Assessment and Interpretation of Circulating 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in the Clinical Environment
Section snippets
Methods of 25(OH)D quantitation
The assessment of circulating 25(OH)D started its journey approximately 4 decades ago with the advent of the competitive protein-binding assay (CPBA).3 From that early time to the present we have progressed to radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC/MS). A brief description of each technique is given here.
Determining analytical recovery of 25(OH)D2 and 25(OH)D3 in human serum or plasma
Questions constantly arise regarding the various 25(OH)D assay procedures' ability to accurately measure total 25(OH)D (25(OH)D2 + 25 (OH)D3) levels in human samples.8 A brief study recently has described the ability of the DiaSorin Liaison Total-D 25(OH)D Assay System to perform this task as compared with the gold standard HPLC/UV quantitation of 25(OH)D2 and 25(OH)D3.18 Baseline serum samples that contained only 25(OH)D3 were obtained from 9 volunteers. All subjects then consumed 50,000 IU/d
Determining and defining a “normal” circulating 25(OH)D level
To define a “normal” circulating level of a given substance or nutrient, one usually obtains blood samples from a diverse population, measures the substance in question, plots the data by Gaussian distribution, and determines normality. This method works well for nutrients such as folate or vitamin E, and was precisely how normative circulating levels of 25(OH)D were defined in humans beginning about 40 years ago by Haddad and Chyu,3 who sampled a population of “normal” individuals whom were
Clinical reporting of circulating 25(OH)D concentrations
As highlighted earlier, all DiaSorin 25(OH)D assays are approved by the FDA for clinical utility. Thus, the diagnostic 25(OH)D tests sold by DiaSorin and IDS Diagnostics (Fountain Hills, AZ, USA) are under strict FDA control and monitoring for assay performance and reliability. In what is considered a distributing trend, many clinical reference laboratories are replacing these FDA-approved tests with “home-brew” LC/MS methods that are diverse and not under FDA scrutiny. The reasons for this
Methods of 1,25(OH)2D quantitation
Of all the steroid hormones, 1,25(OH)2D represented the most difficult challenge to the analytical biochemist with respect to quantitation. 1,25(OH)2D circulates at picomolar concentrations (too low for direct UV or MS quantitation), is highly lipophilic, and its precursor, 25(OH)D, circulates at nanomolar levels. The development of simple, rapid assay for this compound has proven to be a daunting task.
Determining and defining a “normal” circulating 1,25(OH)2D level
Unlike 25(OH)D, a normal circulating level of 1,25(OH)2D can be determined from a Gaussian distribution of subjects. Over the last 3 decades this has been accomplished, and a normal adult level has been defined as 16 to 56 pg/mL with a mean of 37.6 pg/mL.68 Circulating 1,25(OH)2D is diagnostic for several clinical conditions, including vitamin D–dependent rickets types I and II, hypercalcemia associated with sarcoidosis, and other hypercalcemic disorders causing increased 1,25(OH)2D levels.
Stability of 25(OH)D and 1,25(OH)2D in serum or plasma
Researchers have known for nearly 30 years that endogenous 25(OH)D and 1,25(OH)2D are extremely stable in serum or plasma.70 Lissner and colleagues70 showed that vitamin D metabolites in blood stored at 24°C for up to 72 hours remain intact. Recent studies on the stability of 25(OH)D in plasma or serum that has undergone many freeze-thaw cycles have reported the same stability.71 The author has used the same pooled human 25(OH)D and 1,25(OH)2D internal controls stored at −20°C for more than 10
Standardization of 25(OH)D and 1,25(OH)2D analysis
DEQAS (Internet: www.deqas.org) was founded in 1989 to compare the performance of then-available 25(OH)D tests. DEQAS has since become the largest vitamin D quality assessment program in the world, with approximately 600 participating laboratories worldwide. The organization's major aim today is to assess the analytical reliability of 25(OH)D and 1,25(OH)2D assays. The organization achieves this goal by:
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Distributing serum pools at regular intervals
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Conducting statistical analyses of submitted
Summary
The assessment of circulating 25(OH)D and, to a lesser degree, 1,25(OH)2D is rapidly becoming an important clinical tool in the diagnosis and management of many diverse pathologies. At present, the reference ranges for circulating 25(OH)D and 1,25(OH)2D are 32 to 100 ng/mL and 16 to 56 ng/mL, respectively, and are largely based on clinical data derived from the FDA-cleared DiaSorin assay procedures.
