Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.

BACKGROUND: Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor. METHODS: A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection. RESULTS: The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%. CONCLUSIONS: Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.

Impaired hypothalamic-pituitary-adrenal axis activity in patients with multiple sclerosis.

OBJECTIVE: To investigate hypothalamic-pituitary-adrenal axis activity in well-defined multiple sclerosis (MS) patient subgroups. METHODS: A total of 173 patients with clinically definite MS were studied: 40 with primary progressive, 41 with secondary progressive, 58 with relapsing-remitting in remission, and 34 with relapsing-remitting during acute relapse. Sixty healthy subjects served as controls. No patients were receiving steroid or other immunomodulatory therapy. Plasma cortisol, adrenocorticotropic hormone (ACTH), and dehydroepiandrosterone sulfate (DHEAS), as well as urine cortisol levels, were measured using commercial radioimmunoassays. Glucocorticoid receptor (GR)-binding assay in peripheral blood mononuclear cells (PBMCs) was performed using [(3)H]dexamethasone (Dex). PBMC production of the proinflammatory peptide corticotrophin-releasing hormone (CRH), interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was evaluated using enzyme-linked immunosorbent spot assay. RESULTS: All four groups of patients displayed significantly higher cortisol, ACTH, and DHEAS plasma concentrations and urine cortisol values than controls. Although 62% of MS patients did not suppress Dex, suppression test results did not correlate with IL-1beta, IL-6, IFN-gamma, or TNF-alpha production. GR-binding assays showed no differences in binding sites between patients and controls; however, all MS groups showed decreased GR affinity and sensitivity compared with controls. The numbers of IL-1beta-, IL-6-, and TNF-alpha-secreting cells increased significantly in relapsing-remitting MS patients only during exacerbations; in contrast, IFN-gamma-secreting cells increased during both exacerbations and remission. Finally, PBMC CRH-secreting cell numbers were considerably greater in all forms of MS. CONCLUSIONS: Patients with multiple sclerosis show hypothalamic-pituitary-adrenal axis hyperactivity, with lymphocytes expressing similar glucocorticoid receptor numbers to controls; however, binding affinity and glucocorticoid sensitivity of these lymphocytes seem to be reduced.

Inhibition of ovulation by low-dose mifepristone (RU 486).

Mifepristone (RU 486) is a potent antigestagen and antiglucocorticoid which when given at a dose of 25-600 mg disrupts folliculogenesis, inhibits ovulation and induces menses in healthy women. This study reports the effects of much lower doses of mifepristone than used previously, given for the duration of a complete menstrual cycle. Healthy female volunteers (n = 11) with regular menstrual cycles were given mifepristone at a daily dose of 5 mg (n = 6) or 2 mg (n = 5) for 30 days, beginning immediately after an ovulatory placebo cycle. Mifepristone prevented menstruation for the duration of the treatment period, with recurrence of menses 15-29 days after replacement of mifepristone with placebo. Daily mifepristone given in either 5 mg or 2 mg doses inhibited ovulation, as indicated by the lack of a rise in urinary pregnanediol excretion. The excretion of oestrone glucuronide in urine rose during treatment, suggesting ovarian follicular development. Inhibition of ovulation appeared to result from a failure of the positive feedback effect of oestradiol on the hypothalamo-pituitary axis, as no surges of luteinizing hormone were seen despite pre-ovulatory levels of oestrone glucuronide being measured during exposure to mifepristone. The cycle immediately following treatment was shorter than the pre-treatment cycle, with lower peak levels of pregnanediol glucuronide, suggesting an inadequate luteal phase. Recovery from the effects of mifepristone treatment was more rapid after 2 mg than after 5 mg and one subject conceived in the immediate post-treatment phase, indicating adequate ovulation and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: Physicians at the Centre for Reproductive Biology in Edinburgh, Scotland, followed 11 healthy 29-39 year old women with normal menstrual cycles for 3 consecutive menstrual cycles to examine the effect of 2 mg or 5 mg doses of RU-486 taken daily during 1 menstrual cycle on concentrations of ovarian steroids and luteinizing hormone and to compare this with the menses pattern. The women took either a placebo or low-dose RU--486 daily during the first menstrual cycle. Then they took each day the item they did not take during the first cycle (second cycle). The third cycle involved taking the item they did not take during the second cycle. RU-486 prevented menstruation during the treatment period. Menstruation returned significantly later after cessation of RU-486 treatment than after placebo treatment (cycle lengths in days at 5 mg dose, 52.5 vs. 29.6, p .001; at 2 mg dose, 43.6 vs. 27.4, p .02). The lack of an increase in urinary pregnanediol excretion indicated that low doses of RU-486 inhibited ovulation. Urinary estrone glucuronide levels did rise, however, demonstrating that RU-486 did not interfere with follicular development. No rapid rises of luteinizing hormone occurred, suggesting that failure of the positive feedback effect of estradiol on the hypothalamo-pituitary axis suppressed ovulation. The post-treatment cycle was not as long as the treatment cycle and had reduced peak levels of pregnanediol glucuronide, denoting an inadequate luteal phase. Women who took 2 mg RU-486 recovered more quickly than those who took 5 mg. All the women ovulated during the post-treatment cycle. 1 woman became pregnant during the post-treatment cycle, suggesting satisfactory return to ovulation and luteinization. Pre- and post-treatment adrenocorticotrophic hormone and cortisol levels in the blood indicated that these low doses of RU-486 did not affect the pituitary-adrenal axis. These results suggest that low-dose RU-486 has the potential to be an oral contraceptive. Further studies are needed, however.

