In this study the in-vitro biopotency and glycoform distribution of human recombinant follicle stimulating hormone (FSH, Org 32489) has been assessed. The biopotency of recombinant FSH was studied using animal (rat Sertoli) and human (granulosa-lutein) cell models. Recombinant FSH, as measured in the rat Sertoli cell assay, was more potent than the urinary preparations Metrodin, Metrodin-HP and IS 70/45 with half maximal stimulation (ED50; mean +/- SEM, n > 3) occurring at 2.2 +/- 0.5 IU/I (recombinant FSH), 4.7 +/- 1.1 IU/I (Metrodin), 13.2 +/- 0.7 IU/I (Metrodin-HP) and 6.4 +/- 0.3 IU/I (IS 70/45); the pituitary preparation IRP 83/575 had an ED50 of 10.4 +/- 0.1 IU/I. Using human granulosa-lutein cells, cultured for up to 4 days in the absence of exogenous steroid precursors, recombinant FSH was either without effect (three out of five patients) or inhibited both oestradiol and progesterone secretion. FSH (83/575) was without effect on oestradiol with preparations from any of the patients but slightly stimulated (134 +/- 8%; mean +/- SEM, P < 0.05) progesterone production at the highest dose (80 IU/I). The distribution of FSH isoforms, assessed by polyclonal radioimmunoassay, following chromatofocusing over the ranges pH < 3.5 and pH 3.5-7.0 respectively was recombinant FSH, 12.4 and 87.6%; Metrodin, 19.8 and 80.2%; Metrodin-HP, 50.2 and 49.8%; IS 70/45, 15.0 and 85.0%; IS 83/575, 70.9 and 29.1%. All glycoforms were pI < 7.0 for the five preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Variability in the immunoreactive and bioactive follicle stimulating hormone content of human urinary menopausal gonadotrophin preparations.

The within- and between-batch variation in the immunoreactive and in-vitro bioactive FSH content of Pergonal, Metrodin and Metrodin-HP was investigated. Three batches of Pergonal and Metrodin, consisting of three ampoules in each batch, and three batches of Metrodin-HP, consisting of between one and three ampoules per batch, were selected at random. The follicle stimulating hormone (FSH) content of Pergonal, Metrodin and Metrodin-HP was determined by radioimmunoassay (R-FSH) and the in-vitro rat Sertoli cell bioassay (B-FSH) using the international urinary standard 70/45. The variability in the FSH content of the preparations was evaluated within and between batches by analysis of variance. Within-batch variability of B-FSH was not observed in Pergonal or Metrodin-HP but was seen in two batches of Metrodin in which the potency varied by up to 2.4 fold (P = 0.03). The between-batch R-FSH potencies of Pergonal (P1-P3) varied, with P2 (59.8 +/- 0.6) and P3 (61.7 +/- 0.9) being higher than P1 (47.1 +/- 1.5 mean +/- SEM IU/ampoule, P < 0.01). A similar pattern of variability was observed for B-FSH. For Metrodin, each of the batch R-FSH potencies was dissimilar (P < 0.02), with estimates ranging from 34.9 +/- 1.2 to 64.3 +/- 1.8 IU/ampoule. Furthermore, the extensive within-batch B-FSH variation from two batches confounded any meaningful comparison of between-batch variability. For Metrodin-HP, there was no between-batch B-FSH variation (29.0 +/- 6.1 to 33.0 +/- 0.3 IU/ampoule) and the R-FSH content was also well controlled (41.2 +/- 0.5 to 46.9 +/- 1.1 IU/ampoule).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1995

Age related changes in the pituitary-testicular axis in normal men; lower serum testosterone results from decreased bioactive LH drive.

