Gerard J. van Kamp

Influence of Sex Hormones on Plasma Endothelin Levels

Atherosclerosis and hypertension are more common in men than in women, a difference that may be due, in part, to the actions of their respective sex hormones. Men have more atherogenic lipid profiles than women, and sex hormones play an important role in the development of this difference [1-3]. In addition, some evidence has emerged that androgens can induce insulin resistance [3, 4]; several case reports describe cardiovascular events in young persons after the use of anabolic steroids [5, 6]. Sex hormone-associated differences in lipid profiles and insulin sensitivity may partly explain why premenopausal women are relatively protected against cardiovascular disease. It remains to be determined, however, whether the difference between men and age-matched women in the incidence of atherosclerosis can be explained by differences in lipid profiles and insulin sensitivity alone or whether other mechanisms might be involved. Endothelial cells synthesize many active substances that regulate local blood pressure and maintain the fluidity of blood and the patency of blood vessels [7]. These include vasodilators such as endothelium-derived relaxing factor and prostacyclin (which also inhibit platelet adhesion and aggregation), vasoconstrictors such as endothelin, and larger molecules such as fibronectin. Endothelin may also have mitogenic properties [7, 8]. The release of these substances affects the local environment in the blood vessel; endothelin may also have a systemic function [7]. Some evidence has emerged that endothelin may be involved in the pathogenesis of hypertension [7, 9] and atherosclerosis [10]. Elevated plasma levels of endothelin have been observed in patients with myocardial infarction and diabetes. Whether sex hormones have direct effects on the endothelium is not known. We found that plasma endothelin levels tended to be higher in men than in women and lower in pregnant women than in nonpregnant controls. These considerations prompted us to compare endothelin levels in healthy young men and women and to assess the effects of long-term sex hormone therapy in male-to-female and female-to-male transsexuals on plasma endothelin levels. Methods We measured endothelin levels in 29 healthy young women (18 to 31 years of age; mean age, 24.1 years), 20 pregnant women (20 to 32 years of age; mean age, 26.3 years), and 23 healthy men (23 to 33 years of age; mean age, 24.7 years). To assess the effects of sex hormone therapy on plasma levels of endothelin, we measured endothelin levels before and during sex hormone therapy in 12 male-to-female transsexual patients (17 to 33 years of age; mean age, 28.4 years) and 13 female-to-male transsexual patients (17 to 26 years of age; mean age, 24.4 years). Informed consent was obtained from all participants, and the study was approved by the hospital ethics committee. All patients were within 10% of their ideal body weight (Metropolitan Life Insurance Tables, 1959). None had a personal or family history of diabetes or hypertension or had evidence of cardiovascular disease on routine examination (medical history, physical examination, and electrocardiogram). No hormone preparations (such as oral contraceptives) had been used by any of the participants in the 6 months before the study. All women had a regular menstrual cycle (28 to 31 days) before hormone treatment; blood samples were drawn during the follicular phase of the menstrual cycle (days 3 to 5). Female-to-male transsexuals received intramuscular injections of testosterone esters (Sustanon, Organon, Oss, the Netherlands), 250 mg every 2 weeks. Male-to-female transsexuals received oral ethynylestradiol (Lynoral, Organon), 0.1 mg/d, and cyproterone acetate (Androcur, Schering, Weesp, the Netherlands), 100 mg/d, to counteract the effects of testosterone. Plasma endothelin levels were measured before therapy and after 4 months of hormone use. Blood samples were drawn between 0900 hours and 0930 hours after an overnight fast, patients having rested in the supine position for at least 30 minutes. Samples were immediately placed in ice. Testosterone levels were determined 10 to 14 days after injection, and blood samples were drawn simultaneously with those in which plasma endothelin levels were determined. Plasma was separated within 1 hour and then stored at 20C until assayed. Plasma endothelin was measured by radioimmunoassay (Nichols Institute [formerly ITS], Wijchen, the Netherlands) after extraction on Sep-Pak C18 cartridges (Waters, Milford, Massachusetts), as described previously [17]. Recovery rate for this assay is 92.4%. Intra-assay and interassay coefficients of variation according to the manufacturer are 2.4% and 4.2%, respectively. (We found these values to be slightly higher: 3.6% and 5.1%, respectively.) Sensitivity of the assay is 1 pg/mL; cross-reactivity with endothelin-2 is 52%; with endothelin-3, 96%; and with big endothelin, 7%. Intra-assay variation ranged