The effects of physical exercise on plasma levels of relaxin, NTproANP, and NTproBNP in patients with ischemic heart disease.

The insulin-like and vasodilatatory polypeptide relaxin (RLX), formerly known as a pregnancy hormone, has gained interest as a potential humoral mediator in human heart failure. Controversy exists about the relation between plasma levels of RLX and the severity of heart failure. The present study was designed to determine the course of RLX, atrial, and brain natriuretic peptide (NT-proANP and NT-proBNP) during physical exercise in patients with ischemic heart disease (IHD) and to relate hormone levels to peak cardiac power output (CPO) as a measure of cardiopulmonary function with prognostic relevance. 40 patients with IHD were studied during right-heart-catheterization at rest and during supine bicycle ergometry. RLX, NTproBNP, and NTproANP were determined before, during exercise, and after recovery. NT-proANP and NT-proBNP levels increased during maximal charge, and recovery while RLX levels decreased. Cardiac power output at maximal charge correlated inversely with NTproANP and NTproBNP but positively with RLX. Patients with high degree heart failure (CPO<1.96 W) had higher NTproANP and NTproBNP and lower RLX levels than patients with low degree heart failure. While confirming the role of NTproANP and NTproBNP as markers for the severity of heart failure, the present data do not support the concept that plasma levels of RLX are related to the severity of myocardial dysfunction and that systemic RLX acts as a compensatory vasodilatatory response hormone in ischemic heart disease.

Relaxin as a protective substance in preservation solutions for organ transplantation, as shown in an isolated perfused rat liver model.

Reperfusion injury, a well-known problem in organ transplantation, results from multiple pathologic mechanisms, including platelet/mast cell activation and peroxidation of cell membrane lipids. Relaxin was originally described as an insulin-like hormone produced in the ovaries during pregnancy. It causes vessel dilation and inhibition of platelet and mast cell activation. The present study investigated the protective effect of relaxin against reperfusion injury in liver tissue. We used a model of isolated perfused rat liver to simulate liver transplantation. Organ preservation was performed identical to human transplantation in 20 male Wistar rats. During preservation we applied 64 ng/mL relaxin. In contrast controls (n = 10) had no relaxin treatment. To quantify cell damage, we measured malonyldialdehyde (MDA; end product of lipid peroxidation) and myeloperoxidase activity (MPO; marker for accumulation of neutrophil granulocytes) in the perfusates. The livers were examined immunohistochemically for the same parameters. Relaxin as an additional substance in preservation solutions decreased perfusate MPO and MDA levels by up to 30%, as shown by immunohistochemistry. Our preliminary data suggested that relaxin is a promising agent to reduce hepatocyte damage caused by ischemia-reperfusion injury. Quantitative analysis of MDA and MPO levels in the perfusate is the subject of an ongoing study.

Effect of bone sialoprotein and collagen coating on cell attachment to TICER and pure titanium implant surfaces.

To improve integration between implants and biological tissues, this study compared bone sialoprotein (BSP) as a surface-coating material against the major organic and inorganic components of bone, collagen type I and hydroxyapatite (TICER). The expression of osteocalcin, osteonectin and transforming growth factor ss was evaluated using immunohistochemical staining procedures. The distribution patterns of osteoblasts on the surface of pure titanium with a smooth machined surface and a rough surface (TICER) were determined by image processing using confocal laser scanning microscopy. The results compared to uncoated control materials showed that, at all times investigated, the number of cells on the surface of the TICER and pure titanium samples differed significantly (P<0.1), demonstrating the superiority of TICER over pure titanium in this respect. For pure titanium implants, collagen-precoated surfaces were not beneficial for the attachment of bone-derived cells with the exception of day 3 in vitro (P<0.01). BSP-precoated implant surfaces displayed non-significantly higher numbers of settled cells. BSP-precoated implant surfaces were beneficial for osteoinduction as revealed by osteocalcin and osteonectin expression. BSP precoating of the rough TICER implant surface enhanced the osteoinductive effect much more than did collagen precoating. These results contribute to the consideration of at least two distinct pathways of osseointegration.

Implant surface coatings with bone sialoprotein, collagen, and fibronectin and their effects on cells derived from human maxillar bone.

