Presence of immunoreactive endothelin in human plasma

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl-silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR-hET was 0.6 fmol/ml. Reverse-phase HPLC coupled with radioimmunoassay revealed two major IR-hET components, one corresponding to authentic hET(1-21) and another with more hydrophilicity than hET(1-21). These data indicate that ET is a circulating vasoconstrictor hormone in man.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Regional distribution of immunoreactive endothelin in rats

By use of a specific radioimmunoassay for endothelin (ET), the regional distribution of ET-like immunoreactivity (LI) was studied in rats. The antiserum used cross-reacted equally with synthetic porcine and rat ET. Significant amounts of ET-LI are detectable not only in aorta, but also in kidney, lung, heart, liver and central nervous system. Gel chromatography of the tissue extracts revealed size heterogeneity of ET-LI; one major component eluting close to, but slightly larger than standard rat ET and the other minor component with a larger molecular weight. These data indicate that ET-LI is widely distributed throughout the various rat tissues, suggesting its possible involvement in a variety of organ functions.     

Presence of immunoreactive endothelin in human milk

Endothelin-like immunoreactivity was detected in human milk at a concentration of 6.8 +/- 1.6 pmol/l (mean +/- SEM; n = 16) using a highly sensitive radioimmunoassay. Gel filtration and fast protein liquid chromatography (FPLC) verified the identity of the endothelin. FPLC revealed 4 peaks, one eluting just after the void volume, and the other three in the positions of endothelin-1, -2, and -3, respectively.     

Characterization of immunoreactive endothelin in human urine.

We developed three antibodies, specific and sensitive to endothelin-1 (ET-1), and established two sandwich and three competitive enzyme immunoassays (EIAs). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-1 but not with the antibody against C-terminal peptide of ET-1, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-1.     

Chromatographic characterization of immunoreactive endothelin in rat lung

Using a specific and sensitive radioimmunoassay for endothelin, combined with gel filtration and reverse phase high performance liquid chromatography, the molecular form of immunoreactive endothelin in the rat lung was investigated. On reverse phase high performance chromatography, the major immunoreactive endothelin in the rat lung emerged at a position identical to that of authentic endothelin-1. After oxidation of the immunoreactive endothelin by H2O2, the immunoreactivity migrated to a position identical to that of Met-sulfoxide endothelin-1. These data indicate that the major immunoreactive endothelin in the rat lung is not endothelin-3 (putative rat/human endothelin), but is identical or very similar to endothelin-1 (porcine/human endothelin).     

Radioimmunoassay for endothelin and immunoreactive endothelin in culture medium of bovine endothelial cells

Using a synthetic 21-residue endothelin as antigen, we have produced an antiserum for endothelin and developed a specific and sensitive radioimmunoassay (RIA) for endothelin. The minimum detection limit of the RIA was 1 pg/tube. Immunoreactive (ir-) endothelin was extracted from the culture medium by Bondelute C8 column. The ir-endothelin in the culture medium of endothelial cells (EC) from bovine pulmonary artery and carotid artery was 1.48 ng/ml and 3.31 ng/ml, respectively. Reverse-phase high performance liquid chromatography coupled with the RIA revealed that ir-endothelin in the culture medium comprised one major component corresponding to synthetic endothelin. In addition, the cultured EC of bovine pulmonary artery were specifically stained by immunohistochemical technique. These results suggest that endothelin could be produced in the EC of the pulmonary and carotid arteries besides the aorta. The RIA presented in this study could be an useful tool to investigate the pathophysiologic significance of endothelin.     

Characterization of plasma glycosaminoglycans in hemodialysis patients]

