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Biomedical subjects

L M Kraus

Publications and source records attributed to L M Kraus.

At least 19 recordsLinked to original sources
PubMed2001

Carbamoylation of glomerular and tubular proteins in patients with kidney failure: a potential mechanism of ongoing renal damage.

BACKGROUND: Cyanate formed spontaneously from urea carbamoylates non-protonated amino groups of protein, irreversibly altering function, charge and structure. Carbamoylated proteins in renal tissue have not been examined hitherto. OBJECTIVES: To identify homocitrulline (epsilon-amino-carbamoyl-lysine), a result of in vivo carbamoylation by urea-derived cyanate, from patients with renal disease or in newly transplanted kidneys by immunohistochemistry. To evaluate enzymatic activity of carbamoylated and non-carbamoylated matrix metalloproteinase-2 and correlate this with renal tissue carbamoylated in vivo. DESIGN: Anti-homocitrulline antibody is specific for homocitrulline and was used to identify carbamoylation of epsilon-amino-lysine in renal biopsies from patients with elevated BUN, with isolated proteinuria, and as controls, from normal donors at time of transplantation. Enzymatic activity of matrix metalloproteinase-2 carbamoylated in vitro was evaluated. RESULTS: Homocitrulline was present in glomerular basement membrane (8/10), mesangium (8/10), tubular epithelium and cytoplasm (7/10) and Bowman's capsule (1/10) in patients with elevated BUN. The discordant patterns of glomerular and tubular localization of homocitrulline versus immune complexes indicated that the carbamoylated proteins were not a component of immune deposits but were modified proteins in renal tissue. No homocitrulline was found in transplanted kidneys (14/15) or in proteinuric patients (2/2). Enzymatic activity of both human and rat matrix metalloproteinase-2 was strongly inhibited in a dose-dependent fashion when incubated with cyanate. CONCLUSIONS: In situ carbamoylation in proteins occurred in kidneys of patients with renal dysfunction but not in normal newly transplanted kidneys. Decreased enzymatic activity of carbamoylated enzymes may alter specific renal regulatory mechanisms. Carbamoylated proteins with altered function and charge may represent a previously underestimated mechanism in renal pathophysiology.

Amino Acids↗
PubMed2001

Carbamoylation of amino acids and proteins in uremia.

Cyanate spontaneously transformed from urea increases as renal function decreased. Acting as a potential toxin, the active form of cyanate, isocyanic acid, carbamoylates amino acids, proteins, and other molecules, changing their structure, charge, and function. The resulting in vivo carbamoylation can modify the molecular activity of enzymes, cofactors, hormones, low-density lipoproteins, antibodies, receptors, and transport proteins. Antibodies specific for epsilon-amino-carbamoyl-lysine (homocitrulline) located carbamoylated proteins in situ in neutrophils, monocytes, and erythrocytes. Carbamoylated proteins were found in renal tissue from uremic patients but not in normal transplanted kidneys. The irreversible reaction with cyanate converts free amino acids (F-AAs) to carbamoyl-amino acids (C-AAs). The Carbamoylation Index (CI), C-AA/F-AA, quantifies the decrease of the F-AA pool for each essential amino acid. C-AAs contribute, in part, to malnutrition of uremia. C-AAs interfered with protein synthesis to lower 14C hemoglobin synthesis in human reticulocytes and osteocalcin synthesis in rat osteosarcoma-derived tissue culture. Insulin-sensitive glucose uptake was decreased 33% in cultured rat adipocytes by alpha-amino-carbamoyl-asparagine. alpha-Amino carbamoylation occurs primarily in F-AA, while epsilon-amino carbamoylation of lysine in protein occurs continuously during the protein life span. Protein catabolism releases epsilon-amino-carbamoyl-lysine. Quantitation of alpha versus epsilon carbamoylation may yield a more sensitive measurement of protein intake versus protein catabolism, and could be useful in decisions concerning the time to initiate dialysis or subsequent changes in dialysis prescription. Carbamoylated molecules can block, enhance, or be excluded from metabolic pathways, thereby influencing the fate of noncarbamoylated molecules. Although not an "all-or-none" phenomenon, urea-derived cyanate and its actions are contributing causes of toxicity in uremia.

Amino Acids↗
PubMed1998

The search for the uremic toxin: the case for carbamoylation of amino acids and proteins.

