Expression of 3beta-hydroxysteroid dehydrogenase-5,4-en isomerase activity by infiltrating ductal human breast carcinoma in vitro.

In order to determine whether human mammary tumors could contribute to progesterone synthesis from pregnenolone in breast cancer patients, homogenates of infiltrating ductal primary breast tumors at different stages of malignancy (Stages II and III) obtained from pre- and post-menopausal patients (n = 7, age 37-66 years) were incubated with [7n-3H]pregnenolone as substrate. Controls were heated homogenates instead of fresh homogenates. With the use of reverse-isotope dilution analysis, [3H]progesterone was isolated and characterized. No such metabolite was evident in the control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 0.02 to 4.0%. The results reveal that activity of 3beta-hydroxysteroid dehydrogenase-5,4-en isomerase that metabolizes pregnenolone to progesterone can be identified with the viable homogenates. It is suggested that there exists a potential for substantial progesterone synthesis in vivo. This conversion may be of considerable clinical, therapeutic, and pathophysiological significance in the patient with breast cancer. The biological impact of this conversion should be a high priority research objective.

A delta 4-3-keto pathway for testosterone synthesis in the human spermatozoa.

The ability of human spermatozoa to metabolize pregnenolone to progesterone and progesterone to testosterone was assessed. Sonicated specimens of freshly ejaculated sperm from two groups of husbands (n = 6, age 32-38 years; n = 6, age 30-51 years) of infertile couples in the range of sperm concentration between 237.5 and 568.5, 100.1 and 248.8 millions per ejaculate, were separately incubated with [7n-3H]pregnenolone and [1,2,6,7,16,17-3H]progesterone. Using the classical reverse-isotope dilution technique the desired products [3H]progesterone and [3H]testosterone formed from the respective substrates were isolated and characterized, yielding 1.4 to 12.2% and 3.1 x 10(-2) to 2.0 x 10(-1)%. Such metabolites were not evident in the controls. The results indicate that the human spermatozoa contain the enzymes necessary for the transformation of pregnenolone to testosterone via the delta 4-3-keto route.

Characterization of a delta 5-3 beta-hydroxy pathway of testosterone synthesis in the human spermatozoa.

Sonicated specimens of freshly ejaculated sperm from two groups of husbands (n = 6, age 32-38 years; n = 7, age 27-38 years) of infertile couples in the range of sperm concentration between 237.5 and 568.5; 131.1 and 256.7 millions per ejaculate were separately incubated with [7n-3H]pregnenolone and [1,2,6,7(-3)H]dehydroepiandrosterone. Using the reverse-isotope dilution technique [3H]dehydroepiandrosterone and [3H]testosterone formed from the respective substrates were isolated and characterized, yielding 1.2 x 10(-2) to 4.6 x 10(-20/0) and 7.1 x 10(-2) to 2.5 x 10(-10/0)/ Such metabolites were not evident in the controls. These results provide evidence for metabolic transformation of pregnenolone to testosterone via the delta 5-3 beta-hydroxy route.

Aromatization of [4-14C]testosterone to [14C]estradiol-17 beta by testicular tissue from male-to-female transsexuals on estrogen therapy.

Abnormality in testicular biosynthesis of sex hormones may accompany or cause transsexualism. With a view to determine whether the cells of male transsexual testicular tissue possess aromatic cytochrome P-450 activity, homogenates of the tissue from 4 phenotypic male-to-female transsexuals (2 Chinese and 2 Malays, aged 23-34 years) with XY karyotype who had been on estrogen therapy for 9-17 years until orchidectomy were incubated with [4-14C]testosterone as substrate. Using reverse-isotope dilution analysis, identity was established for [14C]estradiol-17 beta. No such metabolite was found in parallel incubations of heat-denatured enzymes. The extent of enzyme-catalyzed conversion was 9.3 x 10(-3) to 3.4 x 10(-2)%. The specific biosynthetic aromatase cytochrome P-450 activity contained in the tissue system can synthesize [14C]estradiol-17 beta from [4-14C]testosterone. Elevated aromatic expression in situ may have important effects on target tissues.

A comparative study of progesterone synthesis by term human fetal amnion and chorion.

