽

Cited By ~ 1

Author(s):

Emma A. Hurst ◽

Natalie Z. Homer ◽

Richard J. Mellanby

Keyword(s):

Vitamin D ◽

Companion Animals ◽

Vitamin D Metabolites ◽

Vitamin D Status ◽

Vitamin D Metabolism ◽

Hydroxyvitamin D ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Health And Disease ◽

25 Hydroxyvitamin D

The demand for vitamin D analysis in veterinary species is increasing with the growing knowledge of the extra-skeletal role vitamin D plays in health and disease. The circulating 25-hydroxyvitamin-D (25(OH)D) metabolite is used to assess vitamin D status, and the benefits of analysing other metabolites in the complex vitamin D pathway are being discovered in humans. Profiling of the vitamin D pathway by liquid chromatography tandem mass spectrometry (LC-MS/MS) facilitates simultaneous analysis of multiple metabolites in a single sample and over wide dynamic ranges, and this method is now considered the gold-standard for quantifying vitamin D metabolites. However, very few studies report using LC-MS/MS for the analysis of vitamin D metabolites in veterinary species. Given the complexity of the vitamin D pathway and the similarities in the roles of vitamin D in health and disease between humans and companion animals, there is a clear need to establish a comprehensive, reliable method for veterinary analysis that is comparable to that used in human clinical practice. In this review, we highlight the differences in vitamin D metabolism between veterinary species and the benefits of measuring vitamin D metabolites beyond 25(OH)D. Finally, we discuss the analytical challenges in profiling vitamin D in veterinary species with a focus on LC-MS/MS methods.

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COVID-19 Disease Severity and Death in Relation to Vitamin D Status among SARS-CoV-2-Positive UAE Residents

Nutrients ◽

10.3390/nu13051714 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1714

Author(s):

Habiba AlSafar ◽

William B. Grant ◽

Rafiq Hijazi ◽

Maimunah Uddin ◽

Nawal Alkaabi ◽

...

Keyword(s):

Vitamin D ◽

United Arab Emirates ◽

Blood Levels ◽

Vitamin D Status ◽

Hospital Visit ◽

Serum Samples ◽

Hydroxyvitamin D ◽

Increased Risk ◽

Multiethnic Population ◽

25 Hydroxyvitamin D

Insufficient blood levels of the neurohormone vitamin D are associated with increased risk of COVID-19 severity and mortality. Despite the global rollout of vaccinations and promising preliminary results, the focus remains on additional preventive measures to manage COVID-19. Results conflict on vitamin D’s plausible role in preventing and treating COVID-19. We examined the relation between vitamin D status and COVID-19 severity and mortality among the multiethnic population of the United Arab Emirates. Our observational study used data for 522 participants who tested positive for SARS-CoV-2 at one of the main hospitals in Abu Dhabi and Dubai. Only 464 of those patients were included for data analysis. Demographic and clinical data were retrospectively analyzed. Serum samples immediately drawn at the first hospital visit were used to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations through automated electrochemiluminescence. Levels < 12 ng/mL were significantly associated with higher risk of severe COVID-19 infection and of death. Age was the only other independent risk factor, whereas comorbidities and smoking did not contribute to the outcomes upon adjustment. Sex of patients was not an important predictor for severity or death. Our study is the first conducted in the UAE to measure 25(OH)D levels in SARS-CoV-2-positive patients and confirm the association of levels < 12 ng/mL with COVID-19 severity and mortality.

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Surveillance evaluation of the standardization of assay values for serum total 25-hydroxyvitamin D concentration in Japan

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/0004563218765570 ◽

2018 ◽

Vol 55 (6) ◽

pp. 647-656 ◽

Cited By ~ 2

Author(s):

Hiroshi Ihara ◽

Sachiko Kiuchi ◽

Takayuki Ishige ◽

Motoi Nishimura ◽

Kazuyuki Matsushita ◽

...