Plasma levels of 7B2 (a novel pituitary polypeptide) and its molecular forms in plasma and urine in patients with chronic renal failure: possible degradation by the kidney.

Plasma immunoreactive (IR)-7B2 was measured in patients with chronic renal failure (CRF), using a specific radioimmunoassay. The mean (+/- S.E.M.) concentration of plasma IR-7B2 in CRF patients under hemodialysis (502 +/- 36 pg/ml, n = 27) was significantly higher than that in normal subjects (men, 52.9 +/- 1.7 pg/ml (n = 179); women, 55.8 +/- 1.3 pg/ml (n = 198]. Significant correlations between plasma levels of IR-7B2 and those of blood urea nitrogen, creatinine and beta 2-microglobulin were evident in non-dialyzed CRF patients. In the analyses of pooled plasma and urine obtained from normal subjects on gel permeation chromatography, a major peak of IR-7B2 was observed at an apparent molecular weight of 20,000 in the plasma, and at a position of a smaller molecular weight in the urine. These results suggest that 7B2 is degraded mainly in the kidney and that measurement of plasma 7B2 may serve as an appropriate tool for assessing renal function.

Urinary molecular forms of human N-terminal of proopiomelanocortin: possible deglycosylation and degradation by the kidney.

The NH2-terminal fragment (hNT) of proopiomelanocortin is found predominantly as one molecular form of apparent mol wt of 12K in the circulation. Since the kidney may play an important role in the elimination and degradation of proopiomelanocortin-related peptides, we analyzed the urinary forms of immunoreactive hNT (IR-hNT) by molecular sieving and carbohydrate affinity (Concanavalin A-agarose) chromatography. RIA specific for the amino terminal portion and for the gamma 3-MSH (carboxy-terminal portion of hNT) were used in these studies. Molecular sieve chromatography revealed several forms of IR-hNT in the urine from normal subjects, patients with Nelson's syndrome, and patients with ectopic ACTH-secreting tumors. A considerable decrease in IR-hNT and IR-gamma 3-MSH was found in the urine of a patient with ACTH deficiency and normal subjects during glucocorticoid suppression. In urine from normal subjects and a patient with lung cancer not causing Cushing's syndrome, the majority of amino-terminal IR-hNT (66-83%) had apparent mol wts of 3-4K, 6-7K, and 8-10K, and did not cross-react with the gamma 3-MSH antiserum. Ten to nineteen percent of the total IR-hNT was eluted in the position of authentic hNT and reacted with the gamma 3-MSH RIA. In patients with Nelson's syndrome and those with ectopic ACTH syndrome, almost no intact hNT (less than 7% of the total) was present in urine; most of the IR-hNT appeared in the elution volumes with an apparent mol wt of 8-10K. In addition, smaller forms (6-7K and 3-4K) of hNT were also detected in the urine of these patients. The major form of urinary IR-gamma 3-MSH exhibited an apparent mol wt of 7-8K and did not correspond to any of the peaks of IR-hNT. Carbohydrate affinity chromatography (Concanavalin A-agarose) of smaller forms of IR-hNT revealed weak affinity to the lectin, which suggests loss of the carbohydrate moiety during renal excretion. We conclude that hNT in urine is present in extensively cleaved forms and that deglycosylation may be an important step in hNT degradation. These results support a role for the kidney in the catabolism of hNT.