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Biomedical subjects

W A Ratcliffe

Publications and source records attributed to W A Ratcliffe.

At least 73 records · Page 4Linked to original sources
PubMed1987

Inadequate luteal phase usually indicates ovulatory dysfunction: observations from serial hormone and ultrasound monitoring of 115 cycles.

We have studied 100 women with regular menstrual cycles and infertility and tried to assess how frequently an 'inadequate' luteal phase (defined by low-peak progesterone levels) follows 'normal' ovulation. Normal follicular growth on serial ultrasound scan and follicular disappearance or collapse within 48 hours of the recorded LH peak were taken together as convincing evidence of ovulation. Eighty-three of 115 cycles were judged to be ovulatory and 32 to be anovulatory. A peak mid-luteal phase maximum serum progesterone (Po) level of 32 nmol/L (10 ng/ml) was taken arbitrarily as the cut-off level of discrimination between 'adequate' and 'inadequate' corpus luteum function. Serum progesterone was undetectable (less than 2.5 nmol/L) throughout in 2 cycles while the maximum was above 32 nmol/L in 102 and detectable but less than 32 nmol/L in 11. Of the latter only 1 was ovulatory. We conclude that cycles with low luteal phase Po levels represent luteinization without ovulation.

Anovulation↗
PubMed1987

Elevation of free thyroxine measurements in patients without thyrotoxicosis.

The recent development of single step analogue assay techniques to measure free thyroxine in serum has meant that this estimation has become widely used as a first line test of thyroid function. However these assays are subject to in-vitro interference in two ways: where there is a variant serum albumin in familial dysalbuminaemic hyperthyroxinaemia, or where there are circulating thyroid hormone binding antibodies. In both these situations a spuriously high result for free thyroxine is obtained. This may have serious implications for patient management and we describe 10 patients in whom the finding of a misleadingly high free thyroxine concentration led to confusion in diagnosis and, in eight of the 10, to inappropriate antithyroid treatment. Clinicians should be aware of these technical problems and where a result for the free thyroxine concentration seems inappropriate to the patient's clinical state then measurement of thyrotrophin (TSH) by a sensitive immunometric method should be performed. If the results remain confusing the presence of a variant serum albumin or thyroid hormone binding antibodies should be sought.

Adult↗
PubMed1986

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands.

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.

Androstenedione↗
PubMed1986

Comparison of blood spot, salivary and serum progesterone assays in the normal menstrual cycle.

Sensitive direct (non-extraction) radioimmunoassays for assay of progesterone (Po) in saliva and capillary blood dried on filter paper have been developed to facilitate collection of specimens for the assessment of luteal function in the investigation of infertility. The antiserum had been raised to a Po 11 alpha-hemisuccinyl conjugate and the assays employ Po 11 alpha-glucuronyl-[125I]tyramine as tracer with separation by solid phase double antibody. Po levels in saliva and capillary blood were compared with those in serum in matched specimens collected daily throughout the menstrual cycle from six female volunteers. The correlation between blood (y) and serum (x) Po levels (y = 0.439x - 2.98, r = 0.98) was superior to that between salivary (y) and serum levels (x) (y = 0.00637x + 0.020, r = 0.89). Blood spot and serum Po levels were uniformly undetectable throughout the follicular phase, whereas in 2 out of 72 saliva specimens Po was inappropriately raised (60 and 120 pmol/l) possibly reflecting contamination of the specimens. Salivary and blood progesterone assays both lack adequate sensitivity to measure Po reliably in early or late luteal phases when serum levels are less than 12 nmol/l but are well suited to assess luteal function during the mid-luteal phase of the cycle (4-10 d before menses) when serum levels in the six subjects ranged from 22-84 nmol/l, with corresponding blood levels of 6.2-3.5 nmol/l and saliva levels of 130-620 pmol/l. We conclude that blood spot and saliva Po levels both correlate well with Po levels in serum, but that blood spot Po levels are more reliable.

Blood Specimen Collection↗
PubMed1986

Does testosterone affect the normal menstrual cycle?