Keyword(s):

Vitamin D ◽

Pilot Study ◽

Surveillance Study ◽

Serum Samples ◽

Acceptance Criterion ◽

Average Percentage ◽

Hydroxyvitamin D ◽

Consensus Values ◽

Liquid Chromatography Tandem Mass ◽

25 Hydroxyvitamin D

Background To assess the vitamin D nutritional status, serum total 25-hydroxyvitamin D (25(OH)D) concentration is measured. We used six automated 25(OH)D immunoassays (AIAs) available in Japan and certified by the Vitamin D Standardization Program (VDSP) at the U.S. Center for Disease Control and Prevention to assess the concordance of the assay results. Methods Serum total 25(OH)D concentrations in SRM 972a and 20 serum samples from patients were determined using three liquid chromatography-tandem mass spectrometry (LC-MS/MS) and six AIAs (pilot study), and an additional 110 serum samples were assessed by the six AIAs (surveillance study). The assay bias from the results of LC-MS/MS by Chiba University or consensus values (i.e. average of six AIAs) was estimated using the procedure described in CLSI document EP09-A3. Results LC-MS/MS at Chiba University could completely separate 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3, and the observed values including total 25(OH)D in SRM 972a were all within ±1·SD of the assigned values. All AIAs produced results greater than ±3·SD. In the pilot study, four of the six AIAs had an average percentage bias, as estimated by confidence interval (CI), larger than ±5% (acceptance criterion in CLSI); the bias converged from −6.5% to 3.2% after adjustment by LC-MS/MS. In the surveillance study, 25(OH)D concentrations in AIAs all adjusted to LC-MS/MS converged within ±5% from consensus values. However, some AIAs showed negative or positive bias from the consensus values. Conclusions Current AIAs in Japan continue to lack standardization. Manufacturers should implement quality assurance strategies so that their values more closely align to those of standard reference material 972a.

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Oral vitamin D supplementation at five times the recommended allowance marginally affects serum 25-hydroxyvitamin D concentrations in dogs

Journal of Nutritional Science ◽

10.1017/jns.2016.23 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 14

Author(s):

Lauren R. Young ◽

Robert C. Backus

Keyword(s):

Vitamin D ◽

National Research Council ◽

Vitamin D Supplementation ◽

Oral Dosage ◽

Vitamin D Status ◽

Serum Concentrations ◽

Serum Samples ◽

Hydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Privately Owned

AbstractLittle is known regarding optimal vitamin D status in adult dogs. To date no studies on vitamin D supplementation for improving vitamin D status have been reported for adult dogs. The aims of this study were to identify dogs with low vitamin D status and evaluate an oral dosage of cholecalciferol (D3) for effectiveness in increasing vitamin D status. For this, forty-six privately owned dogs were evaluated. Of the dogs, thirty-three (or 71·7 %) had serum 25-hydroxyvitamin D (25(OH)D) concentrations less than 100 ng/ml, a minimum previously suggested for vitamin D sufficiency in dogs. Subsequently, thirteen dogs were enrolled in a supplementation trial. Dogs were given either a D3supplement (n7; 2·3 µg/kg0·75) or olive oil placebo (n6) daily with food. Serum concentrations of 25(OH)D were determined at weeks 1, 3 and 6, and at the trial end. Only at the trial end (weeks 9–10) was 25(OH)D significantly greater (P= 0·05) in supplementedv.placebo dogs. Serum concentrations of 24R,25-dihydroxycholecalciferol determined at the trial end were about 40 % of that of 25(OH)D3and not significantly different between the groups. Concentrations of parathyroid hormone, ionised Ca, P and creatinine measured in initial and final serum samples indicated supplementation caused no toxicity. We conclude that vitamin D3supplementation at a dosage near the National Research Council recommended safe-upper limit was not effective for rapidly raising serum 25(OH)D concentrations in healthy, adult dogs. Further work is needed in evaluating the metabolism of orally administered D3in dogs before dosing recommendations can be made.

