Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Enhancement of lysosomal glycohydrolase activity in human primary B lymphocytes during spontaneous apoptosis.

It has been shown that lysosomes are involved in B cell apoptosis but lysosomal glycohydrolases have never been investigated during this event. In this study we determined the enzymatic activities of some lysosomal glycohydrolases in human tonsil B lymphocytes (TBL) undergoing in vitro spontaneous apoptosis. Fluorimetric methods were used to evaluate the activities of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase, alpha-galactosidase, beta-glucuronidase and alpha-fucosidase. Results show that in TBL during spontaneous apoptosis, there is a significant increase in the activity of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase and beta-galactosidase. Also beta-glucuronidase and alpha-fucosidase activities increase but not in a significant manner. Further studies on beta-hexosaminidases revealed that also mRNA expression of the alpha- and beta-subunits, which constitute these enzymes, increases during spontaneous TBL apoptosis. When TBL are protected from apoptosis by the thiol molecule N-acetyl-L-cysteine (NAC), there is no longer any increase in glycohydrolase activities and mRNA expression of beta-hexosaminidase alpha- and beta-subunits. This study demonstrates for the first time that the activities and expression of some lysosomal glycohydrolases are enhanced in TBL during spontaneous apoptosis and that these increases are prevented when TBL apoptosis is inhibited.

Usefulness of early morning urine estrone-3-glucuronide assay in the monitoring ovarian secretory function in precocious puberty.

To evaluate the usefulness of the urinary estrone-3-glucuronide (EI-3-G) in the monitoring of the ovarian function in girls, we studied 11 girls with idiopathic central precocious puberty (ICPP) treated with LHRH analogs (LHRHa) for 2-5 years. Plasma LH, FSH, 17-beta-Estradiol (E2) levels, early morning urine (EMU) E1-3-G concentrations, were assessed before and 3, 6, 12 months after the onset of treatment. As expected, mean basal plasma LH, FSH and E2 concentrations, as well as mean basal EMU E1-3-G levels were significantly (p < 0.01) higher in patients studied than in normal, age matched, prepubertal controls. Three out of the 11 sexually advanced girls showed undetectable (< 15 pg/ml) basal plasma E2 values. On the contrary, in each patient studied, individual basal E1-3-G levels were higher than in normal age-matched prepubertal girls. LHRHa treatment significantly suppressed both basal and peak stimulated plasma gonadotropins, plasma E2 and EMU E1-3-G. However, while serum E2 levels were below the assay detection limit, not allowing to assess the degree of gonadal suppression, E1-3-G urinary concentrations were detectable in each subject treated, in the range of the normal prepubertal values. EMU E1-3-G determination seems to be a very sensitive and reliable approach to the monitoring of the effectiveness of LHRHa treatment in sexually advanced girls, allowing to detect very low estrogen concentrations and to achieve the desired ovarian suppression.

Estrogenic suppression by different administration schedules of goserelin depot for treatment of endometriosis.

Eighteen patients affected by laparoscopically confirmed endometriosis were randomly assigned to three different schedules of treatment with gonadotropin-releasing hormone agonist (GnRH-a) (goserelin depot formulation 3.6 mg) every 28 days for 6 months. Six women received the first implant in early follicular phase, 4 in late luteal phase and 8 in 3rd and 17th day from onset of menses. Pretreatment and posttreatment laparoscopic score, performed according to the American Fertility Society scoring system, were compared; a significant reduction in the extent of disease was observed in each group investigated (A and C: p < 0.01; B: p < 0.05). In each treatment group after the second GnRH-a implant the mean levels of estrone-3-glucuronide (E1-3G), daily measured in early morning urine specimens during the control cycle and the first three months of therapy, were suppressed to menopausal women range. In group B during the 2nd and 3rd month of therapy, the urinary mean levels of E1-3G were significantly lower than in group A and C. In conclusion the different goserelin depot administration schedules gave similar laparoscopic improvement, in spite of the first GnRH-a administration in luteal phase allowed a more marked estrogenic suppression.

Sex steroid modulation of neurohypophysial hormone receptors in human nonpregnant myometrium.

Neurohypophysial hormone receptors were studied in myometrial specimens obtained from nonpregnant women using binding and in vitro contractility studies. The mathematical modeling of self- and cross-competition curves among [3H]oxytocin (OT), [3H]arginine vasopressin, the V1 vasopressin (VP) antagonist [3H]d(CH2)5TyrMeAVP, the corresponding unlabeled peptides, and the OT agonist [Thr4, Gly7] OT strongly indicates the presence of multiple classes of OT and arginine vasopressin receptors. The latter show the same pharmacological characteristics as the neurohypophysial hormone receptors described by our group for the human pregnant myometrium; in addition, they regulate the contractility of uterine strips. Blocking experiments were performed to evaluate the relative OT and V1 VP receptor distribution in 30 uterine specimens obtained from normal cycling and postmenopausal women. The glucuronoconjugate metabolites of 17 beta-estradiol and progesterone were also measured in 16 patients in early morning urine samples taken the same day as surgery. Our results show that V1 VP receptors are not only present but also biologically active in all the uterine specimens studied with virtually equal density in normal cycling and postmenopausal women. However, their concentrations do not correlate with either estrogen or progesterone urinary levels. The lowest OT receptor density was found at mid-cycle and in menopause, independently of any correlation with the urinary estrogens. Conversely, OT receptors rise sharply in the late luteal phase and during menstruation. In addition they show a positive relationship with glucuronoconjugate metabolites of progesterone levels. These results indicate that progesterone does not inhibit the expression of uterine OT receptors in the human uterus. Furthermore, they imply that neurohypophysial hormones are involved in the control of uterine activity during the menstrual cycle.

