Karen W. Phinney

Development of a Candidate Reference Measurement Procedure for the Determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry

Vitamin D exists in two major forms, vitamin D(3) and vitamin D(2). Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D(3) [25(OH)D(3)] and 25-hydroxyvitamin D(2) [25(OH)D(2)] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) has been developed and critically evaluated. The deuterated compounds 25(OH)D(3)-d(3) and 25(OH)D(2)-d(3) are used as internal standards for 25(OH)D(3) and 25(OH)D(2), respectively. The 25(OH)D(3) and 25(OH)D(2) and their respective labeled internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D(3) and 25(OH)D(2). Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D(3) and 25(OH)D(2) ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D(3) and 25(OH)D(2), respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2-0.6% for 25(OH)D levels >1 ng/g and within 2% for the level of <1 ng/g. Chromatographic separation of 25(OH)D(3) and 25(OH)D(2) from their respective isomers 3-epi-25(OH)D(3) and 3-epi-25(OH)D(2) was achieved. The limit of detection at a signal-to-noise ratio of approximately 3 was 40 pg of 25(OH)D on column (or approximately 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)D(3) and 25(OH)D(2) can be compared and that will serve as a standard of higher order for measurement traceability.

Vitamin D status as an international issue: National surveys and the problem of standardization

Abstract Wide spread variation in measurement results of total 25-hydroxyvitamin D (25(OH)D) confounds international efforts to develop evidence-based clinical guidelines. Accordingly, NIH Office of Dietary Supplements (ODS) in collaboration with CDC National Center for Environmental Health (NCEH), National Institute of Standards and Technology (NIST) and Ghent University established the Vitamin D Standardization Program (VDSP) in November 2010. VDSP objectives include: (1) standardize 25(OH)D concentration measurements in national health surveys around the world, (2) evaluate survey differences, (3) extend standardization efforts to assay manufacturers, and to clinical, commercial, and research laboratories, (4) promote standardization of emerging metabolites of vitamin D status, and (5) enable the use of standardized data in patient care and public health. An interlaboratory comparison study is being conducted to assess measurement variability among current assays. Participants include national health surveys from Australia, Canada, Germany, Ireland, Mexico, South Korea, UK and USA, 15 assay manufacturers, and two external quality assurance programs. CDC will implement a formal laboratory certification program. Standardization activities will use single-donor, fresh-frozen serum collected using the CLSI C37 protocol. Initial assay performance criteria, based on biological variability data, are ≤10 % imprecision and ≤5 % bias in relation to the reference values. An ancillary study on commutability of NIST SRM 972a, external quality assurance testing materials is included. To increase the comparability of existing data from different national surveys, master equations will be developed to facilitate the conversion of already existing national survey data to the NIST-Ghent University reference measurement procedures.

Biomarkers of vitamin B-12 status in NHANES: a roundtable summary

A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.

NHANES Monitoring of Serum 25-Hydroxyvitamin D: A Roundtable Summary

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later.

Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25-hydroxyvitamin D data: a case study of the program's potential for national nutrition and health surveys

BACKGROUND The Vitamin D Standardization Program (VDSP) has developed protocols for standardizing procedures of 25-hydroxyvitamin D [25(OH)D] measurement in National Health/Nutrition Surveys to promote 25(OH)D measurements that are accurate and comparable over time, location, and laboratory procedure to improve public health practice. OBJECTIVE We applied VDSP protocols to existing ELISA-derived serum 25(OH)D data from the Irish National Adult Nutrition Survey (NANS) as a case-study survey and evaluated their effectiveness by comparison of the protocol-projected estimates with those from a reanalysis of survey serums by using liquid chromatography-tandem mass spectrometry (LC-tandem MS). DESIGN The VDSP reference system and protocols were applied to ELISA-based serum 25(OH)D data from the representative NANS sample (n = 1118). A reanalysis of 99 stored serums by using standardized LC-tandem MS and resulting regression equations yielded predicted standardized serum 25(OH)D values, which were then compared with LC-tandem MS reanalyzed values for all serums. RESULTS Year-round prevalence rates for serum 25(OH)D concentrations <30, <40, and <50 nmol/L were 6.5%, 21.9%, and 40.0%, respectively, via original ELISA measurements and 11.4%, 25.3%, and 43.7%, respectively, when VDSP protocols were applied. Differences in estimates at <30- and <40-nmol/L thresholds, but not at the <50-nmol/L threshold, were significant (P < 0.05). A reanalysis of all serums by using LC-tandem MS confirmed prevalence estimates as 11.2%, 27.2%, and 45.0%, respectively. Prevalences of serum 25(OH)D concentrations >125 nmol/L were 1.2%, 0.3%, and 0.6% by means of ELISA, VDSP protocols, and LC-tandem MS, respectively. CONCLUSION VDSP protocols hold a major potential for national nutrition and health surveys in terms of the standardization of serum 25(OH)D data.