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The Effect of Vitamin D Supplementation on its Metabolism and the Vitamin D Metabolite Ratio

Nutrients ◽

10.3390/nu11102539 ◽

2019 ◽

Vol 11 (10) ◽

pp. 2539 ◽

Cited By ~ 3

Author(s):

Vito Francic ◽

Stan R. Ursem ◽

Niek F. Dirks ◽

Martin H. Keppel ◽

Verena Theiler-Schwetz ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Supplementation ◽

Vitamin D Metabolites ◽

Vitamin D Status ◽

Hydroxyvitamin D ◽

Dihydroxyvitamin D ◽

Correlation And Regression Analysis ◽

Metabolite Ratio ◽

25 Hydroxyvitamin D ◽

Additional Value

25-hydroxyvitamin D (25(OH)D) is commonly measured to assess vitamin D status. Other vitamin D metabolites such as 24,25-dihydroxyvitamin D (24,25(OH)2D) provide additional insights into vitamin D status or metabolism. Earlier studies suggested that the vitamin D metabolite ratio (VMR), calculated as 24,25(OH)2D/25(OH)D, could predict the 25(OH)D increase after vitamin D supplementation. However, the evidence for this additional value is inconclusive. Therefore, our aim was to assess whether the increase in 25(OH)D after supplementation was predicted by the VMR better than baseline 25(OH)D. Plasma samples of 106 individuals (25(OH)D < 75 nmol/L) with hypertension who completed the Styrian Vitamin D Hypertension Trial (NC.T.02136771) were analyzed. Participants received vitamin D (2800 IU daily) or placebo for 8 weeks. The treatment effect (ANCOVA) for 25(OH)D3, 24,25(OH)2D3 and the VMR was 32 nmol/L, 3.3 nmol/L and 0.015 (all p < 0.001), respectively. Baseline 25(OH)D3 and 24,25(OH)2D3 predicted the change in 25(OH)D3 with comparable strength and magnitude. Correlation and regression analysis showed that the VMR did not predict the change in 25(OH)D3. Therefore, our data do not support routine measurement of 24,25(OH)2D3 in order to individually optimize the dosage of vitamin D supplementation. Our data also suggest that activity of 24-hydroxylase increases after vitamin D supplementation.

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Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin D

Clinical Chemistry ◽

10.1373/clinchem.2015.241430 ◽

2015 ◽

Vol 61 (8) ◽

pp. 1033-1048 ◽

Cited By ~ 38

Author(s):

Miriam J Müller ◽

Dietrich A Volmer

Keyword(s):

Mass Spectrometry ◽

Vitamin D ◽

Dynamic Range ◽

Biological Fluids ◽

Vitamin D Metabolites ◽

Hydroxyvitamin D ◽

Low Concentrations ◽

25 Hydroxyvitamin D ◽

And Function ◽

Established Role

Abstract BACKGROUND The frequency of measurements of vitamin D in the human population has significantly increased over the last decade because vitamin D has now been linked to many diseases, in addition to its established role in bone health. Usually, serum 25-hydroxyvitamin D concentrations are measured to assess the vitamin D status of individuals. Unfortunately, many studies investigating links between vitamin D and disease also use only this single metabolite. Intricate correlations with other vitamin D metabolites or dynamic effects of downstream metabolites may therefore be overlooked. Fortunately, powerful LC-MS/MS approaches have recently become available that can simultaneously quantify the concentrations of multiple vitamin D metabolites. These approaches are challenging, however, because of inherent instrumental problems with detection of vitamin D compounds and the low concentrations of the metabolites in biological fluids. CONTENT This review summarizes recent mass spectrometry assays for the quantitative measurement of multiple vitamin D metabolites and their application in clinical research, with a particular focus on the low-abundance downstream metabolic species generated after the initial hydroxylation to 25-hydroxyvitamin D. SUMMARY To study the pathobiological effects and function of vitamin D metabolites in disease, in particular in low-abundance species beyond 25-hydroxyvitamin D, we need to know their concentrations. Although detection of these vitamin D species is challenging, a number of recent mass spectrometry assays have successfully demonstrated that LC-MS/MS methods can quantify multiple vitamin D compounds over a wide dynamic range individually or as part of multimetabolite assays.