Ovarian function in premenopausal women affected by breast cancer: the measurement of glucuronoconjugate metabolites of 17 beta-estradiol and progesterone throughout one entire menstrual cycle.

For many years, hypersecretion of estrogens has been suspected of being one of the major risk factors of breast cancer for premenopausal women. Seventeen premenopausal women, who had undergone lumpectomy because of breast cancer (T1a No Mo) 3 yr before entering the study, were compared to 9 normal women of similar age, parity and body weight. A chemiluminescent method was used for the determination of estrone-3-glucuronide (E1-3G) and pregnanediol-3-glucuronide (Pd-3G) in early morning urine samples collected for an entire menstrual cycle of each of the 26 subjects. During the follicular phase, no significant differences in E1-3G and/or Pd-3G excretion were found between the two groups. During the luteal phase the E1-3G/Pd-3G ratio in the early, middle and late luteal phase had significantly increased in the women with breast cancer, in spite of normal Pd-3G excretion. Therefore, the measurement of glucuronoconjugate metabolites of ovarian hormones in overnight urine might be conveniently applied to the study of ovarian function in subjects with breast cancer. Furthermore, the results of this study may indicate that an estrogen/progesterone imbalance is an additional risk factor for the premenopausal breast cancer patient.

Measurement of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide in early morning urine samples to monitor ovarian function.

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects. Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.

Simultaneous determination of testosterone, dihydrotestosterone and 5 alpha-androstan-3 alpha,-17 beta-diol by isotopic dilution mass spectrometry in plasma and prostatic tissue of patients affected by benign prostatic hyperplasia. Effects of 3-month treatment with a GnRH analog.

This article reports an isotope dilution mass spectrometric method for the simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in human plasma and prostatic tissue. After addition of tri-deuterated steroids as internal standards to prostatic tissue homogenates or plasma samples, extraction was performed with diethylether. The extracts were purified by two chromatographic steps (Sep-Pak C 18 cartridge and Sephadex LH-20) and injected into a gas chromatograph coupled with a mass spectrometer after derivatization with heptafluorobutyric acid. This method was highly specific and showed good precision, accuracy, reproducibility and sensitivity. T, DHT, and 3 alpha-diol were measured in human plasma and in prostatic tissue of seven patients with benign prostatic hyperplasia (BPH) treated for 3 months with a long acting GnRH analog before surgery. Plasma levels of T, DHT, and 3 alpha-diol were reduced by GnRH analog treatment to castrate levels. The tissue concentrations of the same steroids, compared with those obtained from 19 untreated patients, showed a mean reduction of about 90% for DHT and 3 alpha-diol, and about 75% for T. These results suggest that about 90% of prostatic DHT and 3 alpha-diol depend on testicular activity because they are dramatically reduced after pharmacologic castration.

Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in diluted overnight urine. An approach to the study of luteal insufficiency.

The determination of the concentrations of estrone-3-glucuronide, pregnanediol-3-glucuronide and luteinizing hormone has been performed in early morning urine samples of 14 normal menstruating women using a timed and measured volume urine collection procedure. In order to investigate the variability of the urinary hormonal concentrations due to day-to-day differences in diuresis, the absolute hormonal concentrations have been corrected either for the urinary creatinine excretion or for the volume of urine voided during the night. The results demonstrate that both correction factors are able to reduce substantially the coefficient of variation values in comparison to the absolute hormonal concentrations. The urinary test of ovarian function has been performed in 11 infertile women affected by luteal insufficiency using the same procedure, and the hormonal profiles showed some alterations in both estrone-3-glucuronide and pregnanediol-3-glucuronide concentrations in comparison to the hormonal profiles of the normal subjects. Such alterations were significant in the single subject when integrated values of the hormonal data in defined time intervals were investigated.

Determination of testosterone and its tissue metabolites (DHT and 3 alpha-diol) in human plasma and prostatic tissue by isotopic dilution mass spectrometry.

5 alpha-Dihydrotestosterone has been widely measured in human prostatic tissue using RIA since it is involved in the pathogenesis of human prostatic hyperplasia and seems to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC-MS method for the simultaneous determination of testosterone (T), 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in prostatic tissue based on the isotopic dilution technique was developed. Tri-deuterated internal standards of each compound were previously synthetized in our laboratory. After extraction and purification on Sep-Pak C18 and Sephadex LH-20, T and its metabolites were measured as heptafluorobutyric ester (HFB) derivatives. Quantitative analysis was performed on a VG 7070 EQ mass spectromer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (mean +/- SD; ng/g tissue) found in nine human hypertrophic prostates were: T: 0.71 +/- 0.43; DHT: 4.46 +/- 1.41; 3 alpha-diol: 0.34 +/- 0.23. Preliminary results obtained from the detection of the three androgens in human prostatic hyperplasia treated for 3 months with GnRH before surgery seem to indicate that DHT concentration decreases more than 10 times. Values obtained (n = 1; ng/g tissue) were: T: 0.194; DHT: 0.255; 3 alpha-diol: 0.015.