Successful treatment of life-threatening pentoxifylline intoxication by high-flux hemodialysis.

High-flux hemodialysis is the method of choice for the treatment of many life threatening intoxications. Reports on intoxication with pentoxifylline are rare, and although pharmacokinetic properties of the drug suggest a potential role for hemodialysis, there are no published reports on extracorporeal treatment attempts. We report the first case of successful treatment of potentially life-threatening pentoxifylline intoxication by high-flux hemodialysis. Based on this single case, dialysis should be considered, especially in anuric patients with pentoxifylline intoxication.

Prenatal benzoate treatment in urea cycle defects.

OBJECTIVE: In patients with severe urea cycle defects (UCD) metabolic decompensation with hyperammonaemia typically occurs during the first days of life resulting in severe neurological damage or death. Benzoate can eliminate nitrogen independent of the urea cycle. Usually, benzoate is started soon after birth, but prenatal administration might improve metabolic stability. DESIGN: Two fetuses with a prenatal diagnosis of UCD (female: citrullinaemia; male: ornithine transcarbamylase deficiency) were loaded with benzoate prenatally via the placenta by infusing their mothers with benzoate. Benzoate concentrations were measured in umbilical cord blood and the blood of the mothers and their newborns. RESULTS: Therapeutic concentrations of benzoate were found in umbilical cord blood and in the children's blood. Thus, benzoate transfer across the placenta was demonstrated. Plasma ammonia and glutamine levels in the postnatal period were within the normal range. CONCLUSIONS: Benzoate infusion of the mother shortly before birth is safe and results in therapeutic levels of benzoate in umbilical cord blood.

Nifedipine concentration in maternal and umbilical cord blood after nifedipine gastrointestinal therapeutic system for tocolysis.

OBJECTIVE: To determine nifedipine concentrations in maternal plasma at steady state, and maternal and umbilical cord plasma at delivery, after tocolysis with nifedipine gastrointestinal therapeutic system (GITS) tablets. DESIGN: Prospective clinical pharmacokinetic study. SETTING: Department of Obstetrics at the Zurich University Hospital. POPULATION: Pregnant women treated for threatened preterm labour. METHODS: GITS dosage titrated to clinical response (30-150 mg/day). Nifedipine concentrations by high-performance liquid chromatography and turbo ion spray tandem mass spectrometry. MAIN OUTCOME MEASURES: Steady-state nifedipine concentrations in maternal blood and nifedipine concentrations in maternal and corresponding umbilical cord blood at delivery. RESULTS: Steady-state nifedipine concentrations (micrograms/l, mean +/- SE) were 54 +/- 6 (all doses, n = 31), 38 +/- 8 (60 mg/day, n = 13), and 92 +/- 12 (150 mg/day, n = 7) (P < 0.002). Umbilical cord and maternal concentrations both declined in a ln-linear regression with elimination half-lives of 20.4 and 17.4 hours. Linear regression showed a correlation between umbilical and maternal concentrations of 0.77 +/- 0.1 (n = 21, mean +/- SE). CONCLUSIONS: Steady-state plasma nifedipine concentrations after repeated dosing with nifedipine GITS 30-150 mg/day in pregnant women with preterm labour do not exceed 100 micrograms/l; fetal levels are 77% of maternal levels.

Effects of combined supplementation with B vitamins and antioxidants on plasma levels of asymmetric dimethylarginine (ADMA) in subjects with elevated risk for cardiovascular disease.

Elevated plasma asymmetric dimethylarginine (ADMA) concentrations have been suggested as a potential risk factor for cardiovascular disease (CVD). Studies indicate a linkage between hyperhomocysteinemia, oxidative stress and ADMA metabolism. We tested the hypothesis that combined supplementation of B vitamins and antioxidants reduces ADMA concentrations in subjects with at least two CVD risk factors. A total of 123 men and women (58+/-8.1 years) were randomly assigned to take either a preparation including B vitamins and antioxidants (verum) or placebo for 6 months in a double-blind design. Blood concentrations of ADMA, symmetric dimethylarginine (SDMA), L-arginine, B vitamins, total homocysteine (tHcy), alpha-tocopherol, antioxidant capacity (TEAC), and oxLDL were measured pre- and post-intervention. Treatment with verum significantly decreased tHcy (-2.14 micromol/L; P<0.001) and significantly increased TEAC values (+39.3 microM; P<0.022), but no effect on ADMA was observed. OxLDL was significantly reduced in verum (-7.3 U/L; P=0.001) and placebo (-9.2U/L; P<0.001). At baseline, significant correlations were found only between ADMA and SDMA (r=0.281; P=0.002), L-arginine/ADMA and SDMA (r=-0.294; P<0.001), L-arginine/ADMA and oxLDL (r=-0.281; P=0.016), and L-arginine/ADMA and age (r=-0.231; P=0.010). Our results indicate that combined supplementation of B vitamins and antioxidants is not an adequate strategy to reduce ADMA plasma levels in subjects with elevated CVD risk.