References (71)
- et al.
The anomalous behavior of exogenous 25-hydroxyvitamin D in competitive binding assays
J Steroid Biochem Mol Biol
(2007) - et al.
Analytical and clinical; validation of the 25 OH vitamin D assay for the LIAISON automated analyzer
Clin Biochem
(2004) - et al.
Measurement of 25(OH)D2 and 25(OH)D3 in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method
Clin Chim Acta
(2008) Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D
J Nutr
(2005)- et al.
Association between serum concentrations of 25-hydroxyvitamin D and gingival inflammation
Am J Clin Nutr
(2005) - et al.
Association between serum concentrations of 25-hydroxyvitamin D and periodontal disease in the US population
Am Clin Nutr
(2004) - et al.
Maternal vitamin D deficiency is associated with bacterial vaginosis in the first trimester of pregnancy
J Nutr
(2009) - et al.
Serum 25-hydroxyvitamin D, ethnicity and blood pressure in the third National Health and Nutrition Examination Survey
Am J Hypertens
(2007) - et al.
Hypovitamninosis D is associated with insulin resistance and beta cell dysfunction
Am J Clin Nutr
(2004) - et al.
25-hydroxyvitamin D, insulin resistance and kidney function in the third national health and nutrition examination survey
Kidney Int
(2007)
The paradoxical effects of vitamin D on type 1 mediated immunity
Mol Aspects Med
Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third national health and nutrition examination survey, 1988-1994
Am J Clin Nutr
Vitamin D insufficiency in children, adolescents, and young adults with cystic fibrosis despite routine oral supplementation
Am J Clin Nutr
A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma
Arch Biochem Biophys
Vitamin D2 is much less effective than vitamin D3 in humans
J Clin Endocrinol Metab
Structure-function relationships in the vitamin endocrine system
Endocr Rev
Competitive protein-binding radioassay for 25-hyroxycholecalciferol
J Clin Endocrinol Metab
Two direct nonchromatographic assays for 25-hydroxyvitamin D
Clin Chem
A rapid assay for 25-OH-vitamin D3 without preparative chromatography
J Clin Endocrinol Metab
Importance of the isolation of 25 hydroxyvitamin D before assay
J Lab Clin Med
Underestimation of serum 25-hydroxyvitamin D by the Nichols Advantage Assay in patients receiving vitamin D2 replacement therapy
Clin Chem
Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status
Clin Chem
Determination of vitamin D status by radioimmunoassay with a 125I-labeled tracer
Clin Chem
Vitamin D measurement may not reflect what you give to our patients
J Bone Miner Res
HPLC measurement for 25-hydroxyvitamin D measurement: comparison with contemporary assays
Clin Chem
C-3 epimers can account for a significant proportion of total circulating 25(OH)D in infants, complicating accurate measurement and interpretation of vitamin D status
J Clin Endocrinol Metab
Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3
Clin Chem
Quantification o f serum 25-hydroxyvitamin D2 and D3 using HPLC-tandem mass spectrometry and examination if reference intervals for diagnosis of vitamin D deficiency
Am J Clin Pathol
In vivo versus in vitro recovery of 25(OH)D2 and D3 in human samples using high-performance liquid chromatography and the DiaSorin Liaison Total-D assay: Limitations of the NIST controls
J Steroid Biochem Molec Biol
The physiologic significance of plasma transport of vitamin D and metabolites
Am J Med
Dietary reference intakes: calcium, phosphorus, magnesium, vitamin D, fluoride
Normal serum vitamin D levels
N Engl J Med
Serum 25-hydroxyvitamin D and risk of postmenopausal breast cancer—results of a large case-control study
Carcinogenesis
Plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and risk of breast cancer
Cancer Epidemiol Biomarkers Prev
Plasma vitamin D metabolites and risk of colorectal cancer in women
Cancer Epidemiol Biomarkers Prev
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This article originally appeared in Endocrinology and Metabolism Clinics of North America Volume 39, Issue 2, June 2010.