Urea and cyanate, spontaneously transformed from urea, are increased with decreased renal function becoming potential toxins. Isocyanic acid, the active form of cyanate, carbamoylates proteins, amino acids and other molecules, changing molecular structure and function in vivo. Carbamoylation can occur at multiple sites with a cumulative effect over the the life-span of the molecule. Carbamoylation converts free amino acids to carbamoyl-amino acids (C-AA). C-AA interfere with protein synthesis and transamination reactions and contribute, in part, to protein-malnutrition. Insulin-sensitive glucose uptake is decreased by carbamoyl-asparagine. Cyanate inhibits superoxide release from neutrophils to an extent that interferes with microbiocidal activity. Antihomocitrulline antibodies identified homocitrulline (epsilon-amino-carbamoyl lysine) in situ in proteins in neutrophils in end stage renal disease. Also in uremic patients, homocitrulline was located in proteins in renal tissue but was not found in normal transplanted kidneys. Carbamoylated human low density lipoprotein interferes with human receptor binding, has decreased clearance, and is auto-immunogenic in animals. Carbamoylated insulin has decreased biological activity and changed immunological reactivity. Carbamoylation at a site of molecular activity can affect molecular function of enzymes, co-enzymes, antibodies, hormones and receptors. Carbamoyl-molecules can block, enhance, or be excluded from metabolic pathways, and can affect binding and trafficking, thereby influencing the fate of non-carbamoylated molecules. Normal renal function removes C-AA. In uremia, C-AA are removed by residual renal function or dialysis. Toxicity of cyanate is not an "all or none" phenomenon, but the actions of cyanate are a contributing factor in uremia. Removal of urea, cyanate and carbamoyl-molecules partially alleviates the morbidity and mortality of renal disease.

Amino Acids↗
PubMed1998

Essential carbamoyl-amino acids formed in vivo in patients with end-stage renal disease managed by continuous ambulatory peritoneal dialysis: isolation, identification, and quantitation.

Carbamoyl-amino acids (C-AA) are formed by reaction of amino acids with cyanate, which is spontaneously formed from urea at body temperature and pH. In vivo derivatized C-AA are not measured by the usual amino acid analysis methods, which require a free amino group for derivatization. Free-amino acids (F-AA) but no C-AA were found in the postabsorptive plasma of eight normal persons with blood urea nitrogen (BUN) levels ranging from 9 to 16 mg/dl. In a longitudinal study of postprandial plasma (n=43), essential amino acids, both C-AA and F-AA, were isolated and quantified by reverse-phase high-pressure liquid chromatography in six patients with end-stage renal disease who were managed by continuous ambulatory peritoneal dialysis. The mean BUN was 61 mg/dl (range, 36 to 79 mg/dl). In uremia, removal of F-AA from the essential amino acid pool to form C-AA is measured by the ratio of C-AA to F-AA (carbamoylation index (CI)). Using the mean value for each essential amino acid, the CIs were as follows: leucine, 4; valine, 3.3; isoleucine, 11.4; threonine, 9; lysine, 2; methionine, 3.5; histidine, 3.5; phenylalanine, 0.5; and tyrosine, 1.3. Carbamoylation of F-AA may account, in part, for the lower than normal levels of F-AA in patients with uremia. The derivatized amino group of C-AA interferes with formation of a peptide bond in protein synthesis, which requires an underivatized amino acid. A decrease in the F-AA pool available for protein synthesis and anabolism in the presence of C-AA may provide additional contributing factors for the development of malnutrition in uremia.

Adult↗
PubMed1994

Urea-derived cyanate forms epsilon-amino-carbamoyl-lysine (homocitrulline) in leukocyte proteins in patients with end-stage renal disease on peritoneal dialysis.

Carbamoylated proteins have been located by using a site-specific polyclonal antihomocitrulline antibody and a fluorescent secondary antibody in leukocytes from patients with end-stage renal disease who were undergoing maintenance continuous ambulatory peritoneal dialysis. A covalent reaction with urea-derived cyanate and the epsilon-amino group of lysine forms homocitrulline residues in carbamoylated proteins. Isocyanic acid, the reactive form of cyanate, is spontaneously formed from urea in aqueous solution at physiologic pH and temperature. In washed, fixed monolayers of cells, an intracellular fluorescent antigen-antibody complex was located throughout the cytoplasm of polymorphonuclear neutrophils (PMNs) and monocytes from 11 patients with blood urea nitrogen (BUN) levels ranging from 32 to 102 mg/dl who were undergoing dialysis for 2 to 135 months. A punctate fluorescence present in the cell surface proteins of living cells demonstrated that lysine residues in the external domain of proteins were carbamoylated, forming homocitrulline. In contrast, we found a perinuclear fluorescence in PMNs in normal subjects with no history of renal insufficiency and BUN levels of 6 to 19 mg/dl. This suggests that homocitrulline is located in carbamoylated proteins within the perinuclear membrane, a structural organelle continuous with the endoplasmic reticulum. It appears that continuous exposure to urea-derived cyanate in low levels results in increasing carbamoylation of stable proteins over the PMN's lifetime. When normal PMNs were exposed to 120 mmol/L cyanate ion in vitro for 10 to 30 minutes, the ability of PMNs to release microbicidal superoxide was strongly inhibited. Thus protein carbamoylation may provide a regulatory mechanism. The altered function of PMNs in renal disease may be due in part to the posttranslational modification of proteins by urea-derived cyanate.

