Metabolism of dehydroepiandrosterone sulfate (DS) in normal women and women with high DS concentrations.

In order to determine the contribution of serum dehydroepiandrosterone sulfate (DS) to estrone (E1) production in normal women and the effect of chronic elevation of the serum DS concentration on DS metabolism, four normal women and four women with high endogenous serum DS were infused with [3H]DS and [14C]E1 or [14C]testosterone for 6 h. Blood samples were analyzed for radioactivity as DS, dehydroepiandrosterone (D), androstenedione, testosterone, and dihydrotestosterone. Urine was collected for analysis of creatinine, 17-ketosteroids (17-KS), and radioactivity as estrone (E1). The serum DS of 12.4 +/- 1.44 mumol/L (mean +/- SE) in the group with high DS was higher than that of 3.96 +/- 1.0 mumol/L (1.46 +/- 0.37 micrograms/mL) in the normals (P less than 0.005). Those with high DS also had increased 17-KS (13.2 +/- 2.0 vs. 5.68 +/- 0.68 mg/day, P less than 0.025) and a higher blood production rate of DS (PBDS) (126 +/- 21 (n = 3) vs. 54.3 +/- 13.8 mmol/day, P less than 0.05) but a lower MCRDS (10.94 +/- 0.61 (n = 3) vs. 13.8 +/- 0.27 L/day, P less than 0.01) than that in normals. In the four normal women the fraction of infused DS converted to estrone ( [rho]BMDS E1) was 0.00078 +/- 0.00018, the amount of E1 produced from serum DS was 41.3 +/- 15 nmol/day, the basal plasma E1 was 102 +/- 18 pmol/L, the MCRE1 was 1340 +/- 181 L/day, the value for blood production of E1 (PBE1) was 129 +/- 12 nmol/day, and the portion of E1 derived from DS was 30.4 +/- 9.4%. Correlation analysis of the data from these eight subjects showed that 17-KS, PBDS, and the serum DS were all correlated with body surface area, body weight, and ponderal index and that 17-KS excretion, PBDS, and serum DS were all correlated with one another. The most important predictors of 17-KS excretion were serum DS (P less than 0.001) and the ponderal index (P less than 0.05).

Sample pretreatment to minimize interferences from whole blood in the radioimmunoassay for cyclosporine.

There is much controversy as to whether the analysis of cyclosporine (CsA) should be performed by radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC), and whether the specimen should be serum or whole blood. Whole-blood specimens present specific advantages, but the presence of hemoglobin (Hgb) and other endogenous compounds can produce major errors in the RIA by "quenching" the analytical signal or by interfering with the antigen-antibody binding in the assay. We have developed a simple pretreatment step to remove the Hgb and other proteins responsible for this error. Red cells in whole blood are hemolyzed with a mixture of acetonitrile and water, the protein precipitated with acetonitrile, and the supernatant assayed by RIA. In a controlled study in which CsA concentration was kept constant and the Hgb concentration varied, the errors in measurement were directly proportional (r = 0.999) to the Hgb concentration. CsA values were spuriously deflated or inflated by 22.7 micrograms/L for each gram per 100 milliliters that the Hgb deviated from the 9.2 g/100 ml Hgb in the CsA calibration standards. In a similar study in which patient samples (n = 57) were assayed with and without pretreatment, the fractional error induced by Hgb was compounded in some patients by additional interferences that also appear to be removed by sample pretreatment. Without the pretreatment, CsA values could be in error by 33% when the Hgb varied 4 g/100 ml, thus providing potentially misleading results to the clinician. An I-125-labeled CsA tracer (purported not to be affected by the "quenching" interference of Hgb) produced consistently higher results when it was substituted for the tritiated CsA tracer contained in the Sandoz kit. In summary, sample pretreatment appears to be the simplest method of effectively removing endogenous interferences and minimizing erroneous results from whole blood submitted to the Sandoz RIA for CsA analysis.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro.

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0-1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [methyl-3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203-218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching addition). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching addition). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Virilism as a late manifestation in the Bardet-Biedl syndrome.