Quantitative determination of forty-eight antidepressants and antipsychotics in human serum by HPLC tandem mass spectrometry: a multi-level, single-sample approach.

This method describes the simultaneous determination of amisulpride, amitriptyline, aripiprazole, benperidol, chlorpromazine, chlorprothixene, citalopram, clomipramine, clozapine, desipramine, doxepin, fluoxetine, flupentixol, fluphenazine, fluvoxamine, haloperidol, hydroxyrisperidone, imipramine, levomepromazine, maprotiline, mianserine, mirtazapine, moclobemide, norclomipramine, nordoxepin, norfluoxetine, nortriptyline, O-desmethylvenlafaxine, olanzapine, opipramol, paroxetine, perazine, perphenazine, pimozide, pipamperone, quetiapine, reboxetine, risperidone, sertraline, sulpiride, thioridazine, trazodone, trimipramine, venlafaxine, viloxazine, ziprasidone, zotepine and zuclopenthixol with a single sample/triple injection approach. Drugs were assigned to subgroups covering low, medium and high concentrations (overall range of therapeutic levels to be considered: 0.5-2000 ng/mL) by further dilution of the supernatant obtained after the first protein precipitation. Chromatographic separation was necessary for isobaric mass fragments and performed on a monolithic C18 column (50mmx4.6mm) with methanol gradient and 5mM acetate buffer at pH 3.9. The injection interval was 8 min. A set of three internal standards was used for quantification of drugs with widely varying hydrophobicity. After electrospray ionization positive ion fragments were detected in the multiple reaction monitoring (MRM) mode with an API 4000 tandem mass spectrometer. Regression parameters of calibration curves and limits of quantification showed good covering of therapeutic and subtherapeutic ranges with an average correlation coefficient of 0.9988. Imprecision and inaccuracy measures were prepared for intra- and inter-assay comparisons at three concentration ranges in all subgroups. Average coefficients of variation were 6.1% for intra-assay and 7.4% for inter-assay comparisons, while average deviations from spiked concentrations were 4.8% for intra-assay and 4.2% for inter-assay comparisons, respectively. Recovery rates, measured as the percent recoveries of spiked serum samples against standard solutions without serum matrix, varied between 92 and 111%, with an average of 101%. As the only exception, the olanzapine response was much higher (185%) in serum matrix than in matrix-free controls.

Measurement of accidental urinary insulin loss from a dislocated intraperitoneal insulin catheter.

We report the case of a type-2 diabetic woman who received continuous intraperitoneal insulin infusion and developed deterioration of metabolic control by accidental insulin loss into urine (54 U per day) as a consequence of catheter migration which probably resulted in bladder wall injury. Due to iodine allergy of this patient, an analyte addition procedure for insulin quantification in urine had to be applied to allow proof of insulin loss from the catheter tip before as well as reversal to zero insulin excretion after implantation of a new intraperitoneal port and a shorter catheter. The lost fraction of insulin accounted nearly completely for the difference between pre- and postoperatively required insulin doses (146 versus 88 U per day).

Intracrinology and the local enzymatic control of hormone distribution and metabolism: dehydroepiandrosterone does not just act as a prohormone for androgens and estrogens.

The study of subcellular environments for the interaction of biomolecules and observations of certain features of hormone actions have nourished the concept of 'intracrinology', which describes hormone actions within a singular cell, in contrast to the well-described autocrine, paracrine and endocrine fashion. Synthesis and metabolism of DHEA make it a likely candidate for intracrine actions in target tissues. Recent experimental findings are reviewed in this context.

Testosterone-induced susceptibility to Plasmodium chabaudi malaria: persistence after withdrawal of testosterone.

