[Show abstract][Hide abstract] ABSTRACT: Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that permits the quantification of major circulating vitamin D(3) metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction, and Diels-Alder derivatization procedure prior to LC-MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D(3) peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D(3) [4β,25(OH)(2)D(3)] and was found at concentrations comparable to 1α,25(OH)(2)D(3). Quantification of 1α,25(OH)(2)D(3) in plasma required complete chromatographic separation of 1α,25(OH)(2)D(3) from 4β,25(OH)(2)D(3). An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD(3), 24R,25(OH)(2)D(3), 1α,25(OH)(2)D(3), and 4β,25(OH)(2)D(3) in healthy individuals. The LC-MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)(2)D(3) as well as monitoring the newly identified circulating metabolite, 4β,25(OH)(2)D(3).
[Show abstract][Hide abstract] ABSTRACT: Estrogens suppress tumor growth in prostate cancer which progresses despite anorchid serum androgen levels, termed castration resistant prostate cancers (CRPC), although the mechanisms are unclear. We hypothesize that estrogen inhibits CRPC in anorchid animals by suppressing tumoral androgens, an effect independent of the estrogen receptor.
The human CRPC xenograft LuCaP 35V was implanted into orchiectomized male SCID mice and established tumors were treated with placebo, 17beta-estradiol or 17beta-estradiol and estrogen receptor antagonist ICI 182,780. Effects of 17beta-estradiol on tumor growth were evaluated and tissue testosterone (T) and dihydrotestosterone (DHT) evaluated by mass spectrometry.
Treatment of LuCaP 35V with 17beta-estradiol slowed tumor growth compared to controls (tumor volume at day 21: 785 +/- 81 mm3 vs. 1195 +/- 84 mm3, p = 0.002). Survival was also significantly improved in animals treated with 17beta-estradiol (p = 0.03). The addition of the estrogen receptor antagonist ICI 182,780 did not significantly change survival or growth. 17beta-estradiol in the presence and absence of ICI 182,780 suppressed tumor testosterone (T) and dihydrotestosterone (DHT) as assayed by mass spectrometry. Tissue androgens in placebo treated LuCaP 35V xenografts were; T = 0.71 +/- 0.28 pg/mg and DHT = 1.73 +/- 0.36 pg/mg. In 17beta-estradiol treated LuCaP35V xenografts the tissue androgens were, T = 0.20 +/- 0.10 pg/mg and DHT = 0.15 +/- 0.15 pg/mg, (p < 0.001 vs. controls). Levels of T and DHT in control liver tissue were < 0.2 pg/mg.
CRPC in anorchid animals maintains tumoral androgen levels despite castration. 17beta-estradiol significantly suppressed tumor T and DHT and inhibits growth of CRPC in an estrogen receptor independent manner. The ability to manipulate tumoral androgens will be critical in the development and testing of agents targeting CRPC through tissue steroidogenesis.
[Show abstract][Hide abstract] ABSTRACT: Digoxin, a commonly prescribed cardiac glycoside with a narrow therapeutic window, is routinely used in pharmacokinetic studies to assess the in vivo activity of the drug efflux pump P-glycoprotein. To minimize adverse events, a sub-therapeutic dose of digoxin is usually administered, producing low plasma concentrations requiring a sensitive detection technique. Commonly available immunoassay techniques do not provide the required sensitivity to measure these low plasma concentrations and are potentially non-specific in certain subject populations. Previously published mass spectrometric techniques require either large plasma volumes or a tandem mass spectrometer. To overcome these challenges we have developed a sensitive and specific LC-MS method for the quantification of digoxin in small volumes of human plasma and urine. Plasma (1 mL) was extracted with methyl t-butyl ether under basic conditions followed by LC-MS detection of the sodium adducts of digoxin (803.4 m/z) and digitoxin (787.4 m/z, internal standard). Linearity and accuracy were demonstrated across a wide range of digoxin plasma concentration (0.05-1.5 ng/mL). This specific, sensitive, validated digoxin LC-MS assay can be used to quantify sub-therapeutic digoxin plasma concentrations in men and women (pregnant and non-pregnant).