Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays.

Background Biotin interference in streptavidin-based immunoassays causes widespread analytical distortions that may lead to clinical confusion, inappropriate patient management and, ultimately, adverse events. Its prevalence has increased recently due to the increased use of high-dose biotin therapy in specific patient groups (notably multiple sclerosis) and possibly the general community. Methods We have developed a method to deplete biotin from samples using the streptavidin-coated magnetic microparticles that are a component of most susceptible assays. Results We show that high concentrations of spiked biotin can be adequately depleted from serum using this approach, and that gross biochemical derangements can be restored to normality. We also show that biotin in samples derived from multiple sclerosis patients receiving 300 mg biotin daily can be adequately depleted to remove associated analytical interference and restore normal results. The method is applicable to competitive and sandwich immunoassays and importantly, because it does not change the volume of the sample, suitable for the measurement of free thyroid hormone assays. Application of the method does not significantly change the precision of measurement, and for the majority of analytes, the accuracy is not substantially altered. Conclusions Adopting this method enables laboratories to confirm biotin interference in the appropriate clinical setting. Moreover, it enables laboratories to remove the interference and report accurate and reliable results, without the need for patients to withhold beneficial therapy prior to blood tests. Until the biotin tolerance of susceptible assays is improved, our method gives laboratories a safe alternative for reporting results using streptavidin-based methods.

Characterization of the scope and magnitude of biotin interference in susceptible Roche Elecsys competitive and sandwich immunoassays.

Background Biotin interference is a significant problem to which at-risk laboratories must now be attuned. We sought to systematically characterize the nature of this interference in Roche immunoassays. Methods Known concentrations of biotin were titrated into serum samples and the effects on competitive and sandwich immunoassays were analysed. The maximum and minimum concentrations examined reflect those likely to be achieved in individuals on 5 to 10 mg supplements at the lower end, and 100 to 300 mg biotin at the high end. Results A high variability in biotin tolerance was observed. Some assays, such as troponin T, TSH and antithyroid antibodies, were extremely sensitive to the lower concentrations of biotin (15.6 and 31.3 ng/mL), whereas the majority of assays were relatively resistant. At concentrations ≥500 ng/mL, all assays showed significant interference from biotin but, again, the magnitude of the interference was variable. The more sensitive assays showed profound analytical bias at biotin concentrations that occur with high-dose therapy. Conclusion Our data demonstrate high variability in biotin tolerance across Roche immunoassays. The shape of the dose-response curves provides more detailed information than the single manufacturer-quoted figure for biotin tolerance. Accordingly, these data may be used by laboratories for more accurate risk assessment in predicting the effects of biotin. Our data may also be extrapolated to guide timing of blood tests in patients on high-dose biotin therapy: it demonstrates the number of half-lives required to withhold biotin in order to decrease its concentration to below a given assay tolerance.

Serum 25-Hydroxyvitamin D Insufficiency in Search of a Bone Disease.

Context: Vitamin D "insufficiency" and "deficiency" are defined as serum 25-hydroxyvitamin D [25(OH)D] levels <75 and <30 nmol/L, respectively. We aimed to determine whether these values signal hypocalcemia and hypophosphatemia, secondary hyperparathyroidism, high bone remodeling, low areal bone mineral density (aBMD), microstructural deterioration, or reduced matrix mineralization density (MMD) and so suggest whether bone fragility is present. Methods: Concentrations of 25(OH)D, calcium, phosphate, creatinine, and parathyroid hormone (PTH) were measured in 11,855 participants. Serum C-terminal telopeptide of type 1 collagen, procollagen type 1 N-terminal propeptide (P1NP), aBMD, and distal radius microstructure and MMD were measured in a second subset of 150 participants. Results: A breakpoint for calcium, PTH, and alkaline phosphatase was identified at a threshold 25(OH)D level <30 nmol/L. There was no plateau beyond 75 nmol/L. In the subgroup with measurements of bone morphology, no associations were detectable between serum 25(OH)D concentration, aBMD, trabecular density, cortical porosity, or MMD. Among 1439 participants with serum 25(OH)D <30 nmol/L, 6.1% had low serum calcium, 3.4% had low serum phosphate, 6.1% had high alkaline phosphatase, and 34.2% had elevated PTH. Most participants did not have any abnormalities. Conclusion: At a 25(OH)D threshold of ≤30 nmol/L, abnormalities in biochemical features support the notion of a "deficiency" state predisposing to bone disease. However, no deleterious effects were found in participants within an insufficiency threshold of a 25(OH)D level of 30 to 75 nmol/L, which challenges the rationale justifying vitamin D supplementation in these individuals.

Effects of combined calcium and vitamin D supplementation on insulin secretion, insulin sensitivity and ß-cell function in multi-ethnic vitamin D-deficient adults at risk for type 2 diabetes: a pilot randomized, placebo-controlled trial.