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Calcium-Regulating Hormones and Minerals From Birth to 18 Months of Age: A Cross-Sectional Study. I. Effects of Sex, Race, Age, Season, and Diet on Vitamin D Status

PEDIATRICS ◽

10.1542/peds.77.6.883 ◽

1986 ◽

Vol 77 (6) ◽

pp. 883-890

Author(s):

P. Lichtenstein ◽

B. L. Specker ◽

R. C. Tsang ◽

F. Mimouni ◽

C. Gormley

Keyword(s):

Vitamin D ◽

Human Milk ◽

Binding Protein ◽

Vitamin D Metabolites ◽

Vitamin D Status ◽

Vitamin D Binding Protein ◽

Hydroxyvitamin D ◽

Dihydroxyvitamin D ◽

1,25 Dihydroxyvitamin D ◽

25 Hydroxyvitamin D

The influence of sex, race, age, season, and diet (cow's milk formula v human milk) on the vitamin D and vitamin D-binding protein status in infants less than 18 months of age was investigated in this crosssectional, prospective study of 198 infants. No differences by sex were observed in serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D, or vitamin D-binding protein concentrations. By race, black infants had significantly elevated serum 1,25-dihydroxyvitamin D levels relative to white infants. By age, vitamin D-binding protein concentrations increased with increasing age. By season, serum 25-hydroxyvitamin D concentrations were low in winter, whereas 1,25-dihydroxyvitamin D and vitamin D-binding protein were high in winter compared with summer. By diet, formula-fed infants had higher serum concentrations of all measured vitamin D metabolites and vitamin D-binding protein than human milk-fed infants. Thus, race, age, season, and diet exert, individually or in combination, different and significant effects on vitamin D metabolites; these should be considered in assessing infant vitamin D status.

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Current Assays to Determine Free 25-Hydroxyvitamin D in Serum

Journal of AOAC International ◽

10.5740/jaoacint.17-0085 ◽

2017 ◽

Vol 100 (5) ◽

pp. 1323-1327 ◽

Cited By ~ 16

Author(s):

Sofie Malmstroem ◽

Lars Rejnmark ◽

John B Imboden ◽

Dolores M Shoback ◽

Daniel D Bikle

Keyword(s):

Vitamin D ◽

Cell Activity ◽

Vitamin D Metabolites ◽

Vitamin D Status ◽

Vitamin D Binding Protein ◽

Hydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Hydroxylated Metabolite ◽

Reliable Methods ◽

Made In

Abstract The 25-hydroxylated metabolite of vitamin D is the best clinical indicator of vitamin D status. For many years, emphasis has been on measuring total levels of 25-hydroxyvitamin D [25(OH)D], but recently, interest in measuring free 25(OH)D as a potentially better marker of vitamin D status has arisen. Since the 1980s when the first measurements of free 25(OH)Dwere made, little progress has been made in the development of rapid, reliable methods to determine the levels of free 25(OH)D. For many years, assessment of free 25(OH)D relied on calculations using levels of total 25(OH)D, albumin, and vitamin D binding protein (VDBP), for which many assays exist. However, because of vagaries in the measurement of VDBP in particular and the assumption of a constant affinity of VDBP for the vitamin D metabolites (which has been shown to be problematic), calculated values have proved suspect. This changed a few years ago when a new immunoassay was developed to measure free 25(OH)D directly. This review examines methods for determiningfree 25(OH)D, the different methods used in clinicalstudies, and the relationships between free 25(OH)D and other vitamin D metabolites and the physiologic functions affected by vitamin D metabolites, such asbone cell activity and turnover. The review also comments on the value of assessing free 25(OH)D and theefforts to standardize the assays.

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Comparison of Vitamin D and 25-Hydroxyvitamin D Concentrations in Human Breast Milk between 1989 and 2016–2017

Nutrients ◽

10.3390/nu13020573 ◽

2021 ◽

Vol 13 (2) ◽

pp. 573

Author(s):

Naoko Tsugawa ◽

Mayu Nishino ◽

Akiko Kuwabara ◽

Honami Ogasawara ◽

Maya Kamao ◽

...