Activity of 3 beta-hydroxysteroid dehydrogenase-5, 4-en isomerase was investigated with [7n-3H] pregnenolone as substrate in the homogenates of amnion and chorion collected from women (n = 6, age 20-34 years) after spontaneous labour at term (37-40 weeks gestation) from uncomplicated pregnancies. Controls were heat-denatured homogenates instead of fresh homogenates. Reverse-isotope dilution analysis gave positive identification of [3H] progesterone in all incubations of viable tissues. This metabolite was not evident in the controls. The extent of enzymic conversion of chorion, 1.4-31.1% was consistently higher than that of the amnion, 0.66-1.7%. The results show that the human fetal membranes can be a source of progesterone during pregnancy. The metabolic capability may have physiological significance in the induction and progression of labour to parturition.

Aflatoxin exposure in Singapore: blood aflatoxin levels in normal subjects, hepatitis B virus carriers and primary hepatocellular carcinoma patients.

Blood screening conducted on Singaporeans over 1991-1992 showed exposure to predominantly aflatoxin B1 and to a lesser extent G1. The extent of exposure to B1 among three groups of residents in Singapore, namely normal subjects (n = 423), hepatitis B virus carriers (n = 302) and primary hepatocellular carcinoma (PHC) patients (n = 58) were extensive as reflected by the positive rates of 15.1, 0.7 and 1.7 per cent respectively. However, the degree of individual exposure to this toxin among the three groups was considered low as shown by the low respective mean blood levels of 5.4 +/- 3.2 (range 3.0-17), 7.7 (range 7.5-7.9) and 7.5 picogrammes per ml of blood. It is not immediately clear whether or not such low levels would precipitate an undesirable health effect. The higher positive rate seen in normal subjects as compared with the other groups could be due to differences in dietary intake of aflatoxin B1, differences in metabolic patterns or both. About 70 per cent of PHC patients studied were carriers. The degree of aflatoxin B1 exposure among normal subjects in Singapore was a factor of 22.1 times less than that in Japan, 40.9 times less than that in Indonesia and 51.3 times less than that in the Philippines. Similarly, the extent of exposure among hepatitis B carriers in Singapore was a factor of 8.2 times, 39.6 times and 24.2 times less than those in the other three Asiatic countries respectively. The results reflected stringent Government control over the quality of food stuff imported into this country. As Singapore imports almost all of its dietary needs from elsewhere, it can afford to be selective at a cost. Aflatoxin M1, a metabolite of B1, was most commonly encountered in the liver tissues of deceased (n = 154) who died of causes other than sickness or disease in 1992-93, consistent with our blood findings of prevalence of aflatoxin B1. High performance liquid chromatography (HPLC) with fluorescence detection using one of the aflatoxins G2 or B2 as an internal standard was used for the detection and quantification of aflatoxins. The use of an internal standard structurally and chemically similar to those required to be quantified minimizes errors in quantifications. This is because differences in the quenching of fluorescence between specimen extracts and spiked-standard extracts were internally standardized and compensated for. The presence of an internal standard also helped to locate aflatoxins of interest more accurately.(ABSTRACT TRUNCATED AT 400 WORDS)

A relationship between sperm count and intracellular progesterone, 17 alpha-hydroxyprogesterone, and cortisol concentrations in human spermatozoa.

In an attempt to determine whether human spermatozoa contain steroid hormones, in this study we have measured the immunoreactive levels of P, 17 alpha-OHP, and F in the extracts of sonicated specimens of sperms obtained as ejaculates from husbands of infertile couples. The results of this study demonstrate for the first time that human spermatozoa contain definite amounts of the measured steroids and suggests an association between steroid levels and sperm count. These steroids identified intracellularly in the haploid germ cells may play a role in the molecular events associated with fertilization.

Concentrations of intracellular sex steroids in human spermatozoa.

In an attempt to determine whether human spermatozoa contain steroid hormones, the immunoreactive levels of progesterone, testosterone, and estradiol-17 beta were measured in the extracts of sonicated specimens of sperm obtained as ejaculates from husbands of infertile couples (n = 67, age 26-44 years). In the range of sperm concentration between 3.0 and 869.6 million per ejaculate (143.2 +/- 13.6 mean +/- SEM), the concentrations were as follows: progesterone 127.3-6685.4 fmol/ml (1434.5 +/- 178.6), testosterone 98.3-2219.1 fmol/ml (611.6 +/- 49.0), and estradiol-17 beta 33.0-678.1 fmol/ml (206.9 +/- 19.7). Using Pearson's correlation coefficient, the steroid hormone levels correlated significantly with the total sperm number (p < .02). The spermatozoa contained small but measurable amounts of the sex steroids.

