Kegg Pathway: Biosynthesis of steroids

steroids. 2009 Nov 16;
Salameh WA, Redor-Goldman MM, Clarke NJ, Reitz RE, Caulfield MP

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15minutes per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150muL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3ng/dL, and the assay was linear up to 2,000ng/dL. The method compared very well with an established RIA: y=1.02x+1.5, r(2)=0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.

Int J Dev Biol. 2009 Nov 16;
Myatt L, Sun K

The fetal membranes fulfill several functions during pregnancy. In addition to containing the products of conception and amniotic fluid, they presumably have barrier functions and fulfill paracrine signaling functions between the maternal (decidual) and fetal compartments. As the membranes are in an ideal place to receive both maternal and fetal signals and transmit signals to uterine myometrium, there has been a specific focus on the role of membranes in the initiation and maintenance of parturition. In this review, we summarize the data obtained in our laboratories as well as the data reported in the literature particularly with regard to the synthesis of steroids and prostaglandins in the fetal membranes, in signaling fetal maturation and in parturition. The fetal membranes are a major site both of prostaglandin synthesis and of prostaglandin metabolism. In addition, the abundant expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1), which converts biologically inactive cortisone into active cortisol, in the fetal membranes may provide an extra-adrenal source of glucocorticoids for the fetal compartment during gestation. Accumulating evidence indicates that a positive feedback loop involving glucocorticoids, proinflammatory cytokines, prostaglandins, surfactant protein-A (SP-A) and 11beta-HSD1 is formed locally in human fetal membranes towards term or in preterm labor. This positive feedback loop would produce abundant biologically active glucocorticoids and prostaglandins in the fetal membranes or amniotic fluid, which would ultimately promote fetal organ maturation and initiate parturition.

steroids. 2009 Nov 11;
Iguchi Y, Kihira K, Nishimaki-Mogami T, Une M

FXR (farnesoid X receptor) is a bile acid-activated nuclear receptor that regulates not only the Biosynthesis and enterohepatic circulation of bile acids, but also triglyceride, cholesterol and glucose metabolism. FXR-mediated signaling pathways have become promising novel drug targets for the treatment of common metabolic and hepatic diseases. With the aim of uncovering novel modulators of FXR and further elucidating the molecular basis of FXR activation, we investigated the structure-activity relationships of a variety of naturally occurring sterols structurally related to bile acids in terms of their FXR agonist activity. Here, we report that the ability of bile alcohols to activate FXR varied with the position and number of hydroxyl groups existing in the steroid side chain of bile alcohols. In addition, we showed that the shortening of the steroid side chain of bile acids as well as bile alcohols resulted in a decline of the ability of these agents to activate FXR. Thus, we provide new insights into the structure-activity relationships of bile acids and bile alcohols as FXR agonists.

Endocrinology. 2009 Nov 11;
Kocerha J, Prucha MS, Kroll KJ, Steinhilber D, Denslow N

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of mitochondrial transport of cholesterol for steroid Biosynthesis. To investigate the regulation of this protein in lower vertebrates, we cloned the StAR coding region from large-mouth bass for analysis. Induction of the mRNA corresponded with increasing levels of plasma sex steroids in vivo. Cultures of largemouth bass ovarian follicles were exposed to dibutyryl cAMP (dbcAMP), a potent signaling molecule for steroidogenesis. StAR mRNA expression was significantly up-regulated by dbcAMP signaling, suggesting that the 5' regulatory region of the gene is functionally conserved. To further analyze its transcriptional regulation, a 2.9-kb portion of the promoter was cloned and transfected into Y-1 cells, a steroidogenic mouse adrenocortical cell line. The promoter activity was induced in a dose-responsive manner upon stimulation with dbcAMP; however, deletion of 1 kb from the 5' end of the promoter segment significantly diminished the transcriptional activation. A putative retinoic acid-related receptor-alpha/rev-erbalpha element was identified between the -1.86- and -2.9-kb region and mutated to assess its potential role in dbcAMP regulation of the promoter. Mutation of the rev-erbalpha element significantly impeded dbcAMP-induced activation. Chromatin immunoprecipitation and EMSA results revealed rev-erbalpha and retinoic acid-related receptor-alpha enrichment at the site under basal and dbcAMP-induced conditions, respectively. These results implicate important roles for these proteins previously uncharacterized for the StAR promoter. Altogether these data suggest novel regulatory mechanisms for dbcAMP up-regulation of StAR transcription in the distal part of the largemouth bass promoter.