In order to throw further light on the role of androgens in the aetiology of the polycystic ovary syndrome (PCO) we have examined the effect of artificially increasing serum testosterone levels on menstrual function in a group of ovulating women. Six women were studied who had either severe premenstrual syndrome or loss of libido for which they were treated with 100 mg testosterone by s.c. implantation. All had regular menstrual cycles. For 1 month before implantation serum LH, FSH, oestradiol (E2), progesterone and testosterone were measured three times per week. All women showed normal cyclical variation of LH, FSH, E2 and progesterone. Following implantation, three times weekly blood samples were taken during the first and third cycles. No patient had any disturbance of menstrual pattern. All continued to show cyclical changes of LH, FSH, E2 and progesterone. Serum E2 and progesterone were lower but not significantly so in the luteal phase of the treated cycles. This was despite a mean serum testosterone which rose from 1.3 to 7.1 nmol/l at the end of the third week following implantation and to 4.1 nmol/l at the end of the third month. Sex hormone binding globulin levels fell as expected by 18.5% during the first cycle. The lack of significant effect of a markedly elevated serum testosterone level on cyclical hormone changes is indirect evidence that in PCO the primary cause of the menstrual disturbance is not excessive production of ovarian or adrenal testosterone.

Adult↗
PubMed1986

Effects of medroxyprogesterone acetate in women with polycystic ovary syndrome.

LH levels are inappropriately elevated in women with polycystic ovary syndrome (PCO). Regardless of whether this LH elevation is primary or secondary it may be involved in the establishment of chronic anovulation. We have therefore tried selectively to suppress LH by giving medroxyprogesterone acetate (MPA) 5 mg twice daily for 14 d to 13 women with PCO (14 studies) and have examined their pituitary and ovarian response to assess whether follicular development and ovulation would be triggered post-treatment as a result of secretion of endogenous gonadotrophins in a more nearly physiological ratio. Blood samples were obtained before and two to three times weekly during and after treatment for 35-50 d. In two of these cycles MPA was started fortuitously during the luteal phase of a spontaneous cycle. In the remainder, three patterns of response were seen. In 9 cycles, after an initial rise, there was a progressive fall in gonadotrophin levels, LH (mean 48% of basal) more than FSH (mean 68% of basal); after MPA was stopped FSH levels recovered more rapidly than LH. In two cycles (one subject) MPA administration was associated with a progressive rise in serum oestradiol and progesterone. In a further cycle, treatment was also accompanied by an oestradiol rise and was followed by establishment of regular ovulatory cycles. The observed differential suppressive effect on LH and FSH levels suggests that MPA might be of therapeutic value if followed by treatment with anti-oestrogens or exogenous gonadotrophins in PCO.

Adolescent↗
PubMed1986

Thyroid function tests in patients with familial dysalbuminaemic hyperthyroxinaemia (FDH).

Two patients with familial dysalbuminaemic hyperthyroxinaemia (FDH) are described in whom the albumin variant resulted in raised total T4 levels, and artefactually raised free T4 using a 'single-step' technique employing an analogue of T4 as tracer. The first patient was clinically euthyroid and presented with relapse of schizophrenia and abnormal thyroid function tests (total T4 336 nmol/L, total T3 4.2 nmol/L, TSH 1.8 mU/L, free T4 73 pmol/L). These results led to diagnostic confusion and the patient was treated with a short course of anti-thyroid drugs. The second patient had signs and symptoms of thyrotoxicosis at her first visit but was clinically euthyroid 5 months later when she was 10 weeks pregnant. Thyroid function tests were total T4 259 nmol/L, total T3 3.6 nmol/L, TSH 3.8 mU/L, free T4 46 pmol/L. Further studies showed both patients to be biochemically euthyroid. A variant albumin was confirmed in both patients by a screening test for FDH and by reverse-flow electrophoresis. Family studies on 10 relatives of the first patient identified eight with FDH. A simple screening procedure for the indentification of FDH is described and the use of laboratory tests in suspected cases is discussed.

Adult↗
PubMed1985

A study of 19-O-carboxymethyl ether and 19-hemisuccinate derivatives of testosterone: their immunogenicity and use as iodinated radioligands for radioimmunoassay of testosterone.