Response of ovary in young women experiencing laparoscopy under general anaesthesia.

This study investigated whether changes in circulating levels of immunoreactive oestradiol-17 beta (E2), progesterone (P) and testosterone (T) occur in women at follicular (n = 18, age 25 to 39 years) and luteal (n = 17, 25 to 39 years) phases of the normal menstrual cycles, experiencing laparoscopy after intravenous sedation with general anaesthesia. The pre- and intra-operative follicular phase plasma steroid hormone concentrations were 153.5 +/- 84.3 vs 297.4 +/- 220.8 pg/ml for E2, 2.0 +/- 3.2 vs 3.3 +/- 3.8 ng/ml for P and 746.6 +/- 415.9 vs 1325.8 +/- 535.1 pg/ml for T, respectively. The corresponding luteal phase steroid levels were 259.7 +/- 120.2 vs 382.7 +/- 188.7 pg/ml, 7.0 +/- 4.8 vs 9.9 +/- 6.1 ng/ml and 819.4 +/- 355.7 vs 1703.5 +/- 1058.1 pg/ml. Using the Wilcoxon rank sum test, intra-operative hormone levels with the exception of P in the luteal phase were found to be significantly elevated (p < 0.05). The results suggest that laparoscopy under general anaesthesia evokes increased secretion of ovarian hormones, possibly via the activation of hypothalamo-pituitary-ovarian axis.

Toxicology Services in Singapore.

The toxicology laboratory provides a whole range of forensic toxicology services to the doctors, pathologists, law enforcement agencies and the public. This paper, anchored in one hundred and ten years of history, traces the practice and development of this laboratory to the present day of offering services in blood for alcohol determinations, antemortem biological fluids (gastric aspirate, blood, etc.) and postmortem biological fluids and tissues (liver, kidney, etc.) for drugs and poisons screen. This paper spells out in detail the nature of specimens required, the analytical techniques used, the toxins looked for and the medico-legal implications of our analytical findings. A number of recent cases of interest are presented.

Toxicological screen on gastric aspirates: the Singapore experience.

In the early eighties, in response to increasing self-poisoning incidents by pharmaceuticals in Singapore, a comprehensive wide-sweep screen for these drugs in clinical gastric aspirates was devised and implemented by us. The toxicological screen which involved quadruplicate extractions, two in acidic conditions and the other two in alkaline conditions, required the following analyses to be carried out: UV on the acidic fraction, TLC on the basic fraction and mega-bore capillary GC/NPD (nitrogen phosphorus detector) on the combined fraction. The combined fraction if necessary was subjected to GC/MS analysis. Our screen satisfied the need to confirm the analytical outcome of one method by another. Combining the acidic and basic fractions for a single GC analysis saves on instrument time and manpower. All coma and emergency cases were required to undergo GC/MS analysis to prove the identity of toxic agents beyond reasonable doubt. Quality control procedures were instituted at key steps to ensure quality of analytical outcome.

In vitro formation of testosterone via the delta 5-pathway in the human umbilical cord.

Homogenates of two groups of term umbilical cord (n = 6, 37-40 weeks; n = 6, 38-40 weeks) were separately incubated with [7n-3H]pregnenolone and [1,2,6,7-3H]dehydroepiandrosterone. Using the reverse-isotope dilution technique, [3H]dehydroepiandrosterone and [3H]testosterone formed from the respective substrates were isolated and characterized. The extent of enzymic conversions were 0.015-0.28% and 0.044-2.2%. These results provide evidence for the metabolic transformation of pregnenolone to testosterone via the delta 5-3 beta-hydroxy route.

Human umbilical cord as a possible source of progesterone.

Homogenates of human term umbilical cord were incubated with 7n-3H-pregnenolone. Using a reverse-isotope dilution technique, 3H-progesterone was isolated and characterized. The extent of enzymic conversion ranged from 2.5 to 55.4%. The results indicate that the cord can be a source of progesterone during human pregnancy.