J Hepatol. 2009 Oct 21;
Hindorf U, Jahed K, Bergquist A, Verbaan H, Prytz H, Wallerstedt S, Werner M, Olsson R, Björnsson E, Peterson C, Almer SH

BACKGROUND & AIMS: Corticosteroids alone or in conjunction with azathioprine (AZA) is the standard treatment in autoimmune hepatitis (AiH). Individual variations in thiopurine (TP) metabolism may affect both drug efficacy and toxicity. Our aim was to investigate the utility of thiopurine methyltransferase (TPMT) as well as thioguanine nucleotide (TGN) and methylthioinosine monophosphate (meTIMP) metabolite measurements with regard to clinical outcome. METHODS: Two hundred thirty-eight patients with AiH were included in this cross-sectional study. TPMT status was assessed in all patients, while TGN and meTIMP were measured in patients with ongoing TP medication. Clinical outcome was evaluated by liver tests and the ability to withdraw steroids. RESULTS: TPMT genotyping (n=229) revealed 207 (90.4%) wild-type and 22 heterozygous patients. One hundred forty-three patients had ongoing TP therapy with AZA (n=134) or mercaptopurine (MP; n=9); response was judged as complete response (CR) in 113 patients and partial response (PR) in 30 patients. Both TP dose (1.64 vs 1.19mg/kg; p=0.012) and TPMT activity (14.3 vs 13.5; p=0.05) were higher in PR, resulting in similar TGN levels (PR: 121pmol/8x10(8) red blood cells [RBC]; CR: 113pmol/8x10(8) RBC; p=0.33) but higher meTIMP levels in PR (1350 vs 400pmol/8x10(8) RBC; p=0.004). Patients able to withdraw steroids or who were using 5mg prednisolone daily were treated with lower TP doses than patients on higher steroid doses (1.15 vs 1.18 vs 1.82mg/kg; p<0.001). CONCLUSIONS: TP metabolite measurements are of clinical value in AiH patients who do not respond to standard TP treatment and for the identification of a shifted metabolism, which may demand an alternative treatment strategy.

Pharmacol Rep. 2009 Sep-Oct; 61(5): 851-8
Wójcikowski J, Daniel WA

The aim of the present study was to estimate the inhibitory effect of perazine, a phenothiazine neuroleptic with piperazine structure in a side chain, on human CYP1A2 activity measured as a rate of caffeine 3-N- and 1-N-demethylation. Moreover, the influence of perazine on other caffeine metabolic pathways such as 7-N-demethylation (CYP1A2, CYP2C8/9, CYP3A4) and 8-hydroxylation (CYP3A4, CYP1A2, CYP2C8/9) was also determined. The Dixon analysis showed that in both human liver microsomes and Supersomes CYP1A2 perazine potently and to a similar degree inhibited caffeine 3-N-demethylation (K(i) = 3.5 microM) and 1-N-demethylation (K(i) = 5 microM). Perazine moderately diminished the rate of caffeine 7-N-demethylation in Supersomes CYP1A2 (K(i) = 11.5 microM) and liver microsomes (K(i) = 20 microM), and attenuated C-8-hydroxylation (K(i) = 15.5 microM) in Supersomes CYP1A2. On the other hand, perazine weakly inhibited caffeine C-8-hydroxylation in liver microsomes (K(i) = 98 microM). About 80% of basal CYP1A2 activity was reduced by the therapeutic concentrations of perazine (5-10 microM). The obtained results show that perazine at its therapeutic concentrations is a potent inhibitor of human CYP1A2. Hence, taking account of CYP1A2 contribution to the metabolism of endogenous substances (steroids), drugs (xanthine derivatives, phenacetin, propranolol, imipramine, phenothiazine neuroleptics, clozapine) and carcinogenic compounds, the inhibition of CYP1A2 by perazine may be of physiological, pharmacological and toxicological importance.