Testosterone induces susceptibility to Plasmodium chabaudi malaria by imposing restrictions on those mechanisms which mediate resistance controlled by genes of the H-2 complex and the non-H-2 background in mice. This study investigated whether these restrictions are abolished after withdrawal of testosterone. Female mice of the inbred strain C57BL/10 were treated with 0.9 mg testosterone twice a week for 3 weeks and testosterone was then withdrawn for 12 weeks. The treatment raised plasma testosterone levels from 0.18 ng/ml to 3.79 ng/ml. After the testosterone treatment, these levels progressively dropped and reached 0.21 ng/ml by week 12 after testosterone withdrawal. Surprisingly, however, the testosterone-induced susceptibility still persisted. When mice were challenged on week 12 after testosterone withdrawal, P. chabaudi infections were still fatal in testosterone-treated mice, in contrast to self-healing infections in resistant, i.e. untreated, control mice. In addition, testosterone caused a persistent decrease in the levels of total IgG antibodies, especially IgG1 and IgG2b isotypes. In contrast, testosterone-induced changes in spleen cells, such as the reduction in number by 50%, the relative increase in CD8+ cells and the decrease in Ig+ cells, as well as the acquisition of the susceptible phenotype, were completely reversed on week 10 after testosterone withdrawal at the latest. Testosterone did not affect the production of the TH1-signalling cytokine interferon-gamma and the TH2-signalling cytokines interleukin (IL)-4 and IL-10 in response to P. chabaudi malaria. Together, our data indicated that the gene-controlled host resistance to P. chabaudi malaria is subject to superior hormonal imprinting: when once induced by testosterone, mechanisms which suppress resistance thus causing susceptibility persist independently of testosterone.

Improvement of cytochrome P450c17 catalytic processivity as a novel mechanism to attenuate hormonal consequences of enzyme decay in the testes after a gonadotropin surge.

OBJECTIVE: The aim of this study was to gain understanding of the apparent discrepancy between the moderate restriction of testosterone synthesizing capacity and the nearly complete decay of the androgen-producing enzyme, cytochrome P450c17 (CYP17; steroid 17 alpha-hydroxylase/17,20-lyase), in rat testes during the desensitization phase induced by a single, high-dose gonadotropin (human chorionic gonadotropin, hCG) injection. DESIGN AND METHODS: Adult male rats received 25 IU hCG i.v., and purified Leydig cells and crude interstitial cell microsomes were prepared 0, 4, 12, 24, 48, 72, 120 and 192 h afterwards. Component CYP17 activities, i.e. simultaneously catalyzed productive androgen formation and abortive 17 alpha-hydroxyprogesterone release, and their ratio (processivity), were compared with CYP17 levels and testosterone secretion rates. RESULTS: Leydig cells isolated 48 h after the artificial hCG surge produce 62% less testosterone than control cells upon stimulation in vitro, though CYP17 levels are reduced by 97%. Its total activity decreases by 87%, resulting in a 4.5-fold rise in the turnover number; the processivity is additionally improved 5-fold over controls. Parallel changes occur in interstitial cell microsomes; a negative linear correlation exists between the ratio of productive over total CYP17 activities and the actual CYP17 concentrations. CYP17 is partly denatured to P420 during hCG action, but other heme proteins (cytochrome b5) remain unchanged. Animal treatment with estradiol results in CYP17 down-regulation without any concomitant effect on enzyme processivity. CONCLUSION: Improved CYP17 processivity is suggested to be the consequence of (otherwise rate-limiting) improved electron transfer efficiency towards CYP17. It explains the relatively high testosterone secretion during Leydig cell desensitization and is interpreted to be a protective mechanism to confine adverse consequences of enzyme decay.

Novel connections between NADPH-induced lipid peroxidation and cytochrome P450 inactivation, and antioxidant and enzyme protective properties of estradiol in gonadal membranes.