OBJECTIVE: The mechanism underlying the slight hypoandrogenism associated with normal ageing remains unclear. We have therefore examined age related changes in the activity of the pituitary-testicular axis in healthy normal males. DESIGN: Random blood samples were obtained from 219 normal men whose ages ranged from 20 to 79 years. At the time of the study, none of the men had received treatment or had ever had any endocrine dysfunction diagnosed. MEASUREMENTS: Luteinizing hormone was measured in the subjects' plasma using a commercially available immunoradiometric assay (IRMA, Serono Maiaclone) and a fully validated in-vitro bioassay. Testosterone and FSH were measured using standard radioimmunoassays (RIA) whilst sex hormones binding globulin was assayed by an IRMA. RESULTS: Levels of total testosterone (total-T) and bioactive LH fell with age (r = -0.231 and -0.189 respectively) by 5.9 nmol/l and 2.3 IU/l respectively between grouped patients aged 20-39 years (Group A) and 60-79 years (Group C). In contrast, immunoreactive LH remained unchanged. The LH B:I ratio also fell with age (P < 0.0001) being 5.0 +/- 0.3 (group A) and 3.3 +/- 0.2 (group C), representing a fall of 33%. Since immunoreactivity remained constant, this fall primarily represented the decline in LH bioactivity. Bioactive, but not immunoreactive LH correlated to total-T (P = 0.009, n = 209) and the total-T:LH ratios fell by over 30% between groups A and C using the IRMA, but remained unchanged by bioassay. CONCLUSIONS: There is an underlying decline in both total-testosterone and free-testosterone index, and bioactive LH levels with advancing age, suggestive of a hypothalamo-pituitary defect which leads to lower bioactive LH levels which in turn are responsible for the diminished gonadal steroidogenesis. Elucidation of the mechanism underlying this slight decline in hypothalamopituitary testicular activity is complicated by differences between the data obtained by immunoassay or bioassay.

Adult↗

PubMed1995

Histologic evolution of radiofrequency lesions in an old human myocardial infarct causing ventricular tachycardia.

INTRODUCTION: Radiofrequency (RF) ablation of ventricular tachycardia (VT) late after myocardial infarction may be difficult due to characteristics of the infarct containing the reentry circuit. RF lesions in these infarcts in humans have not been characterized. METHODS AND RESULTS: Catheter mapping and ablation of VT originating from an anterior wall infarct was performed 8 days and again 12 hours prior to death. Pacing identified a region of abnormal conduction where RF ablation terminated VT. This region contained strips of myocytes sandwiched between endocardial fibrosis and dense scar. RF lesions ranged from 2 x 2 mm to 5 x 10 mm and were up to 3 mm in depth. Acute lesions showed superficial thrombus and early coagulation necrosis without inflammation. Older lesions showed coagulation necrosis, sparse neutrophil infiltrate, minimal granulation tissue, hemorrhage, and mixed inflammatory infiltrate along the lumen without re-endothelialization. CONCLUSION: In this patient, RF lesions had sufficient depth but not width to interrupt the thin, but potentially broad, sheets of myocytes in the reentry circuit. In thinned areas, RF lesions can extend to the epicardium. Selecting sites with abnormal electrograms confines RF lesions to the infarct region. Inflammation and hemorrhage could conceivably cause delayed effects of RF.

Aged↗

PubMed1995

Degree of activation of the pituitary-testicular axis in early pubertal boys with constitutional delay of growth and puberty determines the growth response to treatment with testosterone or oxandrolone.