The second case of virilism as a late manifestation of Bardet-Biedl syndrome (BBS) is described, with endocrine and histological evaluation. Both cases manifested ovulatory cycles and developed virilism in adulthood. Elevated plasma testosterone and 17-OH-progesterone were not suppressed by dexamethasone but were suppressed by medroxyprogesterone acetate. Peripheral and ovarian venous blood obtained at the time of surgery demonstrated a marked gradient for testosterone in both ovaries and for progesterone in the ovary bearing the corpus luteum. Histological evaluation of the ovaries demonstrated bilateral ovarian stromal hyperplasia with focal hyperthecosis. Bilateral ovariectomy resulted in complete correction of the endocrine abnormality, although the established hirsutism remains a mark of previous androgen excess.

Role of dehydroepiandrosterone sulfate as a prehormone for ovarian steroidogenesis.

The endocrine effects of induction of ovulation with menotropins were studied in 43 patients: 11 with hypothalamic amenorrhea and 32 with the polycystic ovary syndrome. Patients with polycystic ovary syndrome had higher base-line values of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17 beta-estradiol, dehydroepiandrosterone sulfate, testosterone, and a higher testosterone-free index than those with hypothalamic amenorrhea. During treatment with menotropins, patients with polycystic ovary syndrome had higher values of serum LH, prolactin, dehydroepiandrosterone sulfate, testosterone, percent free testosterone, testosterone-free index, and body weight than those with hypothalamic amenorrhea; serum FSH, dose of menotropins per kilogram body weight, and total follicular volume were higher in patients with hypothalamic amenorrhea than in those with polycystic ovary syndrome. Multiple linear regression after log transformation demonstrated that the testosterone-free index was predicted statistically by total ovarian volume and dehydroepiandrosterone sulfate and that serum 17 beta-estradiol was predicted statistically by total ovarian volume and testosterone-free index. Adding dexamethasone to menotropins in six patients with polycystic ovary syndrome produced significant decreases in 17 beta-estradiol, dehydroepiandrosterone sulfate, testosterone, and testosterone-free index. Higher concentrations of endogenous serum LH and dehydroepiandrosterone sulfate in patients with polycystic ovary syndrome in comparison with those with hypothalamic amenorrhea were associated with higher concentrations of serum testosterone, a lower total follicular volume, and an effective response to menotropins at a lower serum FSH and a lower dose of menotropins per kilogram body weight. These data suggest that serum dehydroepiandrosterone sulfate may be a precursor for ovarian steroidogenesis.

Testosterone free index correlates best with dehydroepiandrosterone sulfate.

Correlation coefficients for dehydroepiandrosterone sulfate (DHEAS) were determined in women on menotropin. DHEAS was significantly correlated with testosterone free index (TFI), 0.78**; percentage free testosterone (%FT), 0.66**; androstenedione (delta 4A), 0.66*; luteinizing hormone (LH), 0.55**; LH/follicle-stimulating hormone (FSH) ratio, 0.55**; 17-OH-progesterone (17-P), 0.55**; testosterone (T), 0.53**; weight (WT), 0.40**, urinary estriol glucuronide (E3G), 0.33*; and free cortisol index (FFI), 0.32*, with 43 df but not with prolactin (PRL), 0.25. Normal male DHEAS (3.5 +/- 1.2, 25) (microgram/ml; mean +/- standard deviation, n) was higher than normal female DHEAS (2.4 +/- 1.1, 27), P less than 0.01 and DHEAS in women on oral contraceptives (1.9 +/- 1.1, 17) was slightly lower than in normal females, P greater than 0.2. In the combined population (male, female, and females on oral contraceptives) DHEAS was correlated with TFI (0.56**), T (0.54**), %FT (0.52**), delta 4A (0.40**), and age (-0.40**) with 66 df and 17-P (0.30*) with 54 df. TFI appears to be one determinant of plasma DHEAS, **P less than 0.01. *P less than 0.05.

Diagnosis-specific serum 17 beta-estradiol (E2) upper limits for treatment with menotropins using a 125I direct E2 assay.