This study uses microsomal membranes from rat testis tissue, including the cytochrome P450c17 (steroid 17 alpha-monooxygenase/17 alpha-hydroxyprogesterone aldolase, catalyzing the conversion of progesterone to androstenedione), to decipher the possible relation of NADPH-induced (no exogenous iron added) lipid peroxidation and cytochrome P450 inactivation and the protective effect of certain steroids. NADPH (300 microM) causes a 3.6-fold stimulation of malondialdehyde formation (thiobarbituric acid-reactive substances) and a 29% cytochrome P450c17 loss within 1 h at 37 degrees C, but has no effect on lipid peroxidation in the presence of the iron chelator desferrioxamine. Hydrogen peroxide has only marginal effects. The antioxidant efficiency of estradiol (IC50 = 13.9 microM) is higher than its cytochrome P450c17 protective efficiency (IC50 = 33.0 microM), whereas androstenedione does not inhibit lipid peroxidation but protects cytochrome P450c17 completely. The human choriogonadotropin-induced degradation of cytochrome P450c17 in incubated decapsulated testes can not be correlated with a stimulation of lipid peroxidation, and it is partially inhibited by estradiol but completely abolished by androstenedione. It is concluded (I) that NADPH stimulates iron-dependent generation of reactive oxygen species by the monooxygenase system even in the presence of certain P450 ligands in the physiological membrane environment, (II) that membrane lipid peroxidation may be suppressed by hydrophobic steroids acting as antioxidants such as estradiol, (III) that steroid ligands stabilize cytochrome P450c17 against inactivation in the presence of NADPH even if they do not act as substrates and do not possess antioxidant activity, and (IV) that the choriogonadotropin-induced down-regulation of cytochrome P450c17 is not due to accumulating steroids acting as "pseudosubstrates" as occasionally supposed.

Protein phosphorylation changes ligand-binding efficiency of cytochrome P450c17 (CYP17) and accelerates its proteolytic degradation: putative relevance for hormonal regulation of CYP17 activity.

Two novel mechanisms of protein kinase function in the complex gonadotropic regulation of the bifunctional cytochrome P450c17 (CYP17), the rate-limiting enzyme of androgen synthesis within the smooth endoplasmic reticulum of gonadal endocrine cells, are reported. In microsomal membranes from rat testes, the maximal type I optical difference spectrum induced by the physiological CYP17 substrate, progesterone, as a measure of spin state transition due to hydrophobic ligand-protein interaction is enhanced by 24% within 15 minutes in the presence of MgATP; the dissociation constant decreases from 71 to 43 nM. Testicular cytosol does not modify this effect which is completely abolished by the protein kinase inhibitor, bisindolylmaleimide, and which does not occur with ketoconazole as ligand. Furthermore, CYP17 degradation by cytosolic protease(s) is 2.5-fold accelerated by ATP; this action is completely reversed by the protein kinase inhibitors bisindolylmaleimide (half-maximal protective concentration 2.04 microM) and KT5720 (99 nM). The former compound also prevents human choriogonadotropin-induced testicular CYP17 inactivation in situ. It is concluded that protein kinase A-catalyzed target phosphorylation integrates the known biphasic steroidogenic response upon hormonal stimulation by initial improvement of substrate accommodation followed by counter-regulatory promotion of CYP17 proteolysis.

Effects of compatible solutes on mammalian cytochrome P450 stability.

The sensitivity of pig cytochrome P450c17 (CYP17), an endoplasmic reticulum membrane-bound enzyme, towards heat denaturation (48 degrees C) was measured by the P450-to-P420 spectral transition indicating conformational labilization of the protein. Both sucrose and glucose have comparable and increasingly protective effects at concentrations ranging from 100 to 800 mM, while ectoine, a novel zwitterionic compatible solute which regulates bacterial osmoadaptation and stabilizes cytoplasmic enzymes, has a strong labilizing effect on CYP17 and favours its proteolytic inactivation possibly by electrostatic derangements. Sucrose or glucose, but not ectoine, can therefore eventually be proposed as compatible stabilizers of cytochrome P450 structures.

Dietary testosterone suppresses protective responsiveness to Plasmodium chabaudi malaria.

This study investigates the effect of orally administered testosterone on serum testosterone levels and immune responses including outcome of Plasmodium chabaudi malaria. Female C57BL/10 mice were fed on a diet impregnated with 17 alpha-methyl-testosterone for 3 weeks. This raised the circulating testosterone levels from 0.28 ng/ml to 2.69 ng/ml on the average. In these mice, blood-stage infections of P. chabaudi resulted in a lethal outcome, whereas protective immunity developed in about 80% of mice fed on control diet without testosterone. Dietary 17 alpha-methyl-testosterone reduced the capacity of peritoneal cells to generate reactive oxygen intermediates after stimulation with C3b-coated zymosan and phorbol-myristate-acetate. Also, mice fed on dietary 17 alpha-methyl-testosterone responded to heat-killed Salmonella typhimurium with a higher increase in serum TNF, whereas the induced increase in the production of IL-10 by spleen cells was largely suppressed and no effect was found with respect to the production of IFN-gamma and IL-4. Our data indicate that the method of oral administration of 17 alpha-methyl-testosterone raises circulating testosterone to levels that impair protective immune responses to P. chabaudi malaria.

