Innovations in serum and urine markers in prostate cancer current European research in the P-Mark project.

An overview is given of serum and urine prostate cancer markers that are currently under investigation and subsequently the P-Mark project is introduced. There are many markers showing promise to overcome the limitations of prostate specific antigen (PSA). Eventually, these markers should be able to increase the specificity in diagnosis, differentiate between harmless and aggressive disease and identify progression towards androgen independence at an early stage. In the P-Mark project, several recently developed, promising markers will be evaluated using clinically well-defined biorepositories. Following successful evaluation, these markers will be validated on a sample set derived from two large, European, prostate cancer studies and used for the identification of special risk groups in the general population. In addition, novel markers will be identified in the same biorepositories by different mass spectrometry techniques.

Luteinizing hormone, its beta-subunit variant, and epithelial ovarian cancer: the gonadotropin hypothesis revisited.

The gonadotropin hypothesis postulates that excessive gonadotropin stimulation results in increased proliferation and subsequent malignant transformation of ovarian epithelium. The authors evaluated this hypothesis by analyzing the association between serum levels of wild-type luteinizing hormone (LH) and ovarian cancer risk. They also examined the relation between a variant of LH containing two missense point mutations (Trp(8)Arg and Ile(15)Thr) in its beta-subunit and ovarian cancer risk. Fifty-eight cases of epithelial ovarian cancer and 116 controls matched on age, menopausal status, and date of blood donation were included in a case-control study nested within the New York University Women's Health Study, a prospective cohort enrolled between 1985 and 1991 in New York City. Wild-type serum levels and variant LH status were determined by immunofluorometric assays in which monoclonal antibodies specific for wild-type and variant LH were used. Compared with women in the lowest tertile of wild-type LH, women in the highest tertile had a lower risk of ovarian cancer, after adjustment for potential confounders (odds ratio = 0.42, 95% confidence interval: 0.09, 2.09). Women heterozygous for variant LH were not at increased risk (adjusted odds ratio = 0.95, 95% confidence interval: 0.27, 3.34). The results suggest that neither wild-type LH levels nor variant LH status is associated with increased risk of epithelial ovarian cancer.

Effects of probucol on rat carotid artery responses to balloon catheter injury.

Balloon catheter injury to the rat common carotid artery has been widely used for testing potential therapies for post-angioplasty restenosis. However, the model has become somewhat discredited because a number of drugs that inhibit intimal thickening, measured 14 days after balloon catheter injury, have been found to be ineffective in clinical trials. Probucol has recently been shown to reduce the incidence of post-angioplasty restenosis in a number of small clinical trials, making it possible to reassess the validity of the rat balloon injury model. The effects of probucol on the underlying causes of intimal thickening in balloon-injured rat carotid arteries were quantified. Probucol inhibited medial smooth muscle cell proliferation by 23% on day 4 after injury (P=0.009), and by 65% on day 10 after injury (P=0.026). Smooth muscle cell migration was reduced by 64% (P=0.008) in probucol-treated animals. In marked contrast, intimal smooth muscle cell proliferation was significantly increased by 41% (P=0.024) by probucol. There was no significant effect on intimal thickening or smooth muscle cell death. These data suggest that drugs that inhibit both medial smooth muscle cell proliferation and migration in the rat balloon injury model may prove useful in the treatment of post-angioplasty restenosis.

Alterations in gonadal steroidogenesis in individuals expressing a common genetic variant of luteinizing hormone.

Some pathologies of the pituitary-gonadal function have recently been found to be due to mutations of the gonadotropin or gonadotropin receptor genes. Although these conditions are extremely rare, they are very informative, by elucidating some less well characterized facets of normal gonadotropin function and the molecular pathogenesis of disturbances in sexual differentiation and fertility. In contrast, there is a common polymorphism in the Luteinizing Hormone (LH) beta-subunit gene, where two point mutations cause two alterations in the amino acid sequence (Trp8 --> Arg and Ile15 --> Thr) and introduce an extra glycosylation signal to Asn13. The carriers of this variant gene are largely healthy, but certain mild differences in their gonadal function have been found, as reflected by alterations in gonadal steroidogenesis, pubertal development and predisposition to diseases such as infertility, polycystic ovarian syndrome, and breast and prostatic cancer. The purpose of this chapter is to review the current knowledge of the occurrence, special functional features and clinical correlates of this LH variant.