Arch Ophthalmol. 2009 Nov; 127(11): 1494-9
Conway BN, Miller RG, Klein R, Orchard TJ

OBJECTIVE: To investigate the role of hemoglobin (HGB) level in predicting proliferative diabetic retinopathy (PDR). METHODS: We assessed 426 individuals without PDR at baseline (213 men; 213 women) from the Pittsburgh Epidemiology of Diabetes Complications Study, an 18-year prospective cohort study of childhood-onset type 1 diabetes. Presence of PDR was determined by stereo fundus photography. Cox proportional hazards modeling with stepwise regression was used to determine the independent association of HGB level with PDR. Analyses were sex specific. RESULTS: There were 206 events. Although the incidence of PDR did not vary by sex (48% in both men and women), in men, HGB exhibited a positive linear relationship with 18-year incidence of PDR (hazard ratio, 1.33; 95% confidence interval, 1.10-1.60; P = .003), while in women, HGB level exhibited a quadratic relationship with PDR (P < .001). After multivariable adjustment for univariately significant covariates, HGB level remained significantly predictive of PDR in both men (P = .004) and women (P = .04). CONCLUSION: Higher HGB level predicts the incidence of PDR in type 1 diabetes mellitus, though the association varies by sex, being linear and positive in men and quadratic in women.

New Phytol. 2009 Nov 5;
Pérez-Bermúdez P, Moya García AA, Tuñón I, Gavidia I

New Phytologist (2009)Summary * The stereospecific 5beta-reduction of progesterone is a required step for cardiac glycoside Biosynthesis in foxglove plants. Recently, we have isolated the gene P5betaR, and here we investigate the function and regulation of P5betaR2, a new progesterone 5beta-reductase gene from Digitalis purpurea. * P5betaR2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. * Like P5betaR, P5betaR2 catalyzes the 5beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5betaR expression does not vary, whereas P5betaR2 is highly responsive. P5betaR2 expression is regulated by ethylene and hydrogen peroxide. * The correlation between P5betaR2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide Biosynthesis, and therefore in the chemical defense of foxglove plants.

N Engl J Med. 2009 Nov 5; 361(19): 1856-63
Achard C, Courtillot C, Lahuna O, Méduri G, Soufir JC, Lière P, Bachelot A, Benyounes H, Schumacher M, Kuttenn F, Touraine P, Misrahi M

Men with mutations in LHB, the gene encoding the beta subunit of luteinizing hormone (LHB), have azoospermia with absent or few fetal Leydig cells. We report a mutation in LHB in a man and his sister. The man presented with absence of virilization, undetectable luteinizing hormone, and a low serum testosterone level. He had complete spermatogenesis with a normal sperm count. The mutant luteinizing hormone had a low level of partial activity in vitro. We concluded that the residual luteinizing hormone activity, resulting in the expression of steroidogenic enzymes in few mature Leydig cells producing small amounts of intratesticular testosterone (20.2 ng per gram), was sufficient for complete and quantitatively normal spermatogenesis.

Crit Care Med. 2009 Oct; 37(10): 2727-32
Yeager MP, Rassias AJ, Pioli PA, Beach ML, Wardwell K, Collins JE, Lee HK, Guyre PM

OBJECTIVE: There is continuing controversy regarding the effect of glucocorticoids on a systemic inflammatory process. Based ona model of glucocorticoid action that includes both pro- and anti-inflammatory effects, we used the human experimental endotoxemia model to test the hypothesis that a transient elevation of plasma cortisol to stress-associated levels would enhance a subsequent (delayed) systemic inflammatory response to bacterial endotoxin. DESIGN: Prospective, randomized, double-blind, placebo-controlled clinical investigation. SETTING: Academic medical center. SUBJECTS: Thirty-six healthy human volunteers. INTERVENTIONS: Participants were randomized to receive a 6-hr intravenous infusion of saline (control), an intermediate dose of cortisol (Cort80; 6.3 mg/hr/70 kg), or a high dose of cortisol (Cort160; 12.6 mg/hr/70 kg) on day 1. On day 2, participants received an intravenous injection of 2 ng/kg Escherichia coli endotoxin followed by serial measurements of plasma cytokine concentrations. MEASUREMENTS AND MAIN RESULTS: Baseline participant characteristics and cortisol and cytokine concentrations were similar in all three groups. The plasma cortisol response to endotoxemia on day 2 was similar in all three groups. The interleukin-6 response to endotoxemia was significantly increased in the Cort80 Group compared with the control Group (p = .004), whereas the interleukin-10 response was significantly suppressed (p = .034). Corresponding results for the Cort160 Group were not significantly different from control Group values. CONCLUSIONS: In this study, transient elevation of in vivo cortisol concentrations to levels that are observed during major systemic stress enhanced a subsequent, delayed in vivo inflammatory response to endotoxin. This appeared to be a dose-dependent effect that was more prominent at intermediate concentrations of cortisol than at higher concentrations of cortisol.