The interaction between implant material and surrounding tissues is believed to play a fundamental role in implant success. Although bone sialoprotein (BSP) has been found to be osteoinductive when coated onto femoral implants, collagen and fibronectin are the most used compounds for preparation of pre-coated cell culture slides at present. In this study, the support of BSP-, collagen- and fibronectin-coated and non-coated implant material for the development of adult human maxillar bone in vitro was studied and compared. The expression of bone turnover markers like BSP and osteocalcin as well as osteonectin, transforming growth factor beta (TGF-beta) and CD90 during different time periods of cell cultivation (3, 5, 10, 15, 20 and 25 days) was visualized immunohistochemically. The distribution patterns of the cells were examined on a rough surface of the titanium-hydroxyapatite dental implant material TICER and on a total smooth surface of the technical implant material glimmer. Significantly different values were found for glimmer at the 15. and the 20. Div, exclusively, indicating that a smooth surface was more improved than a rough ceramic surface by pre-coatings. The White-test using rankings of the median values gave evidence for BSP-coatings at position 1 followed by collagen. Our experiments were designed to use very low concentrated BSP coating solution with the aim to reduce the healing time with a minimal effort and minimal risks for the patients.

Increased serum bone sialoprotein concentrations in patients with Crohn's disease.

INTRODUCTION: Impaired calcium homeostasis and/or the administration of corticosteroids are considered to be among the factors contributing to the pathogenesis of osteopenia in patients with inflammatory bowel disease. There is an increasing evidence suggesting that certain pro-inflammatory cytokines may also directly influence the bone metabolism in these patients. Routine measurement of bone mass and loss usually include dual energy X-ray absorptiometry as well as urinary and serum assessment of collagen crosslinks. More recent studies include likewise the detection of bone sialoprotein into a specific diagnostics of bone turnover. PATIENTS AND METHODS: We investigated 47 patients with inflammatory bowel disease (Crohn's disease N = 41, ulcerative colitis N = 6) and 17 healthy volunteers to assess and compare serum levels of bone sialoprotein and other routine parameters of bone turnover. Bone sialoprotein levels were measured by using a recently described radioimmunoassay. RESULTS: In comparison to the control group, bone sialoprotein and urinary crosslinks were significantly increased only in patients with Crohn's disease, while other markers of bone turnover (e. g. alkaline phosphatase, carboxylterminal propeptide of typ I procollagen, urinary deoxypyridinoline, vitamin D, phosphate and calcium) did not differ significantly between the patients' groups. CONCLUSION: According to these data, increased serum bone sialoprotein concentrations seem to be an additional valuable and sensitive marker of bone resorption in patients with Crohn's disease.

The pregnancy hormone relaxin is a player in human heart failure.

Human congestive heart failure is characterized by complex neurohumoral activation associated with the up-regulation of vasoconstricting and salt-retaining mediators and the compensatory rise of counter-regulatory hormones. In the present study, we provide the first evidence that relaxin (RLX), known as a pregnancy hormone, represents a potential compensatory mediator in human heart failure: plasma concentrations of RLX and myocardial expression of the two RLX genes (H1 and H2) correlate with the severity of disease and RLX responds to therapy. The failing human heart is a relevant source of circulating RLX peptides, and myocytes as well as interstitial cells produce RLX. Elevation of ventricular filling pressure up-regulates RLX expression and the hormone acts as a potent inhibitor of endothelin 1, the most powerful vasoconstrictor in heart failure. Furthermore, RLX modulates effects of angiotensin II, another crucial mediator. Our data identify RLX as a new player in human heart failure with potential diagnostic and therapeutic relevance.

Determination of alpha1-proteinase inhibitor by a new enzyme linked immunosorbant assay in feces, serum and an enterocyte-like cell line.