A new contributing factor to polycystic ovary syndrome: the genetic variant of luteinizing hormone.

Although the etiology of polycystic ovary syndrome (PCOS) is still unclear, LH is considered to play a central role in its pathogenesis. An immunologically anomalous form of LH, with two point mutations in the LHbeta gene, has been recently described. This genetic variant of LH (v-LH), of wide geographic distribution, is functionally different from wild-type (wt) LH. To assess the role of the v-LH in PCOS, we analyzed its frequency in groups of PCOS patients from Finland, The Netherlands, the United Kingdom, and the United States. The LH status was determined by two immunofluorometric assays from a total of 1466 subjects. The carrier frequency of the v-LH allele in the whole study population was 18.5%, being highest (28.9%) in Finland and lowest (11.2%) in The Netherlands. In the individual countries, the frequency of v-LH was similar in obese and nonobese controls, but in The Netherlands and Finland, it was 5- to 7-fold lower in obese PCOS subjects compared with that in the other groups (2-4.5% vs. 10.3-33.3%; P < 0.05). A similar tendency was found in the United States (5.7% vs. 11.1-25.0%), but not in the United Kingdom. The overall high prevalence of v-LH in healthy women and women with PCOS suggests that it is compatible with fertility. The similar frequency of v-LH in healthy nonobese and obese women indicates that obesity per se is not related to the variant. In contrast, the lower frequency of v-LH in obese PCOS patients suggests that v-LH somehow protects obese women from developing symptomatic PCOS. However, the regional differences in this finding between patients with apparently similar diagnostic criteria emphasizes the multifactorial nature of this syndrome, and that its pathogenesis may vary according to the genetic background. Although the definitive role of v-LH in PCOS remains to be proven, its determination may improve the prediction of risk of PCOS, especially in obese women.

Luteinizing hormone and different genetic variants, as indicators of frailty in healthy elderly men.

We investigated the possible clinical correlates between the serum LH concentration and characteristics of frailty and determined the presence and concentration of a genetic LH variant in an independently living population of elderly men. After exclusion of subjects with severe mobility problems and signs of dementia, 403 healthy men (aged 73-94 yr) were randomly selected from a population-based sample. Total testosterone (T), sex hormone-binding globulin (SHBG), and leptin were determined by RIA. Non-SHBG-bound T was calculated. LH and the presence of the genetic LH variant were measured using immunofluorometric assays. The characteristics of frailty were leg extensor strength using dynamometry, bone mineral density of total body and proximal femur, and body composition, including lean mass and fat mass, measured by dual energy x-ray absorptiometry. Disability was further assessed by the Modified Health Assessment Questionnaire and by a measure of physical performance. LH significantly increased with age and inversely correlated with T and non-SHBG-bound T. LH was inversely related to muscle strength and lean mass, and both relations were independent of T. LH was positively related to self-reported disability (Modified Health Assessment Questionnaire); 12.5% of the study population was heterozygous for the LH variant allele. T levels and the degree of frailty were not different in the wild-type LH group compared with the heterozygote LH variant group. A significant positive relation between LH and fat mass as well as leptin was only present in the heterozygote LH variant group. In conclusion, serum LH levels increases with age in independently living elderly men and correlates inversely with a variety of indicators of frailty. The observed relation between LH and frailty, independent of T, suggests that LH reflects serum androgen activity in a different way than T, possibly reflecting more closely the combined feedback effect of estrogen and androgen. A difference in biological response between the two LH forms is suggested, as a difference exists in the relation between LH and fat mass, respectively, and leptin in the heterozygote LH variant subjects vs. the wild-type LH subjects.

Serum levels of beta-subunit of chorionic gonadotropin in patients with pituitary tumors.

