Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry.

The application of gas chromatography and high-pressure liquid chromatography/mass spectrometry techniques for analysis of plasma cholesterol oxidation products is described. Cholesterol oxides that are widely identified in biological samples were subjected to gas (GC) and high-pressure liquid chromatographic (HPLC) separations, and their detection and characterization by mass spectrometry (MS) were compared. Analysis of cholesterol oxides from plasma samples revealed distinct advantages for each method according to the specific cholesterol oxide in question. Whereas HPLC/MS analysis of cholesterol oxides provided less resolution and lower sensitivity as compared to GC/MS, a distinct advantage was evident for direct measurements of cholesterol-7-hydroperoxides and 7-ketocholesterol. These two cholesterol oxides are particularly sensitive to storage in solvents, derivatization procedures, and analytical conditions used for GC analysis, which are minimized or avoided using the HPLC/MS conditions described. Analysis of human and rabbit plasma samples identified cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol); 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol (cholesterol-5 alpha, 6 alpha-epoxide); 5 alpha-cholestane-3 beta, 5,6 beta-triol (cholestanetriol); 3 beta-hydroxycholest-5-ene-7-one (7-ketocholesterol); and 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide) as commonly occurring components (trivial names indicated in parentheses). The latter two compounds were dramatically increased in hypercholesterolemic samples and were found in approximately equal amounts in the free cholesterol and cholesteryl ester fractions. Although most of the plasma cholesterol oxides are found in the dietary cholesterol, others are not, particularly cholesterol-5 beta,6 beta-epoxide, suggesting that at least some of these compounds are formed by in vivo oxidation of cholesterol. Despite the readily measurable levels of the above cholesterol oxides, as well as other less prominent oxides, there was no evidence of cholesterol-7-hydroperoxides associated with plasma free cholesterol. Although several of the plasma cholesterol oxides may derive from cholesterol-7-hydroperoxides, it appears that the latter are either unstable and decompose in plasma, are metabolized to other cholesterol oxidation products, or break down during their isolation.

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Growth hormone response to clonidine in anovulatory infertile women resistant to clomiphene citrate stimulation.

OBJECTIVE: To evaluate the GH response to the clonidine test in a group of infertile women and to determine their ovulatory response to clomiphene citrate (CC) stimulation. DESIGN: Prospective study. SETTING: Reproductive endocrinology unit. PATIENT(S): Thirty-three anovulatory infertile women (age range, 25-36 years) and 9 healthy controls with normal ovulation. INTERVENTION(S): In the early follicular phase, 0.3 mg of clonidine was administered between 8:30 and 9:00 A.M. and blood samples were collected for 120 minutes thereafter for measurement of serum GH levels. Plasma levels of insulin and glucose were measured after a 75-g glucose load, and CC was given at a dosage of 50-250 mg/d for ovulation-induction. MAIN OUTCOME MEASURE(S): Serum concentrations of GH, insulin-like growth factor I, insulin, and insulin-like growth factor binding protein-1. RESULT(S): On the basis of their ovulatory response to CC, 15 patients were considered nonresponsive (group 1) and 18 patients were considered responsive (group 2). Baseline levels of GH, insulin-like growth factor I, and insulin-like growth factor binding protein-1 were similar in the two groups of patients and the controls. The GH response to clonidine was significantly greater in group 2 and in the controls than in group 1. Concentrations of insulin and glucose after the glucose load were not different among the three groups. CONCLUSION(S): Women who were resistant to CC had a reduced GH response to clonidine. These data suggest that adequate GH secretory capacity is important for CC action.

In vitro metabolism of GV150013X by using liver microsomes and liver tissue slices from different species.

The metabolism of GV150013X was studied in vitro using washed liver microsomes and liver tissue slices from different species. This work was carried out in order to compare the metabolite profiles resulting from incubation of GV150013X with human, rat, dog and rabbit liver microsomes and those from rat, rabbit and human liver tissue slices. This compound was found to be converted to at least 8 metabolites by rat and human liver microsomes. In rabbit liver microsomes, the three metabolites M4, M7 and M8, and in dog liver microsomes metabolite M1, were not detected. The main metabolites, M2, M5 and M6, were present in human, rat and rabbit liver tissue slices, while the three metabolites M3, M4 and M8 and metabolite M1 were not detected in rabbit and rat liver tissue slices, respectively. In rat liver tissue slices, the major metabolites plus five minor metabolites, one sulphate conjugate of a monohydroxylated, three trihydroxylated and one dihydroxylated were identified based on HPLC retention time and thermospray-mass spectrometry data. Quantitative species differences among rat, dog, rabbit and human were observed, while qualitative differences only between rabbit and other species were detected.

Metabolism of a novel CCK-B antagonist in rat, dog and human liver microsomes.

1. The in vitro metabolism of a novel CCK-B antagonist ((+)-N-[1-adamantane-1-methyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H- 1, 5-benzodiazepin-3-yl]N'-phenyl-urea; GV150013X) was investigated using rat, dog and human liver microsomes. 2. Four monohydroxy and four dihydroxy metabolites of GV150013X in rat and man were identified by comparison with authentic standards using HPLC and mass spectrometry. 3. The dihydroxy metabolite M1 was not detected in dog liver microsomes mixtures. 4. The formation of dihydroxylated metabolites proceeds via monohydroxylated metabolites M5 and M8 and not directly from GV150013X. 5. A monohydroxy metabolite M5 was the major metabolite in rat and dog, with M5 and dihydroxy metabolites M2 and M3 major metabolites in man.

Trazodone intravenously administered and plasma prolactin levels.