Glycosaminoglycans are heteropolysaccharides composed of disaccharide repeating subunits, each one containing a uronic acid component (glucuronic or iduronic acid) and a hexosamine (N-acetyl-glucosamine or N-acetyl-galactosamine, which may be differently sulphated). The presence of GAGs in human plasma has been demonstrated in several studies; they are bound to plasma proteins through non-covalent linkages. However, very little is known about either their origin or their physiological role. Due to their anionic charge, they may influence some metabolic processes, such as blood coagulation, and they could also have a role in urolithiasis and atherogenesis. Moreover, they may be important in modulating the metabolism of some lipoproteins by affecting the rate of their catabolism. Modifications of GAG pattern have been described in a few pathological conditions such as mucopolysaccharidosis, connective tissue diseases and kidney diseases. A high frequency of accelerated atherosclerosis has been observed in haemodialysis patients (HD), probably associated with the altered lipoprotein profile, which is often described in these subjects. Since GAGs may play a role in lipoprotein metabolism, we isolated and characterized plasma GAGs from a group of HD patients and a group of normal matched subjects. Quantitative analysis of plasma GAGs showed a significant increase of these polysaccharides in the HD group. Circulating levels of GAGs were 8.21 +/- 1.89 micrograms/ml in control subjects, and 15.08 +/- 3.13 micrograms/ml in the HD group (p < 0.0001). The isolation of plasma GAGs by ion-exchange chromatography produced two uronic acid containing families: a low-charge (peak I) and a high-charge (peak II) species. Both of these contained GAGs associated with plasma proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of immunoreactive endothelin in human saliva and rat parotid gland.

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.     

Immunoreactive endothelin in human plasma: marked elevations in patients in cardiogenic shock

Endothelin (ET) is a recently discovered, endothelium-derived peptide that may be the most potent vasoconstrictor yet identified. Although there is much interest in the possible systemic actions of circulating ET in vivo, there is no data on ET levels under physiological conditions, or in cardiovascular disease. We used a radioimmunoassay that was sufficiently sensitive to detect ET immunoreactivity (irET) in the SepPak-extracted plasma from 14 healthy volunteers in a range from 0.03 to 0.69 pg/ml (mean 0.26 +/- 0.236 pg/ml). ET levels were not significantly different from normal in 5 patients with stable congestive heart failure (0.46 +/- 0.36 pg/ml). However, irET was increased markedly in 6 patients in cardiogenic shock (3.65 +/- 1.14 pg/ml), and (less so) in 6 patients on chronic dialysis (1.05 +/- 0.41) and in 4 with pulmonary hypertension (1.52 +/- 0.45) (p less than 0.001). The present results suggest that circulating irET concentration is responsive to altered cardiovascular conditions, and therefore support a potential role for ET as a vasoactive hormone.     

Relationship between zinc levels and plasma leptin in hemodialysis patients

Recent evidences suggested a possible relationship between zinc deficiency and leptin levels in pathogenesis of anorexia in chronic kidney disease. The present study addressed the relationship between zinc and leptin in hemodialysis (HD) patients.MethodsFifty HD patients (54.3 ± 12.7 years old, 62% men) were studied and compared to 21 healthy volunteers (50.7 ± 15.7 years old, 43% men). Biochemical data, serum zinc, plasma leptin, IL-6, TNF-α and C-Reactive Protein levels were determined. Anthropometric parameters, food intake and appetite score were also assessed.ResultsThe leptin levels were higher in HD patients (16.1 μg/mL (0.21–118.25) vs 6.0 μg/mL (0.50–23.10)) in healthy volunteers (p = 0.04), whereas serum zinc levels were lower (54.5 ± 16.3 μg/dL) compared to healthy volunteers (78.4 ± 9.4 μg/dL) (p = 0.0001). The plasma leptin was correlated negatively with plasma zinc (r = ?0.33; p = 0.007), energy (r = ?0.38; p = 0.002) and protein intake (r = ?0.34; p = 0.006) and, positively correlated with BMI (r = 0.54; p = 0.0001), % body fat (r = 0.70; p = 0.0001) and conicity index (r = 0.46; p = 0.001). Plasma zinc was associated with hemoglobin (r = 0.30; p = 0.04) and negatively associated with TNF-α (r = ?0.37; p = 0.002) and C-Reactive Protein (r = ?0.37; p = 0.004). There was no correlation among Zn, leptin and appetite score in these patients.ConclusionThis study showed that low plasma zinc levels are negatively associated with high leptin levels in HD patients.     