from 2% to 8%, and interassay variation ranged from 4% to 9%. In all patients, blood pressure was determined during a 2-hour period using an automatic sphygmomanometer; patients remained at rest during this period. Blood pressure measurements were done every 5 minutes, and the results were averaged. Results are expressed as mean SD. The Student two-tailed t-test for paired data was used to compare measurements within the same group before and during hormone therapy. The Student two-tailed unpaired t-test was used for between-group comparisons. A P value of less than 0.05 was considered statistically significant. Results Plasma endothelin levels were significantly higher in men than in women (5.9 1.2 compared with 4.17 0.67 pg/mL; P < 0.01). Endothelin levels were lower in pregnant women than in nonpregnant controls (2.19 0.73 compared with 4.17 0.67 pg/mL; P < 0.01), suggesting that high levels of estradiol and progesterone found during pregnancy are associated with low endothelin levels (Figure 1). Figure 1. Plasma endothelin levels in 23 men, 29 women, and 20 pregnant women. P P In biological women treated with intramuscular injections of testosterone esters, basal testosterone levels increased from 1.25 0.66 nmol/L to 24.8 13.0 nmol/L (P < 0.01). No significant change was seen in levels of estradiol-17 (247 207 compared with 185 94 pmol/L). Biological men were treated with oral ethynylestradiol, 0.1 mg/d, and cyproterone acetate, 100 mg/d. Testosterone levels decreased from 18.4 8.6 to 1.1 0.4 nmol/L in this group (P < 0.01). Levels of estradiol-17 and ethynylestradiol were not assessed. The biological effects of sex hormone therapy were manifest in both groups. Effects of sex hormone therapy on endothelin levels in both groups are shown in Figure 2. Endothelin levels before treatment tended to be higher in men than in women (8.1 3.1 compared with 6.2 1.1 pg/mL; P = 0.06), confirming our earlier observations. In biological women treated with testosterone, average endothelin levels increased from 6.2 1.1 to 7.8 1.2 pg/mL (P < 0.01) after 4 months of therapy. In men treated with estradiol and cyproterone acetate, endothelin levels decreased from 8.1 3.0 to 5.1 2.0 pg/mL (P < 0.01). Thus, the decrease in endothelin levels in men was greater than the increase in women treated with testosterone (37% and 26%, respectively); antiandrogenic actions of cyproterone acetate may have contributed to the decrease in endothelin levels in men. Figure 2. Effects of cross-gender sex hormone treatment on plasma endothelin levels in transsexual patients. Left panel. P Right panel. Blood pressure was measured in all participants (at rest) during a 2-hour period before and during hormone therapy. No significant alterations were seen; mean systolic and diastolic pressures were unchanged. To investigate the variability of endothelin plasma levels, we measured endothelin levels twice within a period of 2 months in 10 normal men. Endothelin levels were 5.90 0.85 pg/mL at the first measurement and 5.85 1.05 pg/mL at the second measurement (P 0.05), the coefficient of variation being 16.2%. These data suggest that endothelin levels are relatively stable over a period of several months. Discussion To our knowledge, this is the first study to compare plasma endothelin levels in healthy men and women. Our observations suggest that endothelin levels are higher in men than in women and that this difference is mediated by sex hormones. Atherosclerosis is characterized by endothelial injury and the proliferation of intimal smooth-muscle cells, which may be a result of the release of growth factors from the vessel wall [11]. The evidence suggests that endothelin, which is a strong vasoconstrictor, also has mitogenic properties [7, 8]. A correlation between endothelin levels and atherosclerosis has been described [10], suggesting that endothelin might participate in atherogenesis. Thus, if sex hormones affect levels of endothelin, this might be one of the mechanisms by which sex hormones influence the risk for cardiovascular disease. We speculate that a sex-associated difference in levels of endothelin may be one of the mechanisms underlying the difference in the incidence of cardiovascular disease between men and women. In our patients, blood pressure did not change during hormonal treatment. Asscheman and colleagues [12] assessed blood pressure in 425 transsexual patients (303 biological men and 122 biological women) receiving long-term sex hormone therapy. High blood pressure developed in 10 biological men treated with estrogens (2.4% of the total group of 425 patients) and in none of the women treated with testosterone. Studies of the effect of estrogen therapy on blood pressure in postmenopausal women [13, 14] and in women using oral contraceptives [15] have produced conflicting results. Thus, the effect of sex hormone therapy on blood pressure remains unclear. In healthy men, the intravenous administration of endothelin induces an increase in blood pressure and serum p

Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and n‐acetylgalactosamine (GalNAc) peptides

Antibodies (Abs) to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. We compared the specificity of natural and induced MUC1 Abs with the objective of defining an effective MUC1 vaccine for active immunotherapy of adenocarcinoma patients. Serum samples, selected out of a screened population of 492 subjects for their high levels of IgG and/or IgM MUC1 Abs, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients immunized with MUC1 peptides coupled to keyhole limpet hemocyanin (KLH) and mixed with QS‐21. The samples were tested with enzyme‐linked immunoassays for reactivity with (1) overlapping hepta‐ and 20‐mer peptides spanning the MUC1 tandem repeat sequence; (2) two modified 60‐mer peptides with substitutions in the PDTR (PDTA) or in the STAPPA (STAAAA) sequence of each tandem repeat; and (3) four 60‐mer glycopeptides with each 1, 2, 3 and 5 mol N‐acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined, indicating that Abs directed to more than one region of the MUC1 peptide core can coexist in one and the same subject. The most frequent minimal epitopic sequence of natural MUC1 IgG and IgM Abs was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG Abs predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 Abs from breast cancer patients reacted more strongly with the N‐acetylgalactosamine (GalNAc) peptides than with the naked 60‐mer peptide, while reactivity with the GalNAc‐peptides was significantly reduced (2‐tailed p < 0.0001) in the MUC1 IgG and IgM Abs induced by MUC1 peptide vaccination. Whereas in cancer patients glycans appear to participate in epitope conformation, the epitope(s) recognized by MUC1 Abs induced by peptide vaccination are already masked by minimal glycosylation. Therefore, our results indicate that a MUC1 glycopeptide would be a better vaccine than a naked peptide. Int. J. Cancer 86:702–712, 2000.

Endothelial vasoactive mediators in preeclampsia

OBJECTIVE In recent years an increasing amount of evidence supports the concept that preeclampsia is an endothelial disease. The purpose of our study was to evaluate the extent to which endothelial cell dysfunction is involved in pathophysiology of preeclampsia. STUDY DESIGN We studied the urinary excretion of thromboxane B2 and 6-keto-prostaglandin F1 alpha and the venous plasma endothelin levels in 23 preeclamptic patients and in control subjects. In six of these patients and in six controls arterial plasma endothelin levels were also measured. In addition, plasma levels of calcitonin gene-related peptide and plasma fibronectin levels were measured. Results were analyzed by Wilcoxons rank-sum test or signed-rank test. RESULTS In preeclampsia the urinary thromboxane B2/6-keto-prostaglandin F1 alpha ratio (p < 0.001), venous plasma endothelin levels (p < 0.001), and plasma fibronectin levels (p < 0.001) were significantly elevated compared with normotensive pregnancy. Arterial plasma endothelin levels were significantly higher than venous plasma endothelin levels in normotensive and hypertensive patients (p < 0.05). Calcitonin gene-related peptide levels showed a wide range in normotensive pregnancy and in preeclampsia, but the difference was not significant. CONCLUSIONS These results confirm the extensive involvement of the endothelium in the pathophysiology of preeclampsia. Preeclamptic vasoconstriction seems to be mediated by an increase in the vasoconstrictor autocoids thromboxane A2 and endothelin. Production of prostacyclin by the vessel wall and endovascular trophoblast might be just a pivotal escape mechanism of the uteroplacental circulation. Calcitonin gene-related peptide appears not to be involved in the pathophysiology of preeclampsia.

Plasma endothelin levels are increased during septic shock.