OBJECTIVES: To examine whether combined vitamin D and calcium supplementation improves insulin sensitivity, insulin secretion, ß-cell function, inflammation and metabolic markers. DESIGN: 6-month randomized, placebo-controlled trial. PARTICIPANTS: Ninety-five adults with serum 25-hydroxyvitamin D [25(OH)D] ≤55 nmol/L at risk of type 2 diabetes (with prediabetes or an AUSDRISK score ≥15) were randomized. Analyses included participants who completed the baseline and final visits (treatment n = 35; placebo n = 45). INTERVENTION: Daily calcium carbonate (1,200 mg) and cholecalciferol [2,000-6,000 IU to target 25(OH)D >75 nmol/L] or matching placebos for 6 months. MEASUREMENTS: Insulin sensitivity (HOMA2%S, Matsuda index), insulin secretion (insulinogenic index, area under the curve (AUC) for C-peptide) and ß-cell function (Matsuda index x AUC for C-peptide) derived from a 75 g 2-h OGTT; anthropometry; blood pressure; lipid profile; hs-CRP; TNF-α; IL-6; adiponectin; total and undercarboxylated osteocalcin. RESULTS: Participants were middle-aged adults (mean age 54 years; 69% Europid) at risk of type 2 diabetes (48% with prediabetes). Compliance was >80% for calcium and vitamin D. Mean serum 25(OH)D concentration increased from 48 to 95 nmol/L in the treatment group (91% achieved >75 nmol/L), but remained unchanged in controls. There were no significant changes in insulin sensitivity, insulin secretion and ß-cell function, or in inflammatory and metabolic markers between or within the groups, before or after adjustment for potential confounders including waist circumference and season of recruitment. In a post hoc analysis restricted to participants with prediabetes, a significant beneficial effect of vitamin D and calcium supplementation on insulin sensitivity (HOMA%S and Matsuda) was observed. CONCLUSIONS: Daily vitamin D and calcium supplementation for 6 months may not change OGTT-derived measures of insulin sensitivity, insulin secretion and ß-cell function in multi-ethnic adults with low vitamin D status at risk of type 2 diabetes. However, in participants with prediabetes, supplementation with vitamin D and calcium may improve insulin sensitivity. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12609000043235.

Low serum 25-hydroxyvitamin D is associated with increased risk of the development of the metabolic syndrome at five years: results from a national, population-based prospective study (The Australian Diabetes, Obesity and Lifestyle Study: AusDiab).

CONTEXT: Serum 25-hydroxyvitamin D [25(OH)D] concentration has been inversely associated with the prevalence of metabolic syndrome (MetS), but the relationship between 25(OH)D and incident MetS remains unclear. OBJECTIVE: We evaluated the prospective association between 25(OH)D, MetS, and its components in a large population-based cohort of adults aged 25 yr or older. DESIGN: We used baseline (1999-2000) and 5-yr follow-up data of the Australian Diabetes, Obesity, and Lifestyle Study (AusDiab). PARTICIPANTS: Of the 11,247 adults evaluated at baseline, 6,537 returned for follow-up. We studied those without MetS at baseline and with complete data (n = 4164; mean age 50 yr; 58% women; 92% Europids). OUTCOME MEASURES: We report the associations between baseline 25(OH)D and 5-yr MetS incidence and its components, adjusted for age, sex, ethnicity, season, latitude, smoking, family history of type 2 diabetes, physical activity, education, kidney function, waist circumference (WC), and baseline MetS components. RESULTS: A total of 528 incident cases (12.7%) of MetS developed over 5 yr. Compared with those in the highest quintile of 25(OH)D (≥34 ng/ml), MetS risk was significantly higher in people with 25(OH)D in the first (<18 ng/ml) and second (18-23 ng/ml) quintiles; odds ratio (95% confidence interval) = 1.41 (1.02-1.95) and 1.74 (1.28-2.37), respectively. Serum 25(OH)D was inversely associated with 5-yr WC (P < 0.001), triglycerides (P < 0.01), fasting glucose (P < 0.01), and homeostasis model assessment for insulin resistance (P < 0.001) but not with 2-h plasma glucose (P = 0.29), high-density lipoprotein cholesterol (P = 0.70), or blood pressure (P = 0.46). CONCLUSIONS: In Australian adults, lower 25(OH)D concentrations were associated with increased MetS risk and higher WC, serum triglyceride, fasting glucose, and insulin resistance at 5 yr. Vitamin D supplementation studies are required to establish whether the link between vitamin D deficiency and MetS is causal.

Prevalence of vitamin D deficiency and its determinants in Australian adults aged 25 years and older: a national, population-based study.

OBJECTIVE: Vitamin D deficiency is recognized as a global public health problem, but the population-based prevalence of deficiency and its determinants in Australian adults is not known. This study evaluated the vitamin D status of Australian adults aged ≥25 years and risk factors associated with vitamin D deficiency in this population. DESIGN AND PATIENTS: We studied a national sample of 11,247 Australian adults enrolled in the 1999/2000 Australian Diabetes, Obesity and Lifestyle (AusDiab) study drawn from 42 randomly selected districts throughout Australia. MEASUREMENTS: Serum concentrations of 25-hydroxyvitamin D [25(OH)D] were measured by immunoassay. Vitamin D deficiency was defined as a concentration <50 nmol/l. Information on demographic and lifestyle factors was derived from interview-administered questionnaires. RESULTS: The mean serum 25(OH)D concentration was 63 nmol/l (95% CI: 59-67 nmol/l). Only 4% of the population had a level <25 nmol/l, but the prevalence of vitamin D deficiency (<50 nmol/l) was 31% (22% men; 39% women); 73% had levels <75 nmol/l. The prevalence of vitamin D deficiency increased significantly with age, was greater in women, in those of non-Europid origin, in the obese and those who were physically inactive and with a higher level of education. Deficiency was also more common during winter and in people residing in southern Australia (latitude >35°S); 42% of women and 27% of men were deficient during summer-autumn, which increased to 58% and 35%, respectively, during winter-spring. CONCLUSION: Vitamin D deficiency is common in Australia affecting nearly one-third of adults aged ≥25 years. This indicates that strategies are needed at the population level to improve vitamin D status of Australians.