Dialogues Clin Neurosci. 2009; 11(3): 297-303
Wise PM, Suzuki S, Brown CM

The concept that estrogens exert important neuroprotective actions has gained considerable attention during the past decade. Numerous studies have provided a deep understanding of the seemingly contradictory actions of estrogens. We realize more than ever that the effects of estrogens (with and without simultaneous or sequential progestins) are diverse and sometimes opposite, depending on the use of different estrogenic and progestinic compounds, on different delivery routes, on different concentrations, on treatment sequence, and on the age and health status of the women who receive hormone therapy. During the past few years, we have gained an increasing appreciation of the impact of estrogens on the immune system and on inflammation. In addition, we have learned that estrogens cannot only protect against cell death, but can also stimulate the birth of new neurons. Here we posit the concept that estrogen's modulation of the immune status may be the basic mechanism that underlies its ability to protect against neurodegeneration and its powerful neuroregenerative actions. We hope that this update will encourage even richer dialogues between basic and clinical scientists to ensure that future clinical studies fully consider the information that can be derived from basic science studies. Only then will we have a better understanding of the impact of hormones on the menopausal and postmenopausal period in a woman's life.

J Am Coll Cardiol. 2009 Nov 3; 54(19): 1747-62
Triposkiadis F, Karayannis G, Giamouzis G, Skoularigis J, Louridas G, Butler J

Heart failure is a syndrome characterized initially by left ventricular dysfunction that triggers countermeasures aimed to restore cardiac output. These responses are compensatory at first but eventually become part of the disease process itself leading to further worsening cardiac function. Among these responses is the activation of the sympathetic nervous system (SNS) that provides inotropic support to the failing heart increasing stroke volume, and peripheral vasoconstriction to maintain mean arterial perfusion pressure, but eventually accelerates disease progression affecting survival. Activation of SNS has been attributed to withdrawal of normal restraining influences and enhancement of excitatory inputs including changes in: 1) peripheral baroreceptor and chemoreceptor reflexes; 2) chemical mediators that control sympathetic outflow; and 3) central integratory sites. The interface between the sympathetic fibers and the cardiovascular system is formed by the adrenergic receptors (ARs). Dysregulation of cardiac beta(1)-AR signaling and transduction are key features of heart failure progression. In contrast, cardiac beta(2)-ARs and alpha(1)-ARs may function in a compensatory fashion to maintain cardiac inotropy. Adrenergic receptor polymorphisms may have an impact on the adaptive mechanisms, susceptibilities, and pharmacological responses of SNS. The beta-AR blockers and the inhibitors of the renin-angiotensin-aldosterone axis form the mainstay of current medical management of chronic heart failure. Conversely, central sympatholytics have proved harmful, whereas sympathomimetic inotropes are still used in selected patients with hemodynamic instability. This review summarizes the changes in SNS in heart failure and examines how modulation of SNS activity may affect morbidity and mortality from this syndrome.

Zhongguo Zhong Yao Za Zhi. 2009 Jul; 34(13): 1705-11
Shen G, Liang A, Zhao Y, Cao C, Liu T, Li C, Nilsen OG

OBJECTIVE: To explore the interaction between herbal medicines and western drugs based on CYP3A4 enzyme metabolism by using testotesrone as a probe in liver microsome metabolism system in vitro. METHOD: The mixed liver microsome enzymatic system consisting of rat liver microsomes by ultra-high-speed centrifuge was established. The substrate testosterone was added into the system and enzyme CYP3A4 metabolic activity was expressed by the output of 6beta-hydroxy-testosterone which was measured by HPLC method. The proper conditions for testotesrone metabolism in liver microsome system included substrate concentration, incubation time, pH and incubation temperature. When the conditions in vitro were determined, three kinds of Chinese herbal medicinal ingredients (Tetrahydropalmatine, neferine, panax notoginseng saponins) were diluted into different concentrations and incubated with testotesrone in the liver microsomes incubation system, respectively. The results were measured through metabolite production with or without the presence of Chinese medicines. We assessed the Chinese herbal medicinal ingredients effect on the metabolism of CYP3A4 enzyme through 6beta-hydroxy metabolite of testosterone production. RESULT: Liver microsomes were incubated in the system, the testosterone metabolited into 6beta-hydroxy testosterone. The metabolism conditions were proper at the concentration of testosterone 200 micromol x L(-1) which was incubated for 3.5 hours at 37 degrees C in pH 7.0, PBS 0.1 mol x L(-1). The inhibition of tetrahydropalmatine and panax notoginseng saponins on testotesrone were weak with IC50 > 100 micromol x L(-1). The neferine had a little inhibition on testotesrone metabolism, IC50 < 100 micromol L(-1). CONCLUSION: Tetrahydropalmatine and panax notoginseng saponins had no obvious effect on testotesrone metabolism. Neferine had a little effect on testotesrone metabolism. It prompted that drug-interaction could not be apparent between two kinds of Chinese medicines and the CYP3A4 enzyme substrate, Neferine could bring about drug-interaction.