Testosterone 19-O-carboxymethyl ether (T19C) and 19-hemisuccinate (T19H) derivatives were synthesised, and coupled to bovine serum albumin (BSA) or porcine thyroglobulin (PT) for immunogens or to iodohistamine for radioligands. The immunogenicity of these conjugates in mice was compared with those of testosterone 3-O-carboxymethyloxime and 15 beta-thioethyl conjugates. Of 10 immunogens studied, those linked to PT gave the highest antiserum titres and more sensitive standard curves. The site of conjugation (19 or 3 position) had little effect on immunogenicity. Cross-reactivity with 5 alpha-dihydrotestosterone (DHT) was in the range 22-100%, for the 19-linked immunogens. Antisera to T19C and T19H conjugated to PT were then raised in rabbits and characterised with 4 radioligands. Homologous assay systems in which the chemical bridge was identical in immunogen and 125I-radioligand gave the highest antiserum titres but the poorest assay sensitivity while those heterologous with respect to bridge or site gave the most sensitive standard curves. Rabbit antisera to both T19C and T19H immunogens showed good specificities with respect to DHT (range of cross reactions 0.78-21.1%), androstenedione, AN (range 0.45-2.3%) and progesterone, Po (range 0.05-1.4%) with all radioligands. The best assay system employed an antiserum to the T19H-PT immunogen with heterologous radioligand [125I]T19C. It had a detection limit of 15pg/tube and low cross-reactivity with DHT (0.78%), Po (0.3%) and AN (0.43%). We conclude that 19-linked derivatives of testosterone conjugated to PT are good immunogens and the antisera when combined with [125I]testosterone radioligands which are heterologous with respect to the chemical bridge, provide highly specific assays for testosterone with potential for clinical application.

Animals↗
PubMed1985

The assessment of ovulation by a combination of ultrasound and detailed serial hormone profiles in 35 women with long-standing unexplained infertility.

We have examined for the presence of subtle hormonal abnormalities in women with long-standing unexplained infertility. For a full cycle serum LH, FSH, progesterone and oestradiol levels were measured about three times a week, and serial ultrasound scans of the ovaries made until the time of apparent ovulation. The results on 45 cycles in 35 women with unexplained infertility and in three normal volunteers are presented. Normal ovulatory cycles were defined by a length of 26-32 d, and progressive follicular maturation followed by disappearance or abrupt reduction in size of a follicle within 48 h of the recorded LH peak, followed by progressive and sustained rise in serum progesterone levels to more than 25 nmol/l and a luteal phase length of greater than or equal to 13 d. Thirty spontaneous cycles (28 women) were clearly normal while 15 spontaneous cycles (12 women) were abnormal. Abnormalities included luteinization of an unruptured follicle (eight cycles), absence of follicular development (two cycles), poor follicular development (two cycles), persistence of a large ovarian cyst from the preceeding cycle (two cycles) and one aluteal cycle. Six of the abnormal cycles were characterized hormonally by inappropriate elevation of serum LH levels throughout. If this study had been based only on serial ultrasound scans, all results on abnormal cycles might have been misinterpreted. If it had been conducted only with (multiple) progesterone determinations and the level of greater than 25 nmol/l had been taken as indicative of ovulation nine clearly abnormal cycles would have been considered as normal. We conclude that the combination of the hormonal and ultrasound assessment of ovulation increases our confidence for confirmation of normality and reveals various ovulatory disorders which are possibly due to an endocrinological defect or defects.

Adult↗
PubMed1985

On the stability in vitro of bioactive human adrenocorticotrophin in blood and plasma.

The disappearance in vitro of ACTH from whole human blood and plasma held at 22 degrees C has been monitored using a dispersed adrenal cell bioassay and an unextracted radioimmunoassay. In three normal subjects after a metyrapone test and two patients with Addison's disease, endogenous bioactive ACTH levels were unchanged for at least 1 h in blood and 2 h in plasma. Moreover greater than 50% of the bioactive plasma ACTH was still present in the plasma samples from the patients with Addison's disease after 24 h incubation at ambient temperatures. Human pituitary ACTH (1-39), spiked into plasma from dexamethasone suppressed subjects to give a concentration of 250 ng/l, was stable by bioassay for at least 2 h. No loss of biological activity was observed on subjecting plasma from a patient with Addison's disease or spiked plasma to two cycles of thawing at 37 degrees C and freezing at -70 degrees C or thawing at 20 degrees C and freezing at -20 degrees C. Some loss of bioactivity (20%) occurred on subjecting the patient's, but not ACTH-spiked plasma to four cycles of thawing at 20 degrees C/freezing at -20 degrees C. We conclude that bioactive ACTH (endogenous or exogenous) may be more stable in vitro in human blood and plasma than has been previously thought. If our studies can be confirmed in a larger series then it may be that conditions for handling blood specimens for ACTH assays could be reappraised.