Statistical evaluation of 133 cycles of induction of ovulation using generalized linear models demonstrated that the occurrence and severity of ovarian hyperstimulation was influenced by the serum 17 beta-estradiol (E2) concentration (P less than 0.001), conception (P less than 0.001), and the endocrinologic diagnosis, polycystic ovary syndrome (PCO) or hypothalamic amenorrhea (HA) (P less than 0.01). When menotropins were administered between 5:00 P.M. and 8:00 P.M. and blood was drawn at 8:00 A.M., an upper limit for serum E2 in patients with HA of 2417 pg/ml or an upper limit for patients with PCO of 3778 pg/ml gave an approximate 5% risk of severe ovarian hyperstimulation in conception cycles and a 1.3% risk of severe hyperstimulation in nonconception cycles. Comparison of our E2 radioimmunoassay involving extraction and chromatography to the Pantex immunodirect Estradiol 125I kit (Pantex, Santa Monica, CA) demonstrated no detectable systematic error, allowing the use of these limits with either assay. The ovulating injection of human chorionic gonadotropin was given at 5:00 P.M. to 8:00 P.M. on the evening of blood drawing as soon as the first follicle reached an average diameter of 14 mm or greater. The ultrasound parameters allow the chance of pregnancy to be optimized and the chance of multiple gestation to be minimized. Serum E2 monitoring indicates when the risk of ovarian hyperstimulation is too great for human chorionic gonadotropin to be given.

Plasma estradiol is superior to ultrasound and urinary estriol glucuronide as a predictor of ovarian hyperstimulation during induction of ovulation with menotropins.

In order to compare the effectiveness of 8:00 A.M. plasma 17 beta-estradiol (E2), 24-hour urinary estriol glucuronide (E3G), and ultrasound as predictors of ovarian hyperstimulation, 70 cycles of induction of ovulation with 5:00 P.M. to 8:00 P.M. injection of menotropins from 28 subjects were evaluated. Hyperstimulation was four times more frequent in pregnancy than in nonpregnancy cycles (P less than 0.005). The hyperstimulation score (range, 0 to 6) was correlated with plasma E2 (0.63, P less than 0.01), the number of follicles (0.31, P less than 0.05), the duration of treatment (0.31, P less than 0.05), and urinary E3G (0.25, P less than 0.05). Plasma E2 was the best predictor of the hyperstimulation score, and plasma E2 was far superior to both urinary E3G and the number of follicles. Management with ultrasound alone is insufficient to prevent severe ovarian hyperstimulation. With this protocol, human chorionic gonadotropin may be given as soon as the first follicle reaches 1.4 cm in diameter as long as plasma E2 is less than 4000 pg/ml. The values of plasma E2 are dependent on the interval between blood sampling and injection of menotropins.

Immunoreactive beta-endorphin increases after i.v. glucose in obese human subjects.

The plasma beta-endorphin of obese human subjects and non-obese controls was compared following the intravenous infusion of 25 grams of glucose. The plasma beta-endorphin of the obese subjects was significantly higher than controls one hour and four and one half hours after glucose infusion. The increased beta-endorphin of the obese subjects was associated with falling blood sugar.

Sugar, opioids and binge eating.

There is evidence that endogenous opiates are involved in the control of feeding in experimental animals. Several types of experimental obesity are associated with increased opiate production and/or increased numbers and sensitivity of opiate receptors. Research with experimental animals suggests that nutrients, particularly sugar, have an effect on feeding behavior that is mediated by opiates. For instance, the obesity-producing effect of a palatable diet in rodents is blocked by opiate antagonists. Stress induced feeding in rodents leads to preferential sucrose ingestion and is blocked by opiate antagonists and beta-endorphin. The effect of nutrients on the endogenous opiate system of humans is less clear. Clinical experience suggest that carbohydrates (sugar in particular) play a role in binge eating and obesity. Many binge eaters preferentially eat sweets during a binge. Many obese individuals consume more than half of their total daily calories as carbohydrates. Sweet snacking is a frequent behavior at times of stress. Recent evidence suggests that sugar can lead to increased beta-endorphin production in obese subjects.

Correlation of glycosylated hemoglobin measured by affinity vs ion exchange chromatography with mean blood sugar in pediatric IDDM patients.