Plasma prolactin levels during intravenous Trazodone infusion have been evaluated. Although the modifications were not statistically significant, a slight decrease of plasma prolactin levels was noted. This result suggests that Trazodone has no remarkable effect on hypothalamic function.

Effectiveness of norethisterone enantate in the treatment of the climacteic syndrome.

The Authors have studied the effects of Norethisterone enantate given i.m. every one or two months to 75 patients complaining of climacteric syndrome either spontaneous or induced surgically. An overall evaluation of the effectiveness of the treatment allowed the conclusion that with both timing of administration it is possible to obtain in the majority of patients (80%) a very good control of the climacteric symptoms with negligible side effects. A more careful clinical and laboratory evaluation has shown a fast regression of the symptoms already after one month of drug administration. Therefore norethisterone enantate may be considered an effective therapeutic alternative to estrogens and androgens in the management of the climacteric syndrome.

Demonstration of human chorionic gonadotropin during the second half of the menstrual cycle in plasma of regularly menstruating women users of copper IUds.

An attempt has been made of demonstrating the beta subunit of HCG in the plasma of 13 regularly menstruating women wearing a copper IUD. Eighteen menstrual cycles were explored with a total of 94 specimens obtained at 3-day intervals starting from the 16th day of the cycle. A cross reaction with LH was excluded by simultaneous assay of this hormone. Progesteron was also assayed so as to exclude anovulatory cycles. Five out of 12 ovulatory cycles yelded positive results with HCG values ranging from 171 to 692 mIU per ml. We therefore agree with the hypothesis according to which copper IUDs do not interfere with fecundation but with subsequent stages, specifically about the time of nidation of the blastocyst in the uterus.

The effect of lisuride hydrogen maleate in the hyperprolactinaemia-amenorrhoea syndrome: clinical and hormonal responses.

Eleven women with secondary amenorrhoea and hyperprolactinaemia were treated with lisuride, a new semisynthetic ergot derivative. Acute administration of lisuride (100 microgram orally) significantly suppressed serum prolactin (PRL) levels in nine out of eleven subjects. In these nine patients, prolonged treatment with lisuride (50--200 microgram daily) lowered PRL levels into the normal range, menstrual cycles were resumed and eight patients ovulated. Two patients, in whom lisuride failed to lower PRL levels into the normal range, did not resume normal menstruation. These data demonstrate that lisuride may be used clinically to lower PRL levels and to restore cyclic gonadotrophin secretion and ovulation in amenorrhoeic patients with hyperprolactinaemia.

Histological and ultrastructural changes of the endometrium in women using inert and copper-coiled IUDs.

The authors examined biopsy material obtained from women wearing inert and copper-coiled IUDs, under the light and electron microscopes. Examination of this material leads to the conclusion that there are no specific lesion attributable to either type of IUD. It can also be said that lesions characteristic for an inflammatory reaction prevail over all other possible endometrial changes.

The amenorrhoea-galactorrhea syndrome: present diagnostic and therapeutic perspectives. / La sindrome amenorrea-galatorrea: prospettive diagnostiche e terapeutiche attuali

Recent progress in the field of radioimmunology, especially as regards prolactin (PRL) assay, as well as the use of new drugs have opened new perspectives for a better understanding of the "amenorrhoea-galactorrhea syndrome" both from the diagnostic and therapeutic points of view. This syndrome which formerly was considered infrequent, is now observed more and more often, perhaps as a result of the more widespread use of drugs such as phenothiazines, reserpine derivatives, alpha-methyldopa and tricyclic antidepressants, as well as owing to the large-scale use of estrogens and above all of estrogen-progestogen contraceptives. The problems of etiopathogenesis and management of the amenorrhea-galactorrhea syndrome are complicated by the fact that a variety of factors are responsible, as is shown also by the classical nosographic classification with the Forbes-Albright syndrome in the presence of a pituitary tumour, the Chiari-Frommel syndrome of amenorrhea-galactorrhea following pregnancy, and the Argonz-del Castillo-Ahumada syndrome in the absence of lither pregnancy or tumor.

Estradiol and estetrol plasma levels before and after intravenous administration of dehydroepiandrosterone sulfate in normal and pathologic pregnancies.

Intravenous injections of 50 mg dehydroepiandrosterone sulfate (DHEAS) were given to 7 women with normal pregnancies and 5 with pathologic pregnancies and the serum levels of 17 beta-estradiol and estetrol were assayed before and at 15 or 30-minute intervals for three hours after the injection. All tests were carried out during the 25th to 36th week of amenorrhea. Serum estradiol rose rapidly in normal subjects and remained high to the end of the test. In patients with gestational pathology the estradiol pattern was not significantly different from that of the controls. Esterol plasma levels showed a biphasic pattern with an initial rise at 30 min. and a second rise at 90 min. in normal pregnancies, whereas in pathologic pregnancies this response was either lacking completely or was markedly reduced compared to the controls.

Effect of lisuride on inhibition of lactation and serum prolactin.

Lisuride, a new semisynthetic ergot derivative, was given to 53 women to inhibit lactation; 26 women had 300 micrograms daily and 27 had 600 micrograms daily for seven days. Eight lactating women acted as controls. Lisuride effectively inhibited lactation and also suppressed the serum prolactin levels; the latter effect was dose related. Lisuride produced no untoward side effects.

Effect of Lisuride on serum prolactin levels during puerperium.

A new semisynthetic ergot derivative, Lisuride, has been administered to 40 women who wanted to have lactation inhibited. Purpose of the study was to evaluate the drug's effect on serum PRL levels. Treatment was carried out for 7 days with daily doses of 300 or 600 microgram. Lisuride lowered PRL levels, and the decrease was significantly more marked for the 600 microgram dose.