Ligand dependence of cytochrome P450c17 protection against proteolytic inactivation: structural, methodological and functional implications.

Rate constants for the subtilisin-catalyzed proteolytic inactivation of cytochrome P450c17 (CYP17), the endoplasmic reticulum membrane-bound limiting enzyme of gonadal androgen synthesis, have been determined in the absence and presence of various CYP17 ligands and correlated with fractional enzyme saturation (Y). Extrapolation to Y = 1 reveals 15.1-, 4.0- and 7.4-fold enzyme stabilization with progesterone (substrate-type ligand), testosterone (product-type ligand) and ketoconazole (imidazole-type inhibitory ligand), respectively. Structural features of ligand accommodation can therefore be monitored by the susceptibility of target enzymes to proteolysis. It is further proposed that specific protection of a membrane protein by ligand binding during proteolytic digestion may assist in the purification of that protein. Evidence is finally presented that the gonadotropin-induced rapid CYP17 down-regulation is not promoted by an elevation of steroid hormone levels.

Rapid down-regulation of testicular androgen biosynthesis at increased environmental temperature is due to cytochrome P450c17 (CYP17) thermolability in Leydig cells, but not in endoplasmic reticulum membranes.

To identify possible molecular targets in moderate heat-induced, short-term derangements of rat testicular endocrine function, rates of androgen and precursor biosynthesis and key enzyme concentrations were compared at 38 degrees C (normal body core temperature) and 31 degrees C (normal scrotal temperature) in three in-vitro models of decreasing complexity and increasing specificity. In purified Leydig cells and similarly in decapsulated testes, gross testosterone secretion was by 20% higher at 38 degrees C under basal conditions and during the initial phase of stimulation with hCG or cAMP; longer (> 1 hour) exposure to the elevated temperature resulted in a marked decrease (52% after 3 hours) of testosterone response to hCG or cAMP as compared to the corresponding rates at 31 degrees C. This phenomenon was neither due to the development of hormone resistance at the receptor level nor to restricted cholesterol supply and turnover nor to increased testosterone accumulation. Whereas mitochondrial CYP11A (cytochrome P450cscc: cholesterol monooxygenase) was absolutely temperature-insensitive in all systems tested, CYP17 (cytochrome P450c17: steroid-17 alpha-monooxygenase/C17, 20-aldolase) in the smooth endoplasmic reticulum responded with a 57% loss in whole testes and 39% loss in purified Leydig cells upon a 3-hour temperature elevation from 31 degrees C to 38 degrees C. In contrast, CYP17 was stable (4% loss) when tested directly in microsomal membranes. It is concluded that CYP17, but not CYP11A, is very sensitive towards even moderate elevation of environmental temperature, and that this apparent lability is not an intrinsic property of the enzyme protein but rather mediated by heat-activated intracellular factors.

Partial uncoupling of luteinizing hormone and prolactin pulse coincidence in hyperandrogenemic women.

A partly synchronized pulsatile secretion of luteinizing hormone (LH) and prolactin has previously been suggested as an indication of the coupling of the respective pulse generators under certain conditions. In women with hyperandrogenemic chronic anovulation, episodic LH secretion is disturbed. It was, therefore, the aim of the present study to evaluate possible changes in episodic prolactin secretion pattern and in LH/prolactin co-pulsatility, and to relate the results to the accelerated LH pulse frequencies often seen in patients with hyperandrogenemic chronic anovulation. Blood samples of 32 patients with hyperandrogenemia were taken at 10-min intervals between 10.00 and 20.00. Nine regularly cycling women with normal hormone levels served as controls. In the women with hyperandrogenemia, despite an average 41% rise of LH pulse frequency, prolactin pulse frequency decreased slightly by 14% as compared to controls; no correlation between the two parameters was found (r = 0.162). The number of coincident LH and prolactin pulses increased continuously with accelerating LH frequency. The best fitting function was a hyperbola which was limited by the maximal observed prolactin frequency. As a consequence, the fraction of LH pulses that were co-secreted with prolactin episodes decreased with higher LH pulse frequencies, while the fraction of prolactin pulses concomitant with LH pulses increased. Our data provide evidence that in women with hyperandrogenemic chronic anovulation a pathological LH pulse frequency is no longer coupled with pulsatile prolactin secretion, suggesting an isolated alteration of the central neuronal control mechanism for LH secretion.