Addison Disease↗
PubMed1985

Assessment of corpus luteum function by direct radioimmunoassay for progesterone in blood spotted on filter paper.

In this specific, direct RIA for progesterone in capillary blood dried on filter paper, progesterone is eluted, with phosphate buffer containing bovine serum albumin, from 5.9 microL of blood dried on 5.0-mm (diameter) discs of filter paper. The eluate is assayed, with 125I-labeled progesterone-11 alpha-glucuronyl-tyramine as tracer, with separation by a double-antibody solid-phase technique. The sensitivity of the assay is 4.7 pg per tube, corresponding to 2.5 nmol per liter of blood. Within- and between-batch CVs averaged 7.0 and 9.2%, respectively, over the working range of the assay (4.5-64 nmol/L). Concentrations of progesterone in blood spots (y) correlated well with those in serum (x) as measured by an established direct RIA (Clin Chem 28:1314, 1982): y = 0.430x - 2.44 (r = 0.972, n = 104). Progesterone is stable in the blood spots for at least 15 weeks at 25 degrees C. The convenience of multiple sampling of blood by finger prick and the simplicity of the assay make this approach useful in investigating serial progesterone concentrations in outpatients.

Corpus Luteum↗
PubMed1984

Effect of an oral serotonin antagonist, ketanserin, on plasma ACTH concentrations in Nelson's syndrome.

A study was performed to see whether ketanserin, a serotonin antagonist, would reduce the raised concentrations of adrenocorticotrophic hormone (ACTH) in patients with Nelson's syndrome. Six patients who had undergone bilateral adrenalectomy for Cushing's disease and who had Nelson's syndrome were given ketanserin 40 mg twice daily and placebo, for at least two months each, in a double blind crossover study. Ketanserin had no effect on ACTH concentrations. In healthy people serotonin seems to have a stimulatory role in the regulation of ACTH secretion, and the effect of ketanserin in reducing the ACTH response to hypoglycaemia suggested that it might prove useful in Nelson's syndrome. These results show that it is not indicated in these patients.

Adrenocorticotropic Hormone↗
PubMed1984

The effect of ketanserin, a specific serotonin antagonist on the PRL, GH, ACTH and cortisol responses to hypoglycaemia in normal subjects.

The role of serotonin in the prolactin, growth hormone, ACTH and cortisol responses to hypoglycaemia has been investigated in normal subjects using a selective serotonin (5HT2) receptor antagonist, ketanserin. Circulating concentrations of these hormones were measured after administration of insulin (0.1 units/kg body weight iv) to eight normal male subjects with and without simultaneous iv ketanserin (10 mg). Plasma glucose fell to less than 2.0 mmol/1 in all subjects and was unaffected by ketanserin. Ketanserin induced a 50% decrease in the serum prolactin response to hypoglycaemia, 45 and 60 min after administration of insulin (increase in serum prolactin at 60 min: 1145 +/- 295 mU/l without ketanserin; 558 +/- 176 mU/l with ketanserin, P less than 0.05). The peak ACTH response was reduced by 30% (95.3 +/- 33.6 ng/l without ketanserin; 60.0 +/- 22.9 ng/l with ketanserin, P less than 0.05) but the plasma cortisol response was not significantly altered. The serum growth hormone response was unaffected by serotonin blockade. These findings suggest that serotonin, probably acting through 5HT2 receptors, is involved in the stimulation of prolactin and ACTH release but not in the release of growth hormone, during insulin induced hypoglycaemia.

Adrenocorticotropic Hormone↗
PubMed1982

The provision of 125I-labelled tracers for radioimmunoassay of haptens: a general approach?

