Mildred M. Goldman

Simultaneous measurement of thirteen steroid hormones in women with polycystic ovary syndrome and control women using liquid chromatography-tandem mass spectrometry.

Background The measurement of adrenal and ovarian androgens in women with PCOS has been difficult based on poor specificity and sensitivity of assays in the female range. Methods Women with PCOS (NIH criteria; n = 52) and control subjects with 25–35 day menstrual cycles, no evidence of hyperandrogenism and matched for BMI (n = 42) underwent morning blood sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously measure 13 steroids from a single blood sample to measure adrenal and ovarian steroids. Androgen and progesterone results were compared in the same samples using RIA. Results Testosterone, androstenedione, progesterone and 17OH progesterone levels were higher when measured using RIA compared to LC-MS/MS, although the testosterone RIA demonstrated the best agreement with the LC-MS/MS using a Bland-Altman analysis. Results using LC-MS/MS demonstrated that the concentration of androgens and their precursors were higher in women with PCOS than controls [median (2.5, 97.5th %ile); 1607 (638, 3085) vs. 1143 (511, 4784) ng/dL; p = 0.03]. Women with PCOS had higher testosterone [49 (16, 125) vs. 24 (10, 59) ng/dL], androstenedione [203 (98, 476) vs. 106 (69, 223) ng/dL] and 17OH progesterone levels [80 (17, 176) vs. 44 (17, 142) ng/dL] compared to controls (all P<0.02), but no differences in serum concentrations of the adrenal steroids DHEAS, cortisol, corticosterone and their 11 deoxy precursors. Women with PCOS also had an increase in the product:precursor ratio for 3β-hydroxysteroid dehydrogenase [22% (6, 92) vs. 20% (4, 43); p = 0.009]. Conclusion LC-MS/MS was superior to RIA in measuring androstenedione, progesterone and 17OH progesterone levels, while testosterone measurements were better matched in the two assays. Androgen levels were higher in women with PCOS in the absence of a difference in adrenal-predominant steroids. These data support previous findings that the ovary is an important source for the androgen excess in women with PCOS.

The Measurement of 3-Epimer 25-Hydroxyvitamin D by Mass Spectrometry in Clinical Specimens Detects Inconsequential Levels in Adult Subjects

Background Vitamin D is derived from dietary sources or from the action of ultraviolet light on 7-dehydrocholesterol and undergoes a number of enzymatic modifications that lead to the synthesis of active vitamin D metabolites or metabolites with reduced biological activity. Among these, epimerization at the 3-hydroxyl group leads to the synthesis of 3-epimer 25-hydroxyvitamin D (3EVD). Described first in biological system experiments using in vitro incubation of vitamin D in cell culture, this molecule has been reported as having distinct activities when compared with 25-hydroxy vitamin D (25OHVD). Measurements of vitamin D have been conducted using a variety of methodologies and have led to conflicting assessments of the quantities of 3EVD3 that are measured. Method The present article describes the development and use of a simple liquid chromatography–tandem mass spectrometry method validated by the Clinical Laboratory Improvement Amendments to quantitate 3EVD3 in 3528 subjects, including 309 children (162 are <2 years) and 232 pregnant women. Results Our findings demonstrate that, although 3EVD3 constitutes a significant proportion of measureable 25OHVD3 in subjects younger than 1 year, 3EVD3 levels are negligible in most subjects older than 1 year. Conclusions It is important to choose the correct 25OHVD assay dependent on the age of the patient. Patients younger than 1 year should be run on a liquid chromatography–tandem mass spectrometry assay proven to not have potential contributions from any 3EVD present in the sample.