Determination of the oxido-redox status of plasma albumin in hemodialysis patients

The oxido-redox status of plasma albumin in patients treated with hemodialysis was characterized with LC-ESI-MS/MS and was compared with models of oxidative stress. Oxidised albumin was characterized by sulfonation (SO3-) of the SH at Cys 34, unfolding and acidification of the molecule. Albumin in hemodialysis patients presented, instead, only intermediate oxidation products such as sulfenic (SO2), sulfonic (SO)and methionine sulfoxide (C5H9NO2S) involving Cys 165-269 and Met 329-548 but did not present SO3- at Cys 34. Absence of charge and structural alterations compared to the oxidised templates was also confirmed with electrophoretic titration and calorimetry. In conclusion, the oxido-redox status of plasma albumin in hemodialysis patients lacks the hallmarks of the advanced oxidation products. LC-ESI-MS/MS was crucial to characterize albumin in conditions of oxidation stress; surrogate techniques can mirror conformational changes induced by oxidation.     

Multiple immunoreactive forms of calcitonin in human plasma

Purified rabbit serum haptoglobin was partially characterized, and it was found that the hemoglobin-binding property and subunit structure were similar to those of human type 1-1, swine, canine, and equine haptoglobins. However, rabbit haptoglobin was dissociated into subunits and few intermediates only in the presence of urea or sodium dodecyl sulfate without reduction. Thus the absence of interchain disulfide bonds in rabbit haptoglobin is unique among many animal haptoglobins.     

Modified low density lipoproteins activate human macrophages to secrete immunoreactive endothelin

This study attempted to determine if low density lipoproteins (LDL) induce the production of endothelins (ET) by human macrophages. Non-protected LDL from macrophage induced oxidation (n-LDL), copper-oxidized LDL (Ox-LDL), acetylated-LDL (Ac-LDL), butylated hydroxytoluene-LDL (BHT-LDL), BHT-Ac-LDL, polyinosinic acid (PiA, 1.5 micrograms/ml), phorbol myristate acetate (PMA; 0.5 microM) and BHT alone (20 microM) were studied. The different compounds had the following potency to stimulate the ET secretion: PMA greater than Ox-LDL greater than Ac-LDL greater than n-LDL greater than BHT-LDL greater than PiA greater than PiA + Ac-LDL greater than BHT. In conclusion, modified LDL stimulated ET secretion by human macrophages.     

Patterns of immunoreactive pancreatic polypeptide in human plasma

Pancreatic polypeptide (PP) is synthesized as an amino-terminal moiety of a precursor peptide and is released into plasma during stimulation as an amidated hormone (PP1-36). The purpose of this investigation was to ascertain the immunoreactive forms of PP in human plasma using HPLC chromatographic technique. Plasma was obtained from five normal volunteers under various postprandial intervals and from the Blood Bank. PP in each plasma sample was processed for HPLC analysis by immunoprecipitation and/or immunoaffinity extractions. Migration patterns of PP-forms were identified under isocratic elution. This study shows that human plasma contains four distinct immunoreactive (IR) forms of PP during stimulation by a protein-rich meal. These forms are PP1-36 (peak 4), PP3-36 (peak 3) and unidentified material migrating as peak 2 and peak 1. The corresponding migration constants were Kav 0.828 +/- 0.04, Kav 0.790 +/- 0.003, Kav 0.570 +/- 0.009 and Kav 0.409 +/- 0.007, respectively. The predominant fasting from of IR PP chromatographed as peak 1, while peaks 2 and 4 were reduced in amplitude. The 1 h and 3 h postprandial chromatograms of HPLC profiles of plasma PP were similar in shape but lower in relative magnitude and amplitude. The authenticity of peak 4 as the migration of native PP1-36 was confirmed using purified IR native PP1-36 extracted from human pancreas. Partial amino acid sequence analysis of PP peak 3 revealed deletions of two N-terminal amino acid residues. The chemical identities of peaks 1 and 2 are unknown but appear to differ from PP in peaks 3 and 4 by virtue of their migration profiles. It is concluded that there are at least four distinct IR forms of PP in human plasma. Native PP1-36 accounts for less than 1% of total PP after an overnight fast and is about 1/3 of total postprandial IR plasma PP. Discernment of the nature and etiology of forms of PP in plasma may provide a new understanding of the role of PP in mammalian physiology.     

Detection of immunoreactive napsin A in human urine

Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.     