Use of 125iodinated progesterone 11 alpha-glucuronide-tyramine conjugate as radioligand with the majority of antisera raised against progesterone 11 alpha-hemisuccinate-bovine serum albumin is shown to produce steep and sensitive standard curves. Accurate precise and robust assays for progesterone which are clinically useful have been readily developed from such systems. Together with data from other heterologous-bridge systems, the results suggest a hypothesis concerning desirable structural features of the links in heterologous-bridge immunoassays for haptens. The hypothesis should facilitate the establishment of other adequately sensitive external-label assays, especially for gonadal steroids.

Animals↗
PubMed1982

Tamoxifen, aminoglutethimide and danazol: effect of therapy on hormones in post-menopausal patients with breast cancer.

Gonadotrophins, oestradiol, androstenedione, testosterone and dehydroepiandrosterone sulphate (DHAS) were measured sequentially in 72 patients with advanced breast cancer receiving endocrine therapy of various types. Tamoxifen significantly reduced gonadotrophins but did not effect other hormones. Danazol also reduced gonadotrophins. Aminoglutethimide (AGT) reduced oestradiol and DHAS but had not effect on gonadotrophins. The effects of administering tamoxifen, AGT and danazol together (TAD) together were therefore examined. This combination reduced gonadotrophins, oestradiol and DHAS, but no further than tamoxifen and AGT alone. The degree and duration of hormone suppression were similar in both responders and non-responders to tamoxifen, AGT or TAD, though patients responding to AGT showed more complete suppression at the end of the course of treatment, perhaps because they were treated longer. On relapse, adequate gonadotrophin and steroid suppression was demonstrated in patients receiving tamoxifen and AGT respectively. We conclude that (a) response to endocrine therapy is unlikely to be related to the degree of endocrine suppression produced by the therapy; (b) combination endocrine therapy does not further reduce serum-hormone concentrations and (c) relapse is unlikely to be due to escape from the hormone-inhibitory effects of endocrine agents.

Aminoglutethimide↗
PubMed1982

Biochemical and clinical effects of fenclofenac in thyrotoxicosis.

Administration of the anti-inflammatory drug fenclofenac (600 mg bd) for 28 days to four females with thyrotoxicosis resulted in a rapid decline in thyroid hormone levels. The mean total thyroxine (T4) level of 173 +/- 8.6 (SEM) nmol/l before therapy was normalized to 70 +/- 6.4 nmol/l after administration of the drug for 7 days. Free T4 levels also decreased significantly but not to within the reference range in all subjects. Mean total triiodothyronine (T3) and reverse T3 levels declined from 6.2 +/- 0.9 nmol/l and 0.63 +/- 0.2 nmol/l respectively before therapy to 3.8 +/- 0.5 nmol/l and 0.52 +/- 0.1 nmol/l after 7 days' treatment but remained consistently elevated in all subjects. Clinical thyroid status remained unchanged despite these marked reductions in circulating thyroid hormone levels, suggesting that fenclofenac was of no therapeutic benefit.

Adult↗
PubMed1982

Precocious pseudopuberty due to a rare form of congenital adrenal hyperplasia. Biochemical investigation and pitfalls in interpretation of hormone assays.

The biochemical investigation is described of a boy who presented with precocious puberty at the age of 3 years 9 months due to a rare form of congenital adrenal hyperplasia (CAH), steroid 11 beta-hydroxylase deficiency. Serum androgen levels were grossly elevated (17 hydroxyandrogens 10 nmol/l, androstenedione 129 nmol/l), 17 hydroxyprogesterone was modestly elevated (21 nmol/l), while serum gonadotrophins were low and testes were prepubertal in size. The major differential diagnosis was between an androgen-producing tumour and CAH. Initial serum and urine corticosteroid concentrations and their responses to dexamethasone were diagnostically misleading, later found to be due to lack of specificity of the radioimmunoassays and fluorimetric methods employed. Elevated basal plasma ACTH levels and suppression of androgen and ACTH levels by dexamethasone strongly suggested CAH. Definitive diagnosis of an 11 beta-hydroxylase defect was established by capillary column gas liquid chromatography of urine which demonstrated excess androgen and 11-deoxycortisol metabolites but no cortisol metabolites. The diagnosis was confirmed by specific serum assays of 11-deoxycortisol, deoxycorticosterone, and cortisol. The contribution of hormone assays and a protocol for their use in the diagnosis and monitoring of precocious puberty is discussed.

Adrenal Hyperplasia, Congenital↗