Testosterone-induced compared with oestradiol-induced immunosuppression against Plasmodium chabaudi malaria.

Testosterone suppresses immunity against malaria caused by Plasmodium chabaudi in B10 mice. Since this effect is probably not mediated through the classical androgen-receptor response, we investigated whether testosterone might act, after aromatization to oestradiol (OE2), through the oestrogen receptor (ER). Indeed, OE2 was found to act immunosuppressively when used at only about 1% of the immunosuppressive dose of testosterone. This becomes evident as an OE2-induced suppression of self-healing of P. chabaudi infections in female and castrated male B10 mice. The immunosuppressive OE2 effect is associated with a 16-fold increase in the circulating level of OE2 and can be prevented by ER blockers such as tamoxifen and clomifene. In contrast, the immunosuppressive effect of testosterone, which is not associated with any changes in the level of OE2, cannot be abolished by ER blockers or by aromatase inhibitors, such as atamestane and drofazar hydrochloride. Moreover, OE2 and testosterone act differently on spleen cells; OE2 induces a decrease in CD(4+)-T-cells, whereas testosterone causes an increase in CD(8+)-T-cells and a decrease in total nucleated spleen cells. The immunosuppressive effect of testosterone, but not that of OE2, can be adoptively transferred to syngeneic mice by nucleated spleen cells, predominantly T-cells. Our data show that the immunosuppressive activity of testosterone, in contrast to OE2, is not mediated through the ER. The immunosuppressive action of testosterone is therefore thought to be primarily mediated through a non-genomic mechanism.

Norharman (beta-carboline) as a potent inhibitory ligand for steroidogenic cytochromes P450 (CYP11 and CYP17).

Norharman (beta-carboline, a so-called mammalian alkaloid) is identified as a high-affinity type II ligand for two steroidogenic cytochromes P450, viz. CYP11 in rat adrenal mitochondria and CYP17 in rat testicular microsomes. Progesterone binding to CYP17 is competitively inhibited, with Ki = 2.6 microM norharman, whereas harman, tetrahydronorharman and tetrahydroharman are nearly ineffective. The potential role of norharman as an endogenous modulator of steroid hormone biosynthesis and as a basic drug for development of more specific cytochrome P450 inhibitors is emphasized.

Pulsatile luteinizing hormone secretion pattern in hyperandrogenemic women.

OBJECTIVE: To elucidate changes in gonadotropin secretion pattern in patients with hyperandrogenemic chronic anovulation of various origins. DESIGN AND PARTICIPANTS: Hyperandrogenemic patients (n = 32), divided into subgroups according to certain clinical and biochemical criteria, and a control group (n = 9) of regularly cycling women with normal androgen and PRL levels were prospectively investigated. SETTING: Infertility and Biochemical Endocrinology Unit, Department of Obstetrics and Gynecology, University of Düsseldorf, Düsseldorf, Germany. MAIN OUTCOME MEASURES: Blood samples for radioimmunological analyses of gonadotropins and steroids were taken at 10-minute intervals for 12-hour sampling periods. In nonamenorrheic patients, investigations were performed on the 5th day of a cycle. Pulsatile LH and FSH data were analyzed by computerized peak identification programs. RESULTS: In hyperandrogenemic women, mean LH levels were higher than controls, the most elevated concentrations being observed in women with secondary amenorrhea (subgroup 5), in those selected for elevated mean LH levels (subgroup 3), and in those with elevated T and/or androstenedione (A) but normal DHEAS levels (subgroup 1). With the exception of patients with DHEAS elevations but normal T and A levels (subgroup 2), LH pulse frequency and amplitude were increased with most distinct effects occurring in subgroups 3 and 5. Highly elevated T and free T levels were observed in subgroup 5 and in overweight patients (subgroup 6). Estrone (E1) serum concentrations were highest in those subgroups (3 and 5) in which acceleration of LH pulse frequency and increments in LH pulse amplitude were most pronounced; these parameters correlated significantly with E1 levels. CONCLUSIONS: Changes in pulsatile LH release in patients with hyperandrogenemic chronic anovulation correlate primarily with elevated E1 levels, rather than with T or A serum concentrations.