Detection of endothelin 1,2 and endothelin-like immunoreactant in wound surface and plasma in mice with thermal injury

Endothelin is a well known vasoconstructive peptides produced by endothelial cells and has been reported to regulate the systemic circulation. The authors investigated changes in endothelin in plasma and the surface of wounds induced with thermal injury using an experimental ear burn model in mice. At 0, 15, 30, 60, 120 and 180 minutes after thermal injury the plasma endothelin-like immunoreactant levels were 1.50 ± 0.21, 1.86 ± 0.36, 2.81 ±0.55, 2.62 ± 0.27, 1.54 ± 0.14 and 1.25 ± 0.19 fmol/ml (N=8), respectively. Endothelin-like immunoreactant levels in the plasma increased gradually until 30 minutes after the thermal injury. Endothelin-like immunoreactant content in the ear before thermal injury and at 60 minutes after injury were 7.04 ± 0.64 and 8.61 ± 1.24 fmol/ear (N=8), respectively. The change in endothelin-like immunoreactant after thermal injury originated from endothelin 1,2; that is, the endothelin-1,2 content of the burned ear increased significantly 15 and 60 minutes after thermal injury to 12.52 ± 0.68 and 11.58 ± 1.04 fmol/ear, respectively, compared with 1.78 ± 0.91 fmol/ear (N=8) obtained before injury. These results suggested that endothelin 1,2 existed in the region of the wound caused by thermal injury.     

Regional distribution of immunoreactive endothelin in porcine tissue: abundance in inner medulla of kidney

A specific and sensitive radioimmunoassay for endothelin has been developed. Half maximal inhibition of binding of radioiodinated endothelin was observed at 37 pg/tube and endothelin was detectable as low as 1 pg/tube. With this assay, the regional distribution of endothelin was determined in porcine tissue. The highest concentration of immunoreactive endothelin was observed in inner medulla of kidney (6.2 +/- 1.1 pg/mg wet weight), while the concentration in kidney cortex was very low. Immunoreactive endothelin was also found in lung in relatively high concentration. The immunoreactive endothelin in porcine lung and inner medulla of kidney was further characterized by reverse phase high performance liquid chromatography combined with radioimmunoassay.     

Dialyzability and binding of digoxin-like immunoreactive factors (DLIF) with serum macromolecules in uremic patients on hemodialysis

Digoxin-Like Immunoreactive Factors (DLIF) which cross-react with antidigoxin antibodies are present in elevated concentrations in patients on hemodialysis, uremia, hypertensives, liver failure, pre-eclampsia and premature birth. DLIF may have a potential role as a natriuretic hormone with a speculated low molecular weight (less than 1000). We studied the dialyzability and bindings of DLIF with serum components in hemodialysis patients. We analyzed DLIF concentrations in sera and protein free ultrafiltrates of 31 patients and 22 normal volunteers using a fluorescence polarization assay for digoxin. The DLIF concentrations were expressed as nmol/L Digoxin Equivalent. The gel filtration analysis was done using three different Bio-Gel columns with molecular weight cut-offs of 10,000, 20,000 and 40,000. Molecules with lower molecular weight than cut-off were absorbed in the column. Only 3 out of 22 normal volunteers (13.6%) showed measurable DLIF. However 23 out of 31 patients (74.2%) showed measurable DLIF. The concentrations of DLIF were significantly higher in patients with renal failure on hemodialysis (P less than 0.05) by both chi-squared and Fisher's exact test. We observed no statistically significant difference in the concentrations of DLIF in pre and post-dialysis sera, indicating that DLIF were not filtered during hemodialysis. We observed no DLIF activity in the protein free ultrafiltrates of any DLIF positive sera (patients and normal volunteers), indicating that unlike digoxin (where we observed 70-80% of total digoxin concentrations in ultrafiltrates), DLIF were strongly bound to serum components. With Bio-Gel filtration experiments (five different serum pools), we recovered all DLIF activities in the fraction equivalent to the void volume of the column with Bio-Gel P6 and P10 columns, indicating that DLIF were almost completely bound to serum components with molecular weight greater than 20,000. On the other hand, we recovered no DLIF activities in the void volume when the same serum pools were passed through the Bio-Gel P30 column, indicating that DLIF were strongly bound to serum macromolecules with molecular weight less than 40,000. In sharp contrast, when serum containing digoxin was subjected to the same series of experiments, we recovered only 20-30% of digoxin concentrations in void volume with all three columns as expected since digoxin is only 25% bound to albumin (MW 67,000).