Early pubertal boys (testicular volume, 4-6 mL) with constitutionally delayed growth and puberty were randomized to 3 months of treatment after a baseline 12-h overnight hormone profile: group 1 (n = 5), daily placebo; group 2 (n = 5), 2.5 mg oxandrolone daily; or group 3 (n = 6), 50-mg testosterone monthly im injections. LH and GH profiles (15-min samples) were analyzed by peak detection (Pulsar), Fourier transformation, and autocorrelation. FSH and testosterone levels were measured hourly, and insulin, sex hormone-binding globulin, insulin-like growth factor-I, and insulin-like growth factor-binding protein-3 levels were determined at 0800 h. Multiple regression was used to analyze the response to treatment (growth) with respect to baseline features. Endocrine variability was marked. Profiles ranged from unreactive to well established LH pulsatility and adult testosterone levels. The areas under the curve (AUC) for LH, FSH, and testosterone ranged 10-fold (4.4-46.3 IU/L.h), 8-fold (7.9-63.4 IU/L.h), and 45-fold (3.6-161.7 nmol/L.h), respectively. The growth response was individually varied, but significantly increased 0-6 months in the active treatment groups. Age, testicular volume, and LH AUC interacted significantly (r2 = 0.95; P < 0.05). Allowance for these produced a highly significant treatment effect (P = 0.006). Age, testicular volume, LH AUC, and testosterone AUC, but not treatment, significantly increased growth by 0-12 months (r2 = 0.88; P < 0.05). We demonstrate a spectrum of activation of the reproductive axis despite tight clinical staging. This, and not GH status at treatment commencement, influenced the growth response.

Adolescent↗

PubMed1995

Cannabinoids activate an inwardly rectifying potassium conductance and inhibit Q-type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor.

Rat brain cannabinoid receptor (CB-1) was stably transfected into the murine tumor line AtT-20 to study its coupling to inwardly rectifying potassium currents (Kir) and high voltage-activated calcium currents (ICa). In cells expressing CB-1 ("A-2" cells), cannabinoid agonist potently and stereospecifically activated Kir via a pertussis toxin-sensitive G protein. ICa in A-2 cells was sensitive to dihydropyridines and omega CTX MVIIC, less so to omega CgTX GVIA and insensitive to omega Aga IVa. In CB-1 expressing cells, cannabinoid agonist inhibited only the omega CTX MVIIC-sensitive component of ICa. Inhibition of Q-type ICa was voltage dependent and PTX sensitive, thus similar in character to the well-studied modulation of N-type ICa. An endogenous cannabinoid, anandamide, activated Kir and inhibited ICa as efficaciously as potent cannabinoid agonist. Immunocytochemical studies with antibodies specific for class A, B, C, D, and E voltage-dependent calcium channel alpha 1 subunits revealed that AtT-20 cells express each of these major classes of alpha 1 subunit.

Animals↗

PubMed1995

A modification of the pectoralis major myocutaneous flap that reduces the defect at the donor site.

A modification of the pectoralis major myocutaneous flap that reduces the cosmetic defect at the donor site is described.

Humans↗

PubMed1995

Immunometric assays of luteinizing hormone (LH): differences in recognition of plasma LH by anti-intact and beta-subunit-specific antibodies in various physiological and pathophysiological situations.

Restricted immunoreactivity of plasma luteinizing hormone (LH) has been described in some subjects when assayed with certain methods involving antibodies against intact LH. We have compared the performance of the Amerlite LH-30 (A) and Delfia LHSpec (D) assays (which include anti-intact and beta-specific antibodies, respectively) in normal and pathological conditions. As shown previously, results of the two systems were highly correlated with each other and, as we show here, with those of a bioassay. We found eight outliers (results outside the 95% confidence interval of the regression) among 427 samples studied from 121 subjects. Of the outliers, five had Delfia results in a range (< 1 IU/L) that was associated with poor assay precision for that assay, and the ratios of their values by both methods (A:D ratios) were very low. This ratio was affected by endocrine status, e.g., was lower in postmenopausal women than in premenopausal controls, and varied intraindividually within the same menstrual cycle. The restricted immunoreactivity described previously for assays involving anti-intact LH antibodies may, in part, reflect these differences, which, in turn, may reflect the presence of isoforms (e.g., glycoforms) that are differentially recognized by assays that have different antibody configurations.

Antibodies↗

PubMed1995

Coping strategies of caregivers of family members with dementia.