Many studies have shown that normal and tumoral pituitary is able to synthesize chorionic gonadotropin (CG). The aim of the present work was to investigate the circulating levels of free beta-subunit of CG (CG-beta) in a large number of patients with pituitary tumors in basal conditions and after thyrotropin-releasing hormone (TRH) injection. The study includes 27 healthy subjects, 23 patients with prolactinoma, 20 with growth hormone-secreting adenoma and 77 with non-functioning pituitary adenoma (NFPA). The CG-beta was evaluated using a new one-step immunometric assay employing two monoclonal antibodies directed against epitopes present only on the free CG-beta and showing a detection limit of 0.04 U/l and a cross-reactivity with complete CG < 0.01%. In basal conditions, serum CG-beta was undetectable in healthy subjects and in the majority of patients, while in seven patients with NFPA and four with prolactinoma the CG-beta values ranged between 0.05 and 0.72 U/l. In these 11 patients serum levels of intact CG were found within the normal range (normal range < 5 U/l), while two patients with NFPA and one with prolactinoma had levels of free alpha-subunit inappropriately high with respect to gonadotropins and thyrotropin. Injection of TRH caused CG-beta to increase in two out of 16 patients with NFPA, whereas it was ineffective in 12 healthy subjects and 10 patients with prolactinoma. The present data indicate that detectable level of CG-beta not associated with hypersecretion of the intact CG molecule may be observed in about 10% of patients with NFPA or prolactinoma, while abnormal CG-beta responses to TRH are observed infrequently in individual patients with NFPA.

Abnormal response of luteinizing hormone beta subunit to thyrotrophin-releasing hormone in patients with non-functioning pituitary adenoma.

OBJECTIVE: It has been suggested that the response of free beta-subunit of LH (LH beta) to TRH is the most useful in-vivo marker of gonadotroph adenomas in patients with non-functioning pituitary adenomas (NFPA). The aim of the present study was to investigate LH beta secretion in patients with NFPA in whom other markers of gonadotroph adenomas, such as supranormal basal concentrations or responses of intact gonadotrophins to TRH, were absent. DESIGN AND PATIENTS: Serum basal levels of LH beta LH and FSH were evaluated in 80 patients with NFPA showing normal levels of intact gonadotrophin, 20 with PRL-secreting adenomas, 25 with GH-secreting adenomas and 58 healthy subjects. Moreover, LH beta, LH, FSH and alpha-subunit (alpha-SU) were evaluated in 27 patients with NFPA in whom intact gonadotrophin responses to TRH were absent, 8 with PRL-oma, 7 with GH-oma and 17 healthy subjects before and 20, 30 and 60 minutes after the intravenous administration of either 200 micrograms TRH or placebo. A response was considered present when serum LH beta increased by at least 50% above basal levels. MEASUREMENTS: LH beta was evaluated using a new assay based on the sequestration of the combined and free alpha-SU by an anti alpha-SU biotinylated monoclonal antibody (MAb) and the subsequent measurement of the LH beta by an IFMA method employing two MAbs directed towards two different epitopes on LH beta. Intact LH and FSH were assayed with an IFMA method and alpha-SU with an IRMA method. RESULTS: In basal conditions, no significant difference in the mean values of LH beta was observed among patients with different types of tumour and normal controls. In 9 of 27 (33%) patients with NFPA, TRH caused an abnormal elevation of serum LH beta (net increase 410 +/- 403%, range 71-1300) which was completely dissociated from changes in intact gonadotrophins. Of the 5 patients who had a TRH test repeated after transsphenoidal surgery, abnormal LH beta responses disappeared in 2 and were maintained in 3. Disappearance of LH beta response occurred only in patients in whom improvement of visual field and radiological imaging after adenomectomy was observed. In contrast, in all patients with pituitary tumours other than NFPA and healthy subjects a response to TRH was absent (net increase ranging from 0 to 23%). Immunofluorescence, performed on 14 NFPA removed from patients either responsive or unresponsive to TRH, showed a variable proportion of cells positive for LH beta, without a significant difference between the two groups. CONCLUSIONS: These results indicate that measurement of basal LH beta is of poor value in the diagnosis of non-functioning pituitary adenomas and the identification of gonadotroph adenomas among non-functioning pituitary adenomas. Conversely, an abnormal response of free LH beta to TRH occurs in about a third of patients with low/normal basal gonadotrophins unresponsive to TRH stimulation.

Individual differences in lutropin immunoreactivity revealed by monoclonal antibodies.