ObjectiveTo study whether serially measured plasma concentrations of endothelin (a novel, potent, endogenous vasoconstrictor derived from endothelium and macrophages) relate to the pathophysiology and severity of human septic shock. DesignProspective analysis. SettingMedical ICU of a university hospital. PatientsSix patients with septic shock, studied for 8 days after ICU admission. Measurements and Main ResultsThe initial plasma endothelin concentration was increased (14.2 ± 5.2 [SD] vs. normal 4.2 ± 0.7 pg/mL, p < .05) and correlated with the Acute Physiology and Chronic Health Evaluation II score (r2 = .79, p < .05). For pooled data, endothelin levels correlated poorly with leukocyte counts (r2 = .13), mean arterial pressure (MAP) (r2 = .16), and administered doses of dopamine (r2 = .26). In multiple regression analyses, plasma endothelin concentrations were predicted by dopamine doses and not by MAP. Plasma endothelin concentrations predicted the decrease in creatinine clearance, independently from MAP. The pooled value for correlations between endothelin levels and creatinine clearance, during the course of disease in individual patients, was statistically significant (r2 = .31). ConclusionsDuring septic shock, the release or production of endothelin may increase as a consequence of endothelial injury by activated leukocytes and the infusion of catecholamines, and this mechanism may relate to renal vasoconstriction and to the severity of disease. (Crit Care Med 1992; 20:1097–1101)

Serum tumor marker immunoassays in gynecologic oncology: Establishment of reference values

OBJECTIVE Our purpose was to establish reference values for six monoclonal antibody-based serum immunoassays applied in patients with carcinoma of the female genital tract. STUDY DESIGN Sera from 938 healthy women (median age 48.0 years, range 37 to 76 years) were assayed for levels of CA 125, CA 15.3 (two methods), CA M29, CA M26, and mucin-like cancer antigen (MCA). RESULTS Reference values, defined as those including 95% of healthy controls, were in women with unknown menopausal status as follows: CA 125, 37 U/ml; CA 15.3 (Centocor), 33 U/ml; CA 15.3 (Boehringer Mannheim), 28 U/ml; CA M26, 83 U/ml; CA M29, 13 U/ml; and MCA, 19 U/ml. Postmenopausal values were significantly lower for CA 125 and CA M26 and significantly higher for CA M29 and CA 15.3 (both methods). MCA serum levels were age independent. CONCLUSION Reference values found were not in accord with those generally applied in gynecologic oncology. In addition, serum levels were influenced significantly by menopausal status (except with MCA).

Coronary heart disease risk indicators, aerobic power, and physical activity in men with spinal cord injuries

OBJECTIVE To compare the lipid and (apo-)lipoprotein profile and blood pressure of men with long-standing spinal cord injuries (SCI) to those of an age-matched able-bodied (AB) population, and to assess the most important determinants of this profile and blood pressure. DESIGN A cross-sectional study of persons with chronic SCI residing in the community. SETTING Tests were performed in a university research laboratory. SUBJECTS Thirty-seven men (age 37.4 +/- 12.0 yrs) with longstanding (14.7 +/- 8.6 yrs) SCI ranging from level C4/5 to L5 volunteered to participate. Comparisons were made with published data from 3,498 AB men, age 20 to 59 yrs, from the same country. MAIN OUTCOME MEASURES Lipid and lipoprotein profile (total cholesterol [TC], high-, low-, and very low-density lipoprotein cholesterol [HDL-C, LDL-C and VLDL-C, respectively], and triglycerides [TG]), as well as aerobic power, activity level, anthropometric variables, and blood pressure. Multiple regression analyses assessed the most important determinants of the lipid and blood pressure profile. RESULTS None of the lipid variables were related to the lesion level. TC, HDL-C, and TC/HDL-C were not significantly different from the AB population. The most important determinants of TC, LDL-C, and the ratios TC/HDL and HDL-C/LDL-C were age, smoking behavior, and activity level. Aerobic power was not an important determinant of any lipid or (apo-)lipoprotein or blood pressure. CONCLUSION Men with long-standing SCI do not appear to have an essentially different coronary heart disease risk profile compared with AB persons. Modifiable risk factors such as activity level, smoking, alcohol consumption, body mass index, and adipose tissue were more important than lesion level and aerobic power in the determination of the lipid and lipoprotein profile, suggesting several potential interventions.