Elevated peritoneal fluid luteinizing hormone and prolactin concentrations in infertile women with endometriosis.

In this study, we compared (Mann-Whitney U-test) the peritoneal fluid FSH, LH and PRL levels, measured by RIA, at the follicular and luteal phases of the menstrual cycle in women with (n = 43; age 25-44 years) and with no evidence of endometriosis (n = 35; age 25-39 years) who were considered as controls. Both follicular and luteal phase FSH concentrations of women with endometriosis were not statistically different (n = 22 vs 18; 0.32-5.8 vs 0.50-8.2 IU/l, P = 0.247; n = 13 vs 14; 0.6-6.5 vs 0.66-6.7 IU/l, P = 0.604) compared to their respective controls. In contrast to FSH, the concentrations of LH at follicular (n = 19 vs 17; 3.1-34.2 vs 2.3-12.2 IU/l, P = 0.01) and luteal (n = 17 vs 15; 2.1-95.4 vs 1.3-17.9 IU/l, P = 0.02) phases of the test group was significantly elevated at both phases of the cycle. With respect to differences in PRL concentrations at follicular phase no significant change (n = 21 vs 16; 1030-5800 vs 1305-4650 mIU/l; P = 0.255) was observed. The greatest difference in luteal PRL concentrations (P = 0.007) was obtained between the women with endometriosis and controls (n = 17 vs 17; 1895-8600 vs 1041-5000 mIU/l). The results suggest that disordered synchronization of neuroendocrine mechanisms controlling LH and PRL may be the underlying abnormality causing infertility in our group of patients with endometriosis.

Cholesterol side-chain cleavage activity in the human umbilical cord in vitro.

With a view to establish whether cells of the human umbilical cord possess cholesterol side-chain cleavage activity, homogenates of term umbilical cord obtained following spontaneous vaginal delivery from uncomplicated pregnancies (n = 6; 38-40 weeks gestation) were incubated with 26-14C cholesterol. Controls were homogenates heating in a boiling water bath for 10 min. Proof of the existence of cholesterol C-20, 22-desmolase activity in the viable tissue was established by associating radioactivity due to the labelled carbon 26 of 26-14C cholesterol with the p-bromophenacyl ester of isocaproic acid by reverse-isotope dilution analysis. The desmolase efficiency expressed as specific activity of 14C isocaproic acid, dpm g-1 tissue varied from 110 to 351 with a mean of 202. The small but definite conversion indicates that the cholesterol side-chain cleavage reaction previously believed to be exclusive to the steroidogenic pathway operating in the adrenal, gonads and placenta does exist in the human term umbilical cord in vitro.

Upper genital tract fluid oestradiol-17 beta, progesterone and testosterone concentrations during the menstrual cycle.

This study was to obtain evidence whether a cyclical pattern in the concentrations of oestradiol-17 beta, progesterone and testosterone exists in the upper genital tract fluid of women with normal menstrual cycles. The steroid concentrations at proliferative and luteal phases were n = 20 vs 17, 54.5-190.0 vs 44.2-827.1 pg/ml; 0.27-3.3 vs 3.4-46.5 ng/ml and 41.9-734.8 vs 85.7-991.2 pg/ml, respectively. The results were analysed according to Mann-Whitney U test. Significantly higher concentrations of oestradiol-17 beta (p = 0.0036) and progesterone (p = 0.0001) were found. The results suggest the existence of a cyclical variation and point to the ovary as the main source of output of the steroids.

Comparison of AFP and beta-hCG levels in infiltrating duct mammary carcinoma at different stages of malignancy.

In order to assess the likely clinical value of AFP and beta-hCG levels as markers of the extent of spread of infiltrating duct mammary carcinoma, we compared (Mann-Whitney U test) the immunoreactive tumour tissue concentrations of the respective antigens, measured by specific RIA, after mastectomy in Chinese women at stages I (n = 9), II (n = 13) and III (n = 7) with that of normal breast tissue (n = 11). Elevated concentrations of the oncofetal antigens were found at all stages of malignancy. The discovery of an hCG-like material in the extract of normal breast tissues indicates limited value of beta-hCG assay in mammary tumours. However, our findings generally suggest that measurement of the tumour tissue AFP levels warrant consideration as a useful biochemical marker for in-vitro evaluation of tumour spread at all stages of malignancy.