New Assay Simultaneously Measures Vitamin D Metabolites

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Assay Offers Greater Granularity

"I think there is a major need for assays such as this to get into the clinical world, certainly the research world, so we can have additional insight into what is really going on with the relationship of falling vitamin D status today and all of the [associated] outcomes such as falls and fractures," osteoporosis expert Dr. Neil Binkley said in an interview.

"By just measuring 25-hydroxy D, we’re not seeing the whole picture; just like when we measure total cholesterol, we aren’t seeing the whole picture."

Dr. Binkley is associate director of the Institute on Aging at the University of Wisconsin, Madison. He said he had no relevant financial disclosures.



MINNEAPOLIS – A new automated assay can measure concentrations of both 24,25-dihydroxyvitamin D2 and D3 in human serum.

Current assays based on competitive binding experiments can differentiate between 24,25-dihydroxyvitamin D3 and D2, but are time consuming and not easily automated. Also, immunoassays fail to differentiate between 25-hydroxyvitamin D and 24,25-dihydrxoxyvitamin D.

The new, high-throughput mass spectrometry–based assay – developed by researchers at the Mayo Clinic – is able to quantify both analytes.

Patrice Wendling/IMNG Medical Media

Hemamalini Ketha, Ph.D.

Knowledge of the serum concentrations of both 24,25(OH)2D and 25(OH)D3 is particularly important in establishing reduced activity of the primary vitamin D degradation enzyme CYP24A1 in patients with mutations in the CYP24A1 (cytochrome P450 24-hydroxylase A1) gene.

"A lot of physicians want this metabolite to be measured just in case a person might be a candidate for more extensive genotyping studies ... They also want to know the reference range in normal individuals," lead author and research fellow Hemamalini Ketha, Ph.D., said in an interview.

Researchers at the Roswell Park Cancer Institute in Buffalo, N.Y., recently reported that single nucleotide polymorphisms in CYP24A1 may be related to the higher prevalence of estrogen receptor–negative breast cancer in African American women – a group known to have lower levels of vitamin D (Breast Cancer Res. 2012;14:R58).

Dr. Ketha and her colleagues at the Mayo Clinic in Rochester, Minn., used a liquid chromatography tandem mass spectrometry method to measure serum 24,25(OH)2D concentrations in 92 serum samples from men and women with a wide range of 25(OH)D concentrations.

The limit of detection for 24,25(OH)2D3 was 0.08 ng/mL; the limit for 24,25(OH)2D2 was 0.5 ng/mL, Dr. Ketha reported in a poster at the annual meeting of the American Society for Bone and Mineral Research.

The mean concentration of 25(OH)D in the serum samples was 29.36 ng/mL and was 2.59 ng/mL for 24,25(OH)2D3.

Serum 24,25(OH)2D concentrations were linearly correlated with serum 25(OH)D concentrations (R2 = 0.75), she said.

No correlation was observed, however, between 24,25(OH)2D3 and 1,25(OH)2D3 (R2 = 0.0005).

"For diagnostic purposes, the interpretation of concentrations of serum 24,25(OH)2D3 should therefore take the concomitant 25(OH)D3 concentrations into account," the authors concluded.

Dr. Ketha said the assay is rapid and reproducible, and that the Mayo Clinic is planning to make it commercially available through its endocrinology clinical laboratory.

He said he had no relevant financial disclosures. The research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Dr. Ketha said she had no relevant financial disclosures.

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