Endothelin-1 inactivating peptidase in the human kidney and urine.

OBJECTIVE: Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. METHODS: Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37 degrees C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft RESULTS: ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155-1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM 180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM 11.5 micromol/l), endothelin 11 21 fragment (KM 15.3 micromol/l), endothelin antagonist Trp-Leu-Asp-Ile-Ile-Trp (KM 3.1 micromol/I), gastrin (KM 2.2 micromol/l) and cholecystokinin (KM 4.0 micromol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 micromol/l), cyclosporin (IC50 400 micromol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft CONCLUSIONS: Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

Further studies on aminopeptidase-M in blood in children with cholestatic liver diseases and viral hepatitis.

Aim of this study was to determine and further characterize the serum aminopeptidase-M in children with liver diseases. Based on our new assay, we have shown two fractions of the enzyme. Activity of the first fraction is expressed in undiluted serum at pH adjusted from 8.5 (pH of storaged serum) to 7.4. Activity of the second fraction (cryptic activity) appears in the serum (pH 7.4) as a result of dilution and/or addition of aniline naphthalene sulfonic acid. In children with Alagille syndrome, extrahepatic biliary duct atresia, Byler's disease, and acute hepatitis due to hepatitis B virus infection, activities of both fractions are highly elevated as compared to healthy children or those with chronic viral hepatitis. Moreover, serum aminopeptidase-M seems to reflect other aspects of the pathological process than those reflected by the alanine aminotransferase and gamma-glutamyltranspeptidase. Due to increased activity and broad substrate specificity, the enzyme seems to be also a cofactor of cholestasis and hepatitis.

Luteinizing hormone secreting adrenal tumour as a cause of precocious puberty.

A boy aged 6 years presented with genital precocity, enlarged testes and advanced linear growth. An ovoid mass 3-4 cm in diameter was identified by MRI scan in the right adrenal gland. Serum concentrations of LH, testosterone, alpha-subnuit, dehydroepiandrosterone sulphate, androstenedione and oestradiol were persistently elevated. LH was unresponsive to bolus i.v. injection of GnRH or to GnRH analogue therapy. Serum FSH was normal. After removal of the adrenal tumour, serum LH, alpha-subunit, testosterone and adrenal androgen levels fell to normal. In incubation medium of cultured disaggregated tumour cells, LH concentrations were greater than twice the mean serum concentration and 4-5-fold higher than in the medium of cultured non-neoplastic adrenal cells. Specific immunostaining of the tumour was positive for LH and alpha-subunit in many areas and these were not found in the adjacent non-neoplastic adrenal. Testicular biopsy showed almost complete spermatogenesis although germinal cell types were numerically lower than in normal men. These findings are consistent with an adrenocortical adenoma secreting LH being the cause of the patient's precocious puberty.

[Can TNF-alpha and IL-6 be helpful in assessment of chronic hepatitis in children?]. / Czy TNF-alpha i IL-6 moga byc pomocne w ocenie przewleklego zapalenia watroby u dzieci?

The purpose of the work was an assessment of TNF-alpha and Il-6 concentrations in 34 children with diagnosed chronic hepatitis. In all studied patients the values of TNF-alpha and Il-6 concentration were slightly increased. The correlations calculated between TNF-alpha and Il-6 concentrations calculated between TNF-alpha and Il-6 concentrations and laboratory parameters (laboratory indicators of hepatitis activity--AlAT; liver function indicators--prothrombin index, bilirubin concentration, bile acid concentration, alkaline phosphatase activity, anti-pyrin half-life) were non-significant in Spearman non-parametric test (p > 0.005) except for the correlation between albumin and TNF-alpha concentrations. No statistically significant differences of TNF-alpha and Il-6 concentrations were found between groups of patients with active and persistent hepatitis; groups with and without cirrhosis as well as between groups with and without portal hypertension. Normal or slightly increased TNF-alpha and Il-6 concentrations, observed in chronic hepatitis in children should be explained by compensated liver function in such patients.

An aluminum silicate binding assay for quantitation of degradation of cholecystokinin octapeptide and other short peptides.

Most available techniques for the quantitation of enzymatic degradation of peptide hormones are time-consuming and require expensive equipment and/or novel reagents. Our aim here was to develop a rapid and sensitive assay for the measurement of degradation of cholecystokinin octapeptide (CCK-8) as well as other short, hydrophobic peptides. The proposed technique is based on our novel observation that intact CCK-8, but not its degradation product(s), binds to Lloyd reagent, a form of aluminum silicate. When radiolabeled CCK-8 was exposed to rat liver cytosol containing endogenous CCK-degrading activity, there was a time-dependent decrease in the binding of radiolabel to aluminum silicate [from 86 to 8% over 60 min at 37 degrees C]. The decrease in binding closely paralleled the extent of CCK-8 degradation over time as assessed by high-performance liquid chromatography and immunoprecipitation with specific polyclonal antibodies to CCK-8. While aluminum silicate did not efficiently bind to C-terminal and N-terminal CCK tetrapeptides, magnesium silicate bound to both tetrapeptides (> 82%), but not to their radiolabeled degradation products. Both aluminum and magnesium silicate also extensively bound (> 82%) to other peptide hormones including Met-enkephalin, somatostatin, and secretin, but did not bind their degradation products. These binding assays will be useful in studies of peptidases which degrade cholecystokinin or other small, hydrophobic peptides.

[The value of Phadiatop screening tests for diagnosing allergic respiratory diseases in children]. / Wartosc testu przesiewowego Phadiatop w diagnostyce chorób alergicznych dróg oddechowych u dzieci.

Phadiatop test was performed in 174 children aged between 1 and 16 years (mean: 9 6/12 years) referred to the hospital because of the bronchial asthma, obstructive bronchitis and perennial or seasonal allergic rhinitis. Positive results were obtained in over 93% of children with atopic allergy. Comparing with PRIST, multiple allergen assay proved more sensitive (93.5%), specific (90%), and accurate (91.4%). The same data for PRIST were: 91.7%, 80.3%, 87.4%, respectively. False negative and false positive results were also less frequent than for PRIST. Therefore, Phadiatop, is considered an appropriate screening in vitro diagnostic test for inhalant allergy which should be performed at the beginning of respiratory tract diagnosis.