Although alpha(1)-proteinase inhibitor (alpha(1)-PI), the main serine proteinase inhibitor in human plasma, is predominantly liver-derived, the proof of fecal alpha(1)-PI is a maker for intestinal protein loss. Furthermore, alpha(1)-PI is synthesized locally by human intestinal epithelial cell lines (e. g. Caco-2). Therefore, we investigated the diagnostic value of a new enzyme-linked immunosorbent assay (ELISA) to detect alpha(1)-PI in serum, feces, and Caco-2 supernatants in comparison with radial immunodiffusion (RID). alpha(1)-PI concentrations assessed by ELISA were on an average 30 % higher than those measured by RID. Only the ELISA system detected alpha(1)-PI in supernatants of Caco-2 cells. Our data imply first that this ELISA system is more sensitive to assess alpha(1)-PI than other methods, and second that it obviously determines locally synthesized alpha(1)-PI which can not be liver-derived. However, further examinations are necessary to distinguish between enterocyte-derived or systemic alpha(1)-PI and its diagnostic relevance in bowel disease.

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis.

Bone sialoprotein (BSP) is a noncoflagenous bone matrix protein that is important for both mineralization and cell-cell interactions. Tissue studies in primary breast cancers have shown that immunohistochemical expression of BSP is associated with a high incidence of bone metastases in the course of the disease. We used a RIA to investigate the importance of serum BSP as a marker for subsequent bone metastases. Between 1994 and 1996, preoperative blood samples were collected from 388 consecutive patients with nonmetastatic breast cancer and from 30 control patients with benign breast disease. Serum BSP concentrations were measured in a blinded fashion by RIA. The cutoff for elevated serum BSP values was 24 ng/ml, ie., two SDs above the normal mean value. Serum BSP was correlated with the risk of metastasis and analyzed with regard to its prognostic value. After a median follow-up period of only 20 months, 28 patients had developed metastases. Fourteen patients had bone metastases only, 9 visceral metastases only, and 5 a combination of osseous and visceral metastases. Of the 19 women with skeletal metastases, 17 had preoperative serum BSP values in excess of 24 ng/ml (median BSP values: 48.3 ng/ml for isolated metastatic bone disease, 30.6 ng/ml for combined metastases), whereas none of the women with visceral metastases only had elevated serum BSP concentrations (median BSP value: 12.3 ng/ml). The median serum BSP value in the control group (benign breast disease) was 8.8 ng/ml serum BSP; levels correlated with the size of the primary tumor, but not with any other prognostic factors. Using a multivariate regression analysis, serum BSP was found to be the most important independent prognostic factor for the development of skeletal metastasis (P < 0.001; relative risk, 94); its specificity was 96.7%, and its sensitivity was 89.5%. Our study shows that patients with preoperatively elevated serum BSP levels are at high risk of subsequent bone metastases in the first years after primary surgery. The mechanism of BSP in the pathogenesis of skeletal metastases is unclear. Because BSP contains an integrin recognition sequence, its expression in tumor cells may facilitate their adhesion to the bone surface. However, it is possible that a proportion of circulation BSP is derived from normal or tumor-induced bone turnover. Breast cancer patients with elevated serum BSP levels may benefit from osteoprotective adjuvant therapy with bisphosphonates.

The effect of estradiol on urodilatin production in postmenopausal women.

Urodilatin, which is structurally homologous to atrial natriuretic peptide, is most probably synthesized in the kidney. It inhibits water and sodium reabsorption and causes renal vasodilatation. However, little is known about the regulation of its synthesis, especially with regard to the influence of hormones. Transdermal administration of estradiol with low dosage producing physiologic conditions, and oral administration of estradiol with high pharmacologic dosage were investigated for their effect on urodilatin production in postmenopausal women measuring urinary excretion. Both routes of administration of estradiol increased urodilatin excretion after 28 days of treatment, the increment being statistically significant only for the transdermal route. The increased urodilatin production registered after transdermal estradiol replacement therapy may suggest that estradiol has a vasodilatory effect on the kidney.

Urinary and plasma urodilatin measured by a direct RIA using a highly specific antiserum.

