Steroid reference intervals in women: influence of menopause, age and metabolism.

OBJECTIVE: To investigate the impact of age, obesity and metabolic parameters on 13 circulating steroids in reproductive and menopausal age. To define reference intervals (RIs). DESIGN: Cross-sectional. METHODS: Three hundred and twenty five drug-free, healthy and eumenorrheic women were selected from the general population. Independent relationships of LC-MS/MS-determined steroid levels with age, BMI and metabolic parameters were estimated. Reference sub-cohorts were defined for calculating upper and lower limits in reproductive age, menstrual phases and menopause, and these were compared with limits in dysmetabolic sub-cohorts. RESULTS: Lower androgens, pro-androgens and estrogens, but higher cortisol and metabolites were found in menopausal compared to reproductive age women. Androgens and precursors decreased during reproductive age (P < 0.001-P = 0.002) but not after menopause. 17OH-progesterone decreased with BMI (P = 0.006) and glucocorticoids with waist circumference (P < 0.001P = 0.002) in reproductive age, but increased with triglycerides (P=0.011P=0.038) after menopause. Inverse associations of dihydrotestosterone with BMI (P=0.004) and HDL-cholesterol (P=0.010), estrone with total cholesterol (P=0.033) and estradiol with triglycerides (P=0.011) were found in reproductive age. After menopause, estrone increased with waist circumference (P<0.001) and decreased with insulin resistance (P=0.012). Ovarian steroid RIs were estimated in menstrual phases and menopause. Age- and reproductive status-specific RIs were generated for androgens, precursors and corticosteroids. Lower limits for reproductive age cortisol (P=0.020) and menopausal 11-deoxycortisol (P=0.003) in dysmetabolic sub-cohorts were reduced and increased, respectively, compared to reference limits. CONCLUSIONS: Obesity and dysmetabolism differently influence circulating steroids in reproductive and menopausal status. Age, menstrual and menopausal status-specific RIs were provided by LC-MS/MS for a broad steroid panel.

The standardization of nonsterile compounding: a study in quality control and assessment for hormone compounding.

Sterile and nonsterile compounding of medication has attracted much attention over the last few years due to the onset of various infections and negative compounding practices. This paper reports on the standardization of compounded hormones utilizing the Wiley Protocol, which provides nonsynthetic bioidentical estradiol, progesterone, dehydroepiandrosterone, and testosterone in a transdermal topical cream base for women and men in a standardized dosing regimen. Here, we present data from 2008 through 2012, which details the process of standardization and quality testing of the hormones through submission of random compounded samples for quality control and assessment. Pharmacies delivering the Wiley Protocol were required to follow the same compounding formulation, as well as submit random samples for quarterly testing. Sample concentrations were tested using high-performance liquid chromatography. We found that pharmacies that submitted samples had a 91% passing rating with a percent of target of 98.6% +/- 8.4%. It was also determined that pharmacies that prepared more compounded cream had a higher passing rating than those that prepared limited quantities. We found that standardization across multiple pharmacies could be achieved through quarterly testing of submitted samples by a third-party laboratory when following necessary procedures as defined by the Wiley Protocol. It was also determined that experience and training were a critical factor in the mixing of compounded prescriptions, with high consistency and accuracy providing patient safety.

Biochemical reference intervals for sex hormones with a new AutoDelfia method in aged men.

BACKGROUND: Our aim was to establish sex hormone reference intervals measured with a new AutoDelfia immunoassay method for aged men free of medication and/or conditions known to influence sex hormone levels. METHODS: The reference population consisted of 466 individuals between 64 and 97 years (mean 72 years) and a mean body mass index (BMI) of 26.9 kg/m(2). RESULTS AND CONCLUSIONS: Because age correlated significantly with most sex hormones studied, we calculated reference intervals for three age groups (64-69, 70-74 and > or =75 years). In clinical practice, single ranges can be used for men aged 64 years or over for testosterone, estradiol and follicle-stimulating hormone (FSH) with the AutoDelfia method. For free testosterone and luteinizing hormone (LH), separate reference intervals should be used for men aged 64-74 years and those aged 75 years or over. For sex hormone-binding globulin, two separate reference intervals by age (64-69 and > or =70 years) are also needed for aged men. LH and FSH reference ranges should be judged with caution, because they may be too high due to cases of subclinical hypogonadism included in the reference population.

Determination of delta4-3-ketosteroids based on oxime formation by difference circular dichroism spectroscopy.

delta4-3-Ketosteroids exhibit an intensive negative Cotton effect on the circular dichroism (CD) spectra in the wavelength range for the n-pi* electronic transition (270-350 nm). With hydroxylamine hydrochloride, delta4-3-ketosteroid compounds can be transformed into oxime derivatives. Following oxime formation, positive ellipticity with low intensity can be registered in this wavelength range. The quantitative determination of delta4-3-ketosteroids is based on the considerable difference between the ellipticities before and after oxime formation. The difference ellipticity for the six ketosteroids examined (norethisterone, levonorgestrel, levonorgestrel acetate, methyltestosterone, testosterone phenylpropionate, nortestosterone phenylpropionate) varies linearly with the concentration in the interval 6 x 10(-6)-3 x 10(-3) mol/L. The method can be well applied to determination of delta4-3-ketosteroid contamination of norgestimate [(+)-13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn4-en-20-yn-3-one oxime acetate]; 0.02-10% impurity can be measured.

Steroid profiling in the study of rat testicular steroidogenesis.

Twenty authentic steroids, derivatized as O-methyl oximes (MO), trimethylsilyl (TMS) ethers or as MO-TMS ethers have been subjected to capillary gas chromatography using two different columns. Virtually all of the steroid derivatives have been resolved, one difficult pair to separate being 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol on the non-selective phase OV-1. Where syn and anti forms of MO derivatives arose, these were also resolved under the conditions utilised. This technique of 'steroid profiling' has been applied to the separation and quantification of metabolites of pregnenolone which were formed during incubations of the microsomal and cytosolic fractions from rat testes. The majority of the metabolites were found in the microsomal incubation. These compounds included some odorous 16-androstenes as well as other C21 and C19 steroids, the formation of which was consistent with the 5-ene and 4-ene pathways of testosterone biosynthesis being operative. In addition, evidence was obtained for 16 alpha-hydroxylation of C21 steroids. Very much less metabolic activity was found in the cytosolic fraction of rat testes. Metabolic pathways have been proposed which both confirm and extend earlier work. We conclude that the rat testis can only form some of the odorous, possibly pheromonal, 16-androstenes and that these are quantitatively less important than in the porcine testis.