We screened 11 monoclonal anti-lutropin (LH) antibodies for use in two-site immunometric combinations, with time-resolved fluorescence as the detection principle. Seven immunofluorometric assays (IFMA), six detecting only the intact hormone and one detecting both the intact hormone and free beta subunit, were compared with each other and with a conventional radioimmunoassay involving a polyclonal antiserum. Two monoclonal antibodies that exclusively reacted with intact LH showed restricted reactivity with LH in 25% of the samples tested. The LH of one individual was not detected at all by one of the antibodies. The existence of two distinct populations suggests the existence of a genetic variant of LH. A comparison of the results obtained by IFMAS with those by radioimmunoassay showed marked discrepancies in the low range, with IFMAS measuring much lower values. The excellent correlation between the various monoclonal IFMA strongly suggests that the discrepancies seen are caused by the insufficient sensitivity and matrix effects of the radioimmunoassay.

Ultrasensitive two-site immunometric assay of human lutropin by time-resolved fluorometry.

A rapid, sensitive two-site immunometric assay for human lutropin (LH), with two monoclonal antibodies directed against the beta subunit, has been developed with time-resolved fluorescence as the detection principle. The cross-reactivities with human thyrotropin and human follitropin are negligible and that with human choriogonadotropin is 1% in the standard two-step protocol. Free beta subunits of LH are also detected. Besides the standard procedure (45 + 15 min), more rapid alternative protocols (15 + 15 min or 30 min in one step) have also been evaluated. The minimum detectable dose per well is approximately 1 amol (10(-18) mol), corresponding to less than 0.01 int. unit/L, for a sample volume of 25 microL and less than 0.001 int. unit/L for 200-microL samples. The intra- and interassay CV is less than 6% for LH concentrations from 0.2 to 250 int. units/L. With 200-microL sample volumes, intra-assay variation is less than 6% at 0.04 int. unit/L and interassay variation is 7% at 0.05 int. unit/L. The very high sensitivity and reproducibility of the presented method enables assay of low prepubertal and of suppressed LH concentrations, and represents a clear improvement over currently available immunoassays.

Sympathetic innervation of the cardiovascular system in the giraffe.

The extreme blood pressure gradients in the giraffe, produced by gravity and behaviour, present a special challenge to blood flow and vascular capacity regulation, e.g. via sympathetic nerves. We report the distribution of nerves in vascular tissue from giraffe extremities and neck based on immunofluorescence against specific antisera to dopamine-beta-hydroxylase, neuropeptide Y, neurofilament, and synapsin I. Sympathetic innervation of gravitation-dependent arteries in the limbs was restricted to the adventitia-media border, while in carotid arteries fibers penetrated deep into the media. Surprisingly, limb veins appeared to be devoid of sympathetic innervation, while jugular veins had a sparse innervation. The morphological response, medial hypertrophy, that parallels the hydrostatic pressure gradients in the circulation combines with the thick skin and tight underlying fascia, the 'g-suit', and an exquisitely distributed sympathetic innervation pattern to provide an effective array of mechanisms for cardiovascular regulation in the adult giraffe.

Pitfalls in the Dextran-coated charcoal assay of estrogen receptors in breast cancer tissue.

This study investigated the influence of the degree of concentration of breast tumor cytosols on the apparent estrogen receptor content as measured by the Dextran-charcoal assay. It was found that the dilution of cytosols to 1-2 mg protein/ml frequently but not always causes highly underestimated receptor concentrations. This could not be explained by the protein loss through adsorption to the charcoal. The effect was also studied in the presence of gelatin, sodium molybdate or with limited trypsinization of the incubation mixture. Addition of 1 mg/ml gelatin in the Dextran-charcoal suspension was very useful in most cases in preventing dilution induced losses in receptor sites. Both trypsinization and addition of sodium molybdate produced increases in receptor concentrations that were not as susceptible to inactivation through dilution of the cytosol. These data suggest that the observed high variability in the dilution induced receptor losses can be explained by receptor heterogeneity: some receptor form(s) are either readily absorbed to or "stripped" by the charcoal particles. As a conclusion we recommend that in order to optimize the estrogen receptor assay as regards both binding sites and affinities the cytosol concentrations should be maintained as high as possible and a protein expander be included in the Dextran-charcoal suspension. Though sodium molybdate frequently gives considerable increases in estrogen binding sites it occasionally has an opposite effect. For this reason we hesitate to recommend its use in routine assays of estrogen receptors.