Rosemary L. Schleicher

Serum 25-hydroxyvitamin D status of the US population: 1988–1994 compared with 2000–2004

BACKGROUND Changes in serum 25-hydroxyvitamin D [25(OH)D] concentrations in the US population have not been described. OBJECTIVE We used data from the National Health and Nutrition Examination Surveys (NHANES) to compare serum 25(OH)D concentrations in the US population in 2000-2004 with those in 1988-1994 and to identify contributing factors. DESIGN Serum 25(OH)D was measured with a radioimmunoassay kit in 20 289 participants in NHANES 2000-2004 and in 18 158 participants in NHANES III (1988-1994). Body mass index (BMI) was calculated from measured height and weight. Milk intake and sun protection were assessed by questionnaire. Assay differences were assessed by re-analyzing 150 stored serum specimens from NHANES III with the current assay. RESULTS Age-adjusted mean serum 25(OH)D concentrations were 5-20 nmol/L lower in NHANES 2000-2004 than in NHANES III. After adjustment for assay shifts, age-adjusted means in NHANES 2000-2004 remained significantly lower (by 5-9 nmol/L) in most males, but not in most females. In a study subsample, adjustment for the confounding effects of assay differences changed mean serum 25(OH)D concentrations by approximately 10 nmol/L, and adjustment for changes in the factors likely related to real changes in vitamin D status (ie, BMI, milk intake, and sun protection) changed mean serum 25(OH)D concentrations by 1-1.6 nmol/L. CONCLUSIONS Overall, mean serum 25(OH)D was lower in 2000-2004 than 1988-1994. Assay changes unrelated to changes in vitamin D status accounted for much of the difference in most population groups. In an adult subgroup, combined changes in BMI, milk intake, and sun protection appeared to contribute to a real decline in vitamin D status.

Case-control study of an acute aflatoxicosis outbreak, Kenya, 2004

Objectives: During January–June 2004, an aflatoxicosis outbreak in eastern Kenya resulted in 317 cases and 125 deaths. We conducted a case–control study to identify risk factors for contamination of implicated maize and, for the first time, quantitated biomarkers associated with acute aflatoxicosis. Design: We administered questionnaires regarding maize storage and consumption and obtained maize and blood samples from participants. Participants: We recruited 40 case-patients with aflatoxicosis and 80 randomly selected controls to participate in this study. Evaluations/Measurements: We analyzed maize for total aflatoxins and serum for aflatoxin B1–lysine albumin adducts and hepatitis B surface antigen. We used regression and survival analyses to explore the relationship between aflatoxins, maize consumption, hepatitis B surface antigen, and case status. Results: Homegrown (not commercial) maize kernels from case households had higher concentrations of aflatoxins than did kernels from control households [geometric mean (GM) = 354.53 ppb vs. 44.14 ppb; p = 0.04]. Serum adduct concentrations were associated with time from jaundice to death [adjusted hazard ratio = 1.3; 95% confidence interval (CI), 1.04–1.6]. Case patients had positive hepatitis B titers [odds ratio (OR) = 9.8; 95% CI, 1.5–63.1] more often than controls. Case patients stored wet maize (OR = 3.5; 95% CI, 1.2–10.3) inside their homes (OR = 12.0; 95% CI, 1.5–95.7) rather than in granaries more often than did controls. Conclusion: Aflatoxin concentrations in maize, serum aflatoxin B1–lysine adduct concentrations, and positive hepatitis B surface antigen titers were all associated with case status. Relevance: The novel methods and risk factors described may help health officials prevent future outbreaks of aflatoxicosis.

Serum vitamin C and the prevalence of vitamin C deficiency in the United States: 2003–2004 National Health and Nutrition Examination Survey (NHANES)