Affinity chromatography provides a more specific estimate of glycosylated hemoglobin (GlyHb) than does ion exchange chromatography (HbA1). However, whether GlyHb correlates closer than HbA1 with mean blood glucose has not been established. GlyHb and HbA1 were measured in pediatric IDDM patients attending a clinic (n = 285 visits) over a one year period and correlated with the mean of a patient's blood glucose measurements from records of home blood glucose monitoring. Mean GlyHb was higher than mean HbA1 (10.8% vs 9.6%) as was its standard deviation (2.2% vs 1.5%). While both GlyHb (r = 0.75) and HbA1 (r = 0.65) were strongly correlated with estimates of mean blood glucose, the correlation with GlyHb was significantly stronger than with HbA1 for the entire spectrum of metabolic control (P = 0.03), as well as for a segregated group of 'poorly controlled' patients with mean blood glucose greater than 150 mg/dl (P = 0.04). The results suggest that GlyHb is more accurate than HbA1 for estimating metabolic control and that GlyHb shows greater discriminating power than HbA1, especially at high concentrations of blood sugar. The mean blood glucose can be estimated from the equation: mean blood glucose (mg/dl) = (11.3 x GlyHb) + 32.

Use of particle-loaded membranes to extract steroids for high-performance liquid chromatographic analyses improved analyte stability and detection.

Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using the novel particle-loaded octyl (C8)-bonded silica in PTFE membrane. Extracts are directly injected, without further concentration, onto a narrow (2.0 mm) or conventional (4.6 mm) bore octyldecyl (C18) HPLC column. Method performance data demonstrate linearity from 0.4 microgram/dl (low limit of detection) up to at least 60 micrograms/dl. Extraction recoveries exceeded 85% and precision (between-run) R.S.D.s averaged < 5%. Interferences were minimal and selectivity was improved over conventional immunochemical steroid assays. When compared to large particle sorbents packed in columns or to traditional liquid-liquid extractions, the membrane extracted steroids in less time, used less reagent, and had smaller elution volumes, thereby obviating steroid instability/adsorption problems associated with traditional concentrating techniques required to improve analytical sensitivity.

New markers in serum for lymphocyte activation for predicting allograft rejection. Neopterin and soluble interleukin-2 receptor.

Investigations have been reported on the use of neopterin and soluble interleukin-2 receptors as rejection markers. Each of these analytes is available as part of an easy-to-perform analytic method. Each is available in a diagnostic kit that is accurate, precise, and sensitive enough to provide good clinical information. Both of these materials have a significant relationship with the cell-mediated immune response, particularly at the level of T-cell activation. Neopterin does increase significantly in cases of transplant rejection but shows greater response to viral infection. Soluble interleukin-2 receptors increase during periods of transplant rejection but also respond during T-cell activation brought on by bacterial or viral infection. The use of these markers is best described for kidney transplants and less well documented for liver and heart transplants.

Misleading elevation of the free thyroxine index in nursing home residents.

The significance of an elevated free thyroxine index (FTI) as an indicator of hyperthyroidism was studied while screening 651 elderly nursing home residents. Eleven subjects had FTI elevations. Most of these patients were chronically ill and/or malnourished. Clinical assessment, repeated FTI determinations, and subsequent measurements of levels of triiodothyronine, free thyroxine by equilibrium dialysis, and thyrotropin (thyroid-stimulating hormone) by sensitive assay showed that all subjects with FTI elevation were euthyroid. The FTI elevation in the chronically ill institutionalized elderly patient is not necessarily an expression of hyperthyroidism.

Concentrations of cyclosporin A and its metabolites in human tissues postmortem.

We report concentrations and distribution of cyclosporine A (CsA) and individual metabolites associated with various organ tissues and whole-blood specimens collected at autopsy from seven transplant patients who received CsA therapy. Solid-phase extraction (SPE) and specific high-performance liquid chromatographic (HPLC) procedures were used to separate and quantitate the cyclosporines. Patterns of deposition were unique for the various tissue types. Metabolites M17, M1, M18, and M8 (in addition to CsA) were the principal compounds detected in significant quantities. On a per weight basis, the sum concentration of CsA and metabolites in organ tissues was up to 53 times greater than in companion whole-blood specimens. Metabolite M17 prevailed in most tissues, except in fat and pancreas, where CsA was predominant. Overall, pancreas specimens contained a greater concentration of cyclosporines (per kilogram of tissue), followed consecutively by spleen, liver, fat, kidney, lung, bone marrow, heart, and whole blood. No CsA-related compounds were detected in brain or spinal cord tissue.