An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

Introduction: About one-third of breast and ovarian carcinoma patients have circulating antibodies reactive with polymorphic epithelial mucin (MUC1), either free or bound to immune complexes. While the presence of these immune complexes has prognostic significance in breast cancer patients, the significance of free MUC1 antibodies is less clear. The objective of this study was to develop a reliable assay for the accurate determination of circulating free antibodies to MUC1. Material and Methods: We developed an enzyme-linked immunosorbent assay (ELISA) (PEM.CIg) employing a 60 mer peptide (a triple tandem repeat sequence of the MUC1 peptide core) conjugated to bovine serum albumin and peroxidase-labeled antihuman immunoglobulin G or M antibodies. The assay was standardized and its analytical performance evaluated. A total of 492 serum samples were obtained from 40 healthy men, from 201 healthy women (including 55 women without a history of pregnancy and 45 pregnant women), and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients. MUC1 serum levels were determined with commercial CA 15-3 tests. Results: Circulating antibodies to MUC1 are present both in healthy subjects and in breast cancer patients. The within- and between-assay coefficients of variation were, respectively, 2 and 12% for the IgG determinations and 1.2 and 3% for the IgM determinations. Correlation coefficients for serially diluted serum samples ranged from 0.9998 to 0.9920 for IgG and from 0.9996 to 0.9818 for IgM determinations. The reactivity of serum samples was partially blocked by the addition of various MUC1 peptides and by MUC1 mucin. The inhibiting effect of modified 60 mer peptides suggests the presence of antibodies directed to more than one epitope. Conclusions: The PEM. CIg assay is a reliable ELISA for measuring free MUC1 antibodies in serum. We are in the process of relating the results obtained in the breast cancer group to disease outcome to evaluate its prognostic significance. In addition, the assay may become a useful tool for vaccine therapy monitoring.

The Utility of Lipid-Associated Sialic Acid (LASA or LSA) as a Serum Marker for Malignancy

The utility of the lipid-associated sialic acid (LASA or LSA) test as a serum marker for malignancy is reviewed. The name LASA or LSA test is confusing because it suggests that only or mainly lipid-bound sialic acid is measured. In reality, glycoprotein-bound sialic acid is determined predominantly. The assay appears to have a particularly high positivity rate in leukemia, Hodgkins disease, melanoma, sarcoma, advanced ovarian carcinoma and oropharyngeal tumors, suggesting that LASA may serve as a valuable marker in these malignancies. As a consequence of the rise of sialic acid-rich acute-phase proteins, such as alpha 1-acid glycoprotein, in inflammatory diseases the specificity of LASA and therefore its diagnostic accuracy is low. LASA can be useful for monitoring cancer patients during treatment, especially in combination with other tumor markers.

Increased angiotensin-converting enzyme activity in cerebrospinal fluid of treated patients with Parkinson's disease

In the human brain angiotensin converting enzyme (ACE, EC 3.4.15.1) exists in two forms of activity [il. ACE is mainly found in a membrane-fund fraction of cortical epithelial cells, catalysing the conversion of inactive an~otensin I to hypertensive angiotensin II [2}. A soluble ACE form is located neuronally within the corpus striatum and may hydrolyse neuropeptides with a messenger function [3]. ACE activity in normal human lumbar cerebrospinal fluid (CSF), may largely reflect ACE activity in non-cortical regions of the brain, for instance the basal ganglia [4]. It is therefore of interest to evaluate the activity of ACE in neurological disease, such as Parkinson’s disease (PD), with a degeneration of the nigrostriatal dopaminergic neurons and consequent striatal dopamine depletion. Previous studies in PD patients have shown either decreased [5] or normal [6] CSF ACE activity. These authors, however, have not studied untreated and treated PD patients separately. Recently, increased CSF ACE activity in schizophrenic patients

Genetic and biochemical markers for Alzheimer's disease: recent developments

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of unknown aetiology, characterized by irreversible cognitive and physical deterioration. It is a major cause of morbidity and death in the elderly and a growing public health problem as life expectancy in the general population increases. AD is both genetically and phenotypically a heterogeneous disorder. An early-onset, familial type is recognized, as well as a later-onset, sporadic type. The diagnosis is made on clinical grounds, with the aid of a small number of additional investigations, using consensus criteria. 1 However, at autopsy about 10± 20% of clinically diagnosed AD patients are found to have conditions other than AD. Therefore, genetic and/or biochemical markers that support the clinical diagnosis and can distinguish AD from cognitive symptoms attributable to ageing and from other dementias will be of great value. The identi® cation of such accurate markers for the early diagnosis of AD is mandatory for the development of ef® cient pharmacological treatment, since therapy should be initiated at an early stage of the disease, before extensive and irreversible brain damage has occurred.