BACKGROUND Vitamin C (ascorbic acid) may be the most important water-soluble antioxidant in human plasma. In the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994), approximately 13% of the US population was vitamin C deficient (serum concentrations <11.4 micromol/L). OBJECTIVE The aim was to determine the most current distribution of serum vitamin C concentrations in the United States and the prevalence of deficiency in selected subgroups. DESIGN Serum concentrations of total vitamin C were measured in 7277 noninstitutionalized civilians aged > or =6 y during the cross-sectional, nationally representative NHANES 2003-2004. The prevalence of deficiency was compared with results from NHANES III. RESULTS The overall age-adjusted mean from the square-root transformed (SM) concentration was 51.4 micromol/L (95% CI: 48.4, 54.6). The highest concentrations were found in children and older persons. Within each race-ethnic group, women had higher concentrations than did men (P < 0.05). Mean concentrations of adult smokers were one-third lower than those of nonsmokers (SM: 35.2 compared with 50.7 micromol/L and 38.6 compared with 58.0 micromol/L in men and women, respectively). The overall prevalence (+/-SE) of age-adjusted vitamin C deficiency was 7.1 +/- 0.9%. Mean vitamin C concentrations increased (P < 0.05) and the prevalence of vitamin C deficiency decreased (P < 0.01) with increasing socioeconomic status. Recent vitamin C supplement use or adequate dietary intake decreased the risk of vitamin C deficiency (P < 0.05). CONCLUSIONS In NHANES 2003-2004, vitamin C status improved, and the prevalence of vitamin C deficiency was significantly lower than that during NHANES III, but smokers and low-income persons were among those at increased risk of deficiency.

Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method

BACKGROUND Measurement of vitamin D molecules are important in the management of patients with bone disease. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 25OHD(3) and 25OHD(2) in human serum and compared it to the traditionally used DiaSorin radioimmunoassay (RIA). METHODS Serum samples (200 microl) were treated with acetonitrile and centrifuged to remove protein. An online solid-phase extraction procedure was used. Calibration solutions (5-100 ng/ml) of 25OHD(2) and 25OHD(3) were prepared using 4% albumin in phosphate-buffered saline. Chromatography: C18 column, isocratic ethanol/water (83/17, v/v). Mass spectrometry system: atmospheric pressure chemical ionization in the positive ion mode. Transitions: 25OHD(3), m/z 401.4-->383.4; 25OHD(2), m/z 413.4-->395.4; and the internal standard hexadeuterated-25OHD(3), m/z 407.7-->389.7. RESULTS Detection limits were 0.49 ng/ml (25OHD(3)) and 1.86 ng/ml (25OHD(2)). Intra- and inter-assay coefficients of variation (CV) were <7% and <11%, respectively, for 25OHD(3) and <9% and <16%, respectively, for 25OHD(2). Recovery averaged (SD) 99% (2%) for 25OHD(3) and 95% (0.8%) for 25OHD(2). In a method comparison of 551 specimens from the National Health and Nutrition Examination Survey, the LC-MS/MS method gave values that were on average 13% higher (95%CI: 11-15%) than RIA results. CONCLUSIONS This high throughput candidate reference method requires minimal sample preparation and is suitable for routine use for analysis of vitamin D status.

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.

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.

Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum.

BACKGROUND An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D(2) (25OHD(2)), 25-hydroxyvitamin D(3) (25OHD(3)) and the C-3 epimer of 25OHD(3) (epi-25OHD(3)) in human serum. METHODS Tri- and hexa-deuterated internal standards were added to serum (100 μl) to monitor recovery. Liquid-liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8-100 nmol/L 25OHD(2,) 12-150 nmol/L 25OHD(3), and 4-50 nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2), m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3. RESULTS Recovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20 nmol/l. Bias averaged <5%. Detection limits were <5 nmol/l. Median (nmol/l) 25OHD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (

Is there a reverse J-shaped association between 25-hydroxyvitamin D and all-cause mortality? Results from the U.S. nationally representative NHANES.

CONTEXT A reverse J-shaped association between serum 25-hydroxyvitamin D (25[OH]D) concentration and all-cause mortality was suggested in a 9-year follow-up (1991-2000) analysis of the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994). OBJECTIVE Our objective was to repeat the analyses with 6 years additional follow-up to evaluate whether the association persists through 15 years of follow-up. PARTICIPANTS The study included 15 099 participants aged ≥ 20 years with 3784 deaths. MAIN OUTCOME MEASURE Relative risk (RR) of death from all causes was adjusted for age, sex, race/ethnicity, and season using 2 Poisson regression approaches: traditional categorical and cubic splines. Results were given for 9 25(OH)D levels: <20, 20 to 29, 30 to 39, 40 to 49, 50 to 59, 60 to 74, 75 to 99 (reference), 100 to 119, and ≥ 120 nmol/L. RESULTS The reverse J-shaped association became stronger with longer follow-up and was not affected by excluding deaths within the first 3 years of follow-up. Similar results were found from both statistical approaches for levels <20 through 119 nmol/L. Adjusted RR (95% confidence interval [CI]) estimates for all levels <60 nmol/L were significantly >1 compared with the reference group. The nadir of risk was 81 nmol/L (95% CI, 73-90 nmol/L). For 25(OH)D ≥ 120 nmol/L, results (RR, 95% CI) were slightly different using traditional categorical (1.5, 1.02-2.3) and cubic splines approaches (1.2, 0.9-1.4). The association appeared in men, women, adults ages 20 to 64 years, and non-Hispanic whites but was weaker in older adults. The study was too small to evaluate the association in non-Hispanic black and Mexican-American adults. CONCLUSIONS A reverse J-shaped association between serum 25(OH)D and all-cause mortality appears to be real. It is uncertain whether the association is causal.