J Environ Sci (China). 2009; 21(7): 900-6
Liu ZH, Ito M, Kanjo Y, Yamamoto A

An estrogen receptor (ER)/androgen receptor (AR) ligand competitive binding assay (ER/AR-binding assay) and chemical analyses were used to evaluate the endocrine disrupting chemicals (EDCs) behavior of two municipal wastewater treatment plants (WWTPs) (K and S). In the influents, estrone (E1), androsterone (A), androstenedione (AD), BPA (bisphenol A), NP (nonylphenol) and daidzein (DZ) were detected in high amounts with subsequent 24 h-average concentrations of 350, 1000, 29, 1300, 3900, and 5700 ng/L in K-WWTP and of 310, 620, 59, 1600, 2600, and 8400 ng/L in S-WWTP. The estrogenic (androgenic) activity as 17beta-estradiol (E2) equivalents (EEQ) or testosterone (Te) equivalents (TEQ) was consequently 620 ng E2/L (570 ng Te/L) and 580 ng E2/L (800 ng Te/L) for the two WWTPs. The removal efficiencies of the above mentioned sole target chemicals were 51%-100% for K-WWTP and 55.6%-100% for S-WWTP. The removal efficiencies of EEQ were about 73% for both WWTPs, while the removal efficiencies of TEQ were 62.1% for K-WWTP and 98.4% for S-WWTP. In addition, chemical-derived EEQ were about 1.2%-52.4% of those by ER-binding assay for K-WWTP and the corresponding ratios were 1.3%-83.3% for S-WWTP, while chemical derived TEQ were less than 3% of values measured by the AR-binding assay for both WWTPs.

Horm Metab Res. 2009 Oct 27;
Ziegler CG, Mohn C, Lamounier-Zepter V, Rettori V, Bornstein SR, Krug AW, Ehrhart-Bornstein M

Endogenous cannabinoids are important signaling molecules in neuroendocrine control of homeostatic and reproductive functions including stress response and energy metabolism. The hypothalamic paraventricular and supraoptic nuclei have been shown to release endocannabinoids, which act as retrograde messengers to modulate the synaptic release of glutamate during stress response. This study endeavors to elucidate possible interaction of the endocannabinoid system with the regulation of adrenocortical function at the adrenal level. Human adrenocortical NCI-H295R cells and normal human adrenal glands were used to study the possible effects of anandamide and cannabinoid receptor 1 (CB1) antagonist SR141716A on aldosterone and cortisol secretion. Our data indicate the expression of CB1 in human adrenal cortex and adrenocortical NCI-H295R cells; CB2 was not expressed. Furthermore, anandamide inhibited basal release and stimulated release of adrenocortical steroids (corticosterone and aldosterone); this effect was reversed by CB1 antagonist (SR141716A). Therefore, the endocannabinoid system at the level of the adrenal, can directly influence adrenocortical steroidogenesis.

Ugeskr Laeger. 2009 Oct 26; 171(44): 3197-200
Hitz MF, Jensen JE

Climate changes have many unbeneficial effects on human health, but may also have beneficial effects. An increased mean temperature reduces the incidence of death due to hypothermia and cardiovascular disease and may increase exercise frequency. As the ozone layer decreases, the synthesis of vitamin D in the organism will also increase. An increased level of plasma vitamin D has beneficial effects on bone- and muscle health, seems to reduce cancer incidence and mortality and reduces the prevalence of autoimmune- and cardiovascular disease.