Urodilatin (95-126) (URO) appears to play a major physiologic role in fluid homeostasis and produces major changes when administered intravenously. Here we describe a monospecific, high-affinity antiserum against URO with no cross-reactivity (<0.01%) against the structural highly homologous atrial natriuretic peptide 99-126 (ANP-99-126), ANP analogs, and related peptides such as brain natriuretic peptide. A competitive RIA was developed, based on this antiserum. Urine samples with or without ethanol extraction and plasma samples without pretreatment were analyzed by the RIA, which had a detection limit of 10.5 ng/L, a linear measuring range between 10.5 and 1000 ng/L, and recoveries of 93-102% in urine and 90-104% in plasma. The intraassay CVs were 8.2% and 8.1% for urine samples with 269 and 669 ng/L URO; the interassay CV was 9.7% at 839 ng/L. Using this assay, we present URO data for urine from healthy volunteers receiving low and routine sodium diets and from clinical urine specimens; we also present pharmacokinetic data for URO in plasma from patients suffering from bronchial asthma and treated by URO infusion.

The influence of norethisterone acetate on urinary urodilatin excretion in postmenopausal women.

We have previously found that transdermal estradiol application significantly stimulated the urinary excretion of urodilatin, a newly discovered renal peptide with diuretic properties. It is well established that the addition of progestogen is necessary in hormone replacement therapy in women with an intact uterus. This study was designed specifically to examine the effect of progestogen norethisterone acetate (NETA) in postmenopausal women. NETA given alone orally in dosages of 1 mg/d and 2 mg/d for 10 days, as used for the progestogen-challenge test, did not increase urodilatin excretion. NETA in combination with estradiol, administered orally and transdermally in the second half of a 4-week estradiol treatment cycle, did not significantly change urodilatin excretion. The results of the present study indicate that NETA addition to estrogen replacement therapy may antagonize the stimulating estradiol effect on urodilatin production as registered in our earlier study.

Improved purification of human bone sialoprotein and development of a homologous radioimmunoassay.

Bone sialoprotein (BSP) is a phosphorylated skeletal glycoprotein. Here we describe a new procedure for the purification of BSP involving wide-pore reversed-phase HPLC, and the development of a homologous RIA for human BSP. The immunoassay showed linearity between 3 and 120 micrograms/L, a lower detection limit of 0.7 micrograms/L, and a mean recovery rate of 99.4%. Intraassay variation was 7.0% (mean = 10.9 micrograms/L) and 6.1% (mean = 38.8 micrograms/L), and interassay variation was 9.2% (mean = 11.1 micrograms/L) and 9.4% (mean = 39.0 micrograms/L). No cross-reactivity was detected with osteocalcin, osteonectin, or bone alkaline phosphatase. Preliminary clinical evaluation in healthy controls (n = 90) showed a mean serum BSP concentration on 12.1 +/- 5.0 micrograms/L (+/- SD). BSP was significantly increased in patients with Paget disease of bone, primary and secondary hyperparathyroidism, and also in subjects with renal failure without skeletal involvement. Impairment of hepatic function did not affect serum BSP concentrations.

Bone sialoprotein in serum of patients with malignant bone diseases.

Bone sialoprotein (BS), a protein synthesized by osteoblasts and osteoclasts and highly modified posttranslationally, constitutes a predominant fraction of the noncollagenous organic matrix in human bone. We report an assessment of serum concentrations of BS in patients with malignant bone diseases. In patients with bone metastases (according to scintigraphic criteria), serum BS concentrations were greater than in patients without bone metastases (P <0.05). However, ROC curve analysis revealed that serum BS was inferior to serum bone alkaline phosphatase in discriminating between patients with and without bone metastases. Patients with bone metastases showed a weak correlation between serum BS concentrations and bone formation markers. Only "traditional" markers of bone formation-but not BS-were correlated with urinary deoxypyridinoline (P <0.01). Liver and kidney dysfunction had no significant influence on BS values in these patients (as assessed by analysis of variance; P >0.05). In multiple myeloma patients treated with corticosteroids and bisphosphonates, BS concentrations were lower than in tumor patients without bone metastases (P <0.001), and the correlation between BS concentrations and the number of bisphosphonate courses applied was significant (r = -0.578; P <0.05). In postmenopausal women, serum BS concentrations averaged 142% greater than in premenopausal women. Further studies should be done, therefore, to elucidate whether serum BS is able to predict high bone turnover after menopause.

[Relaxin in amniotic fluid and serum of pregnant patients]. / Relaxin im Fruchtwasser und Serum von Schwangeren.