Data derived from a national sample of 89 caregivers of non-institutionalized family members with dementia were examined in order to identify the specific coping strategies caregivers utilized and whether the identified strategies were associated with negative or positive outcomes. Results from this study indicated that caregivers predominantly used problem-focused strategies. Further analysis demonstrated that employing more positive coping strategies did not necessarily result in a reduced sense of burden.

Adaptation, Psychological↗

PubMed1994

Proton uptake by cytochrome c oxidase on reduction and on ligand binding.

On reduction, cytochrome oxidase was found to take up 2.4 +/- 0.1 protons in the pH range 7.2-8.5, of which 2 are associated with the binuclear centre, and the remaining fractional proton with haem a/CuA. Ligation to oxidised cytochrome oxidase of the azide, formate, fluoride or cyanide anions is accompanied by uptake of one proton. In the case of the reduced enzyme, no protonation changes are observed on binding O2 (Hallén S. and Nilsson T. (1992) Biochemistry 31, 11853-11859) or CO. Cyanide binding to reduced oxidase is, in contrast, still accompanied by uptake of a proton. These findings are discussed in terms of our previously-published proposal for the ligand chemistry of the binuclear site. The results overall suggest a principle of electroneutrality of redox and ligand state changes of the binuclear centre, with charge compensations provided only by protonation reactions.

Animals↗

PubMed1994

Evidence for a role of protein kinase C in the sustained activation of rat dorsal horn neurons evoked by cutaneous mustard oil application.

The intracellular mechanisms involved in the sensitisation of spinal dorsal horn neurons brought about by sustained or repeated nociceptive inputs are unknown. The present experiments addressed any role of protein kinase (PKC) in sustained nociceptive responses of rat dorsal horn neurons by: (i) ionophoretic administration of PKC inhibitors whilst recording activity evoked by repeated cutaneous application of mustard oil; and (ii) assessing subcellular translocation of PKC evoked in spinal cord by cutaneous application of mustard oil. Both marked attenuation of mustard oil-induced neuronal activity by PKC inhibitors and selective translocation of PKC in spinal cord tissue ipsilateral to mustard oil application strongly supported a critical role of PKC in sustained nociceptive responses to mustard oil.

Administration, Cutaneous↗

PubMed1994

Oral ondansetron pharmacokinetics: the effect of chemotherapy.

The effect of a typical 5-day chemotherapy treatment with cisplatin (20-40 mg/m2 per day) and 5-fluorouracil (5-FU, 1 gm/m2 per day) on the pharmacokinetics of ondansetron was investigated. Twenty cancer patients received 8 mg of ondansetron in three periods, including an oral tablet on day 1, an intravenous infusion on day 4, and an oral tablet on day 5. Absolute bioavailability after the oral dosing on day 1 was 87.5 +/- 31.3%, and on day 5 was 85.2 +/- 22.1% (P > .05). Mean values of AUC, Cmax, Tmax, and half life on days 1 and 5 were 399 +/- 275 and 381 +/- 222 ng.hour/mL, 53.3 +/- 26.8 and 43.6 +/- 21.7 ng/mL, 1.9 +/- 1.4 and 2 +/- 1.4 hours, and 5.21 +/- 1.78 and 6.19 +/- 1.99 hours, respectively. These values were not significantly different (P > .05). In summary, this study showed that cisplatin and 5-FU did not significantly alter the pharmacokinetics of oral ondansetron in cancer patients during the 5 days of chemotherapy. Oral bioavailability of ondansetron appeared to be greater in cancer patients than in healthy subjects.

Administration, Oral↗

PubMed1994

Evidence for a role of metabotropic glutamate receptors in sustained nociceptive inputs to rat dorsal horn neurons.

Several antagonists at metabotropic glutamate (mGlu) receptors, when applied ionophoretically, inhibited the excitation of single dorsal horn neurons elicited by cutaneous administration of the C fibre-selective algogen, mustard oil. The selectivity and stereospecificity of AP3 isomers at mGlu, compared to NMDA receptors was confirmed on responses to agonists and matched by their effects on mustard oil-evoked activity.

Animals↗