25-hydroxyvitamin D status of healthy, low-income, minority children in Atlanta, Georgia.

OBJECTIVES: The goals were to determine the prevalence of vitamin D deficiency among minority children in a southern US city, to examine differences in serum 25-hydroxyvitamin D levels between non-Hispanic black and Hispanic children, and to determine dietary sources of vitamin D. METHODS: Low-income, minority children (N = 290; mean age: 2.5 ± 1.2 years) were recruited during well-child clinic visits. Serum 25-hydroxyvitamin D and calcium levels were measured and dietary information was assessed. RESULTS: The mean 25-hydroxyvitamin D3 level was 26.2 ± 7.6 ng/mL, whereas 25-hydroxyvitamin D2 was not detected. Overall, 22.3% of children had deficient serum 25-hydroxyvitamin D3 levels (≤20 ng/mL), 73.6% had less-than-optimal serum 25-hydroxyvitamin D levels (≤30 ng/mL), and 1.4% had low serum calcium levels (≤9 mg/dL). A significantly larger proportion of non-Hispanic black children, compared with Hispanic children, had vitamin D deficiency (26% vs 18%; P < .05). Age and season of recruitment were significantly associated with vitamin D deficiency and low serum calcium levels. Older children (≥3 years) were less likely to have vitamin D deficiency (odds ratio [OR]: 0.89 [95% confidence interval [CI]: 0.81–0.96]; P < .001). Study enrollment during spring and summer reduced the likelihood of vitamin D deficiency by ∼20% (spring, OR: 0.85 [95% CI: 0.73–0.98]; P = .03; summer, OR: 0.82 [95% CI: 0.73–0.92]; P < .01). Fortified milk provided most dietary vitamin D (62%), with Hispanic children reporting greater intake. CONCLUSIONS: Suboptimal vitamin D status was common among apparently healthy, low-income, minority children. Age and season were significant predictors of vitamin D deficiency.

Effects of Delayed Sample Processing and Freezing on Serum Concentrations of Selected Nutritional Indicators

BACKGROUND Environmental conditions during sample processing, shipping, and storage are often suboptimal, particularly in less developed countries. We used samples from US volunteers to investigate the effects of delayed whole blood (WB) processing and delayed freezing of serum on selected nutritional indicators. METHODS WB tubes (n = 35) were either stored at 32 degrees C for up to 3 days before serum separation or centrifuged within 2 h of collection; serum samples were stored at 11 degrees C for up to 14 days to simulate delayed shipping. We assessed analyte stability by comparing results with data from optimally prepared/stored serum samples (<2 h on the clot, frozen at -70 degrees C) and by using clinical-acceptability criteria based on combined analytical imprecision and intraindividual biologic variability. RESULTS Clinically acceptable changes in concentration varied from 3%-15%. Delayed WB processing did not unacceptably affect concentrations of carotenoids and vitamins B(12), D, and E; however, we obtained clinically unacceptable changes for ferritin (+9%), soluble transferrin receptor (sTfR) (+5%), and folate (-30%) after 1 day, and for vitamin A (-10%) after 3 days. Delayed freezing of serum did not affect concentrations of ferritin, sTfR, carotenoids, and vitamins A, B(12), and E; however, we obtained clinically unacceptable changes for vitamins C (-20%) and D (+7%) after 7 days and for folate after 14 days (-22%). CONCLUSIONS Despite substantial delays in WB processing or in the freezing of serum samples, most nutritional indicators showed remarkable stability. This information is important for both the design of field studies and the use of residual samples subjected to suboptimal preanalytical factors.