Relaxin was measured in serum and amniotic fluid of 136 pregnant women between the 12th and 38th gestational week by means of a new human relaxin-RIA. The pregnancies consisted of 111 pathology-free single fetuses, 10 with rhesus incompatibility, 7 with chromosomal aberration and 8 with sonographic diagnosed abnormalities. Relaxin could be detected in all samples tested the levels being ten times lower in amniotic fluid compared to serum. Serum relaxin levels showed a slight but not statistically significant decrease with increasing gestational age, in amniotic fluid relaxin values were consistent over the course of pregnancy. The ratio of amniotic fluid to serum relaxin displayed a statistically significant increase from the 12th to 23rd week of pregnancy. Individual courses of relaxin concentration in amniotic fluid revealed only low intra-individual variations but distinct inter-individual differences.

Urinary excretion of insulin after estradiol treatment of postmenopausal women.

The influence of estradiol treatment on the urinary excretion of insulin was investigated in postmenopausal women. Thirteen women were treated with transdermal estradiol and 12 with oral estradiol for 4 weeks. With transdermal, but not with oral administration, a significant increase of urinary insulin excretion was registered.

Serum immunoreactive bone sialoprotein as a new marker of bone turnover in metabolic and malignant bone disease.

Bone sialoprotein (BSP) is a phosphorylated glycoprotein with a M(r) of 70-80 kDa that accounts for approximately 5-10% of the noncollagenous proteins of bone. Due to its relatively restricted distribution to mineralized tissues, BSP may serve as a potential marker of bone metabolism. Employing a recently developed RIA, serum BSP was measured in 133 healthy subjects, aged 20-80 yr, and in patients with primary hyperparathyroidism (pHPT; n = 26), Paget's disease of bone (PD; n = 14), untreated multiple myeloma (MM; n = 32), and breast cancer with bone metastases (BC; n = 19). Results were compared to clinical and laboratory data, including serum total alkaline phosphatase as a marker of bone formation, and the urinary cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) as markers of bone resorption. In healthy adults, serum BSP values ranged between 5.0-21.6 ng/mL (5-95% interval), with a median of 10.5 ng/mL (total group). In healthy females, a linear correlation was found between serum BSP and age (r = 0.51; P < 0.001), with significantly higher values in postmenopausal than in premenopausal women (13.3 +/- 4.8 vs. 9.0 +/- 3.8; P < 0.01). In the healthy group, BSP values did not change with body mass index, lumbar bone mineral density, serum calcium, serum creatinine, or serum total alkaline phosphatase levels. In contrast, a weak, but significant, correlation was observed between serum BSP and the urinary excretion of PYD and DPD. Compared to those in healthy controls, serum BSP levels were significantly higher in patients with pHPT, PD, MM, or BC (P < 0.01 for all groups). These differences remained after analyses were adjusted for age and sex. In pHPT, serum BSP levels were closely correlated to urinary PYD and DPD (r = 0.87 and 0.83, respectively; P < 0.01), whereas in PD, no correlation was observed between any of the bone markers. Serum BSP levels were highest in patients with MM, and there was a significant difference between early and advanced stages of the disease (30.2 +/- 8.0 vs. 64.3 +/- 6.8; P < 0.01). In a subgroup of 15 patients with metastatic BC, iv bisphosphonate treatment resulted in a rapid reduction of serum BSP levels to 40% of the baseline values within 4 days of treatment. In conclusion, BSP appears to be a sensitive marker of bone turnover, and the present data suggest that its serum levels predominantly reflect processes related to bone resorption.

Urinary excretion of relaxin after estradiol treatment of postmenopausal women.

The influence of estradiol treatment on the urinary excretion of relaxin, a hormone in earlier years only found during pregnancy and presently associated with functions in the cardiovascular system, was investigated in postmenopausal women. Thirteen postmenopausal women were treated with transdermal estradiol and 12 women with oral estradiol for 4 weeks. A new radioimmunoassay for human-relaxin (rec-hRLX-2) was used. With transdermal, but not with oral administration, a significant increase of urinary relaxin excretion was registered. Further experiments are necessary to elucidate the source of urinary relaxin and its role in the hormone replacement therapy of postmenopausal women.