Development and Certification of a Standard Reference Material for Vitamin D Metabolites in Human Serum

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Healths Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.

Biomarkers of folate status in NHANES: a roundtable summary

A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Summary of an NIH Workshop to Identify Research Needs to Improve the Monitoring of Iodine Status in the United States and to Inform the DRI

The Office of Dietary Supplements (ODS) at the NIH sponsored a workshop on May 12-13, 2011, to bring together representatives from various NIH institutes and centers as a first step in developing an NIH iodine research initiative. The workshop also provided an opportunity to identify research needs that would inform the dietary reference intakes for iodine, which were last revised in 2001. Iodine is required throughout the life cycle, but pregnant women and infants are the populations most at risk of deficiency, because iodine is required for normal brain development and growth. The CDC monitors iodine status of the population on a regular basis, but the status of the most vulnerable populations remains uncertain. The NIH funds very little investigator-initiated research relevant to iodine and human nutrition, but the ODS has worked for several years with a number of other U.S. government agencies to develop many of the resources needed to conduct iodine research of high quality (e.g., validated analytical methods and reference materials for multiple types of samples). Iodine experts, scientists from several U.S. government agencies, and NIH representatives met for 2 d to identify iodine research needs appropriate to the NIH mission.

Assessing Vitamin D Status: Time for a Rethink?

Vitamin D status is universally assessed by measuring circulating total 25-hydroxyvitamin D [25(OH)D]3, the sum of 25(OH)D3 and 25(OH)D2. However, despite the widespread acceptance of this biomarker of vitamin D status, studies have suggested that measurement of 25(OH)D alone may not be sufficient to understand the relationship between vitamin D exposure and certain health outcomes. As a result, attention has turned to other aspects of vitamin D biology. Approximately 90%–95% of 25(OH)D is tightly bound to a specific α globulin, vitamin D binding protein (VDBP), which is structurally related to serum albumin. Of the remaining 25(OH)D, about 1% is unbound and the rest loosely bound to serum albumin. A current hypothesis is that the free and albumin-bound moieties are responsible for delivering 25(OH)D to the cell, with the VDBP-bound 25(OH)D acting as a reservoir of the metabolite. In humans, common genetic polymorphisms produce 3 major circulating variants of the VDBP (Gc1F, Gc2, and Gc1S), which differ in their affinity for 25(OH)D. The prevalence of these polymorphs has been shown to vary across different ethnicities and populations, with the Gc1F variant generally more common among individuals of African ancestry. In a recent study reported in the New England Journal of Medicine (1), Powe et al. investigated the prevalence of the different VDBP phenotypes (Gc1F, Gc2, and Gc1S) in a cohort of black and white Americans and examined the relationship with 25(OH)D concentrations as well as other parameters. Unlike previous investigations, this study included measurements of the concentration of serum VDBP. In this work, black Americans were found to have a preponderance of the high-affinity Gc1F phenotype, with homozygotes having about half the concentration of total VDBP found in whites, in whom the Gc1S variant predominated. The mean concentration of total 25(OH)D was also lower in blacks …