Am J Med. 2009 Nov; 122(11): 1056-60
Lee P, Greenfield JR, Seibel MJ, Eisman JA, Center JR

BACKGROUND: Obesity is associated with hypovitaminosis D. Whether body mass index (BMI) determines the replacement dose of vitamin D to achieve sufficiency is unclear. OBJECTIVE: To determine the relationship between BMI and serum 25-OH vitamin D concentrations and whether the increase in serum 25-OH vitamin D concentrations with vitamin D replacement is dependent on BMI. METHODS: Retrospective review of anthropometric data and serum 25-OH vitamin D concentrations in 95 patients attending an outpatient clinic in a tertiary hospital. In a second component of the study, 17 hospital inpatients with severe vitamin D deficiency (serum 25-OH D concentrations<6 ng/mL [15 nmol/L]) were supplemented with 10,000 units vitamin D(3)/day orally for 1 week. Biochemistry and anthropometric measurements were compared before and after vitamin D replacement. RESULTS: Serum 25-OH vitamin D concentrations correlated negatively with BMI in the 95 outpatients (r(2) = 0.11, P <.01). In the longitudinal study, BMI correlated positively with serum intact parathyroid hormone (r(2) = 0.84, P <.01) and negatively with 1.25-(OH)(2) vitamin D (r(2) = 0.19, P=.06) at baseline. Serum 25-OH D concentrations achieved following 1 week of vitamin D(3) replacement correlated negatively with BMI (r(2) = 0.63, P <.01). CONCLUSION: Efficacy of vitamin D supplementation is dependent on BMI. Overweight and obese patients with hypovitaminosis D might require higher doses of vitamin D to achieve vitamin D repletion compared with individuals with normal body weight.

steroids. 2009 Oct 23;
Scarth JP, Spencer HA, Hudson SC, Teale P, Gray BP, Hillyer LL

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.

J Am Coll Cardiol. 2009 Oct 27; 54(18): 1674-82
Mak GJ, Ledwidge MT, Watson CJ, Phelan DM, Dawkins IR, Murphy NF, Patle AK, Baugh JA, McDonald KM

OBJECTIVES: This study was designed to evaluate the impact of eplerenone on collagen turnover in preserved systolic function heart failure (HFPSF). BACKGROUND: Despite growing interest in abnormal collagen metabolism as a feature of HFPSF with diastolic dysfunction, the natural history of markers of collagen turnover and the impact of selective aldosterone antagonism on this natural history remains unknown. METHODS: We evaluated 44 patients with HFPSF, randomly assigned to control (n = 20) or eplerenone 25 mg daily (n = 24) for 6 months, increased to 50 mg daily from 6 to 12 months. Serum markers of collagen turnover and inflammation were analyzed at baseline and at 6 and 12 months and included pro-collagen type-I and -III aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha. Doppler-echocardiographic assessment of diastolic filling indexes and tissue Doppler analyses were also obtained. RESULTS: The mean age of the patients was 80 +/- 7.8 years; 46% were male; 64% were receiving an angiotensin-converting enzyme inhibitor, 34% an angiotensin-II receptor blocker, and 68% were receiving beta-blocker therapy. Pro-collagen type-III and -I aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha increased with time in the control group. Eplerenone treatment had no significant impact on any biomarker at 6 months but attenuated the increase in pro-collagen type-III aminoterminal peptide at 12 months (p = 0.006). Eplerenone therapy was associated with modest effects on diastolic function without any impact on clinical variables or brain natriuretic peptide. CONCLUSIONS: This study demonstrates progressive increases in markers of collagen turnover and inflammation in HFPSF with diastolic dysfunction. Despite high background utilization of renin-angiotensin-aldosterone modulators, eplerenone therapy prevents a progressive increase in pro-collagen type-III aminoterminal peptide and may have a role in management of this disease. (The Effect of Eplerenone and Atorvastatin on Markers of Collagen Turnover in Diastolic Heart Failure; NCT00505336).

Ann Neurol. 2009 Oct; 66(4): 564-8
Eikermann-Haerter K, Baum MJ, Ferrari MD, van den Maagdenberg AM, Moskowitz MA, Ayata C

Familial hemiplegic migraine type 1 (FHM1), a severe migraine with aura variant, is caused by mutations in the CACNA1A gene. Mutant mice carrying the FHM1 R192Q mutation exhibit increased propensity for cortical spreading depression (CSD), a propagating wave of neuroglial depolarization implicated in migraine aura. The CSD phenotype is stronger in female R192Q mutants and diminishes after ovariectomy. Here, we show that orchiectomy reciprocally increases CSD susceptibility in R192Q mutant mice. Chronic testosterone replacement restores CSD susceptibility by an androgen receptor-dependent mechanism. Hence, androgens modulate genetically-enhanced CSD susceptibility and may provide a novel prophylactic target for migraine.