Keyword(s):

Vitamin D ◽

Breast Milk ◽

Sun Exposure ◽

Stepwise Multiple Regression ◽

Human Breast Milk ◽

Vitamin D Status ◽

Human Breast ◽

Hydroxyvitamin D ◽

Lactating Mothers ◽

25 Hydroxyvitamin D

Background: Breast milk is considered the optimal source of nutrition during infancy. Although the vitamin D concentration in human breast milk is generally considered poor for infants, vitamin D in breast milk is an important source for exclusively breastfed infants. Increases in vitamin D insufficiency and deficiency in lactating mothers may reduce vitamin D concentrations in breast milk. This study aimed to compare vitamin D and 25-hydroxyvitamin D (25OHD) concentrations in breast milk collected in 1989 and 2016–2017 and simultaneously analyze them with liquid chromatography-tandem mass spectrometry (LC-MS/MS); the association between the lifestyle of recent lactating mothers (2016–2017) and vitamin D status in human breast milk was also evaluated. Method: Lactating mothers were recruited from three regions of Japan in 1989 (n = 72) and 2016–2017 (n = 90), and milk from 3–4 months was collected in summer and winter. The samples were strictly sealed and stored at −80℃ until measurement. Breast milk vitamin D and 25OHD concentrations were analyzed by LC-MS/MS. Vitamin D intake, sun exposure, and sunscreen use of the lactating mothers in 2016–2017 were assessed. Results: Both vitamin D and 25OHD concentrations in breast milk were higher in the summer regardless of the survey year. Significantly lower vitamin D and 25OHD concentrations were observed in 2016–2017 compared with 1989 in summer, but no survey year difference was observed in winter. The stepwise multiple regression analyses identified season, daily outdoor activity, and suntan in the last 12 months as independent factors associated with vitamin D3 concentrations. Conclusion: The results suggest that low vitamin D status in recent lactating mothers may have decreased vitamin D and 25OHD concentrations in breast milk compared with the 1980s. These results are helpful for developing public health strategies to improve vitamin D status in lactating mothers and infants.

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Vitamin D Metabolites and Clinical Outcome in Hospitalized COVID-19 Patients

Nutrients ◽

10.3390/nu13072129 ◽

2021 ◽

Vol 13 (7) ◽

pp. 2129

Author(s):

Sieglinde Zelzer ◽

Florian Prüller ◽

Pero Curcic ◽

Zdenka Sloup ◽

Magdalena Holter ◽

...

Keyword(s):

Vitamin D ◽

Degradation Products ◽

University Hospital ◽

Vitamin D Metabolites ◽

Serum Concentrations ◽

Serum Samples ◽

Liquid Chromatography Tandem Mass ◽

Relevant Role ◽

Metabolite Ratio ◽

The University

(1) Background: Vitamin D, a well-established regulator of calcium and phosphate metabolism, also has immune-modulatory functions. An uncontrolled immune response and cytokine storm are tightly linked to fatal courses of COVID-19. The present retrospective study aimed to inves-tigate vitamin D status markers and vitamin D degradation products in a mixed cohort of 148 hospitalized COVID-19 patients with various clinical courses of COVID-19. (2) Methods: The serum concentrations of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 25,26(OH)2D3 were determined by a validated liquid-chromatography tandem mass-spectrometry method in leftover serum samples from 148 COVID-19 patients that were admitted to the University Hospital of the Medical Uni-versity of Graz between April and November 2020. Anthropometric and clinical data, as well as outcomes were obtained from the laboratory and hospital information systems. (3) Results: From the 148 patients, 34 (23%) died within 30 days after admission. The frequency of fatal outcomes did not differ between males and females. Non-survivors were significantly older than survivors, had higher peak concentrations of IL-6 and CRP, and required mechanical ventilation more frequently. The serum concentrations of all vitamin D metabolites and the vitamin D metabolite ratio (VMR) did not differ significantly between survivors and non-survivors. Additionally, the need for res-piratory support was unrelated to the serum concentrations of 25(OH)D vitamin D and the two vitamin D catabolites, as well as the VMR. (4) Conclusion: The present results do not support a relevant role of vitamin D for the course and outcome of COVID-19.

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