Adult↗
PubMed1991

Tyrosine and N-carbamoyl-tyrosine in end-stage renal disease during continuous ambulatory peritoneal dialysis.

Lower-than-normal tyrosine concentrations of unexplained pathogenesis in plasma and intracellular body water have been reported in patients with chronic renal failure. We found a derivative of tyrosine that is not measured by the usual methods of amino-acid analysis because its alpha-amino group is blocked and cannot react to form other derivatives. An in vivo covalent reaction with urea-derived cyanate forms alpha-amino-carbamoyl-tyrosine (N-C-Tyr) in patients with end-stage renal disease. A longitudinal study of patients with end-stage renal disease who were treated with continuous ambulatory peritoneal dialysis shows that plasma that is obtained within 4 hours of the morning meal contains 70.1 +/- 6 mumol/L of tyrosine (mean +/- SEM) and 77.2 +/- 12 mumol/L of N-C-Tyr (mean +/- SEM). Thus there is a molecule of N-C-Tyr for each molecule of tyrosine present. The carbamoylation index or ratio of N-C-Tyr to tyrosine, blood urea nitrogen, episodes of peritonitis, and changes in dialysis protocol were compared. A reduction in the number of peritoneal dialysis exchanges resulted in parallel increases in carbamoylation index and blood urea nitrogen. Altering dialysis by increasing the number of exchanges or adding supplemental hemodialysis resulted in a decrease in the carbamoylation index with a delayed decrease in blood urea nitrogen. We found a significant increase of N-C-Tyr (p = 0.005) and of the carbamoylation index (p = 0.004) during six episodes of peritonitis compared with 10 periods of no peritonitis in two patients who had multiple episodes of peritonitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1979

Antisickling agents: effects of carbamyl phosphate or cyanate on survival, erythrocytes, and leucocytes in the mouse.

Equal mole doses of the anions of disodium carbamyl phosphate (carbamyl P) or sodium cyanate, antisickling agents, have been compared in C57B1 mice. Using 15 mice per group, two groups were given the equivalent ip dose of carbamyl P or cyanate anion (7 mmoles/kg/day) in a divided dose, in the morning and six hours later, for 17--18 days. The control group received sodium chloride (13.8 mmoles of Na+ or Cl-/kg/day). Surviving mice per group were sodium chloride, 15/15; disodium carbamyl P, 14/15; and sodium cyanate, 0/15, all mice died by day 2. Surviving mice appeared normal throughout the study, and no abnormalities were seen at necropsy. The hematologic measurements were the same for sodium chloride or disodium carbamyl P, including hemoglobin, packed cell volume, erythrocyte counts, leucocyte counts, and differential counts. The mean hemoglobin carbamylation was 1.24 (+/- 0.06 SE) moles of valine hydantoin/mole of hemoglobin tetramer in mice receiving disodium carbamyl P for 18 days, sufficient for antisickling activity. The enzymatic degradation of carbamyl P to NH3, CO2, and Pi was measured in serial blood samples in additional C57B1 and DBA/2J mice following ip injections of carbamyl P or cyanate. Both NH3 and Pi increased immediately after giving carbamyl P, but no increase occurred after cyanate administration. Thus enzymatic degradation of carbamyl P occurs in vivo and appears to be an important detoxification mechanism. When equivalent mole doses of anion are administered, disodium carbamyl P is less toxic than sodium cyanate in mice.

Ammonia↗
PubMed1977

Characterization of in vivo effects of dilithium carbamyl phosphate on dog hemoglobin structure and function.

The effect of oral administration of dilithium carbamyl phosphate on adult beagle dogs is dose related. The levels of carbamylation as mol valine hydantoin/mol hemoglobin tetramer reaches a plateau of 0.3-0.38 with daily doses of 100 mg/kg body weight. Related changes in oxygen binding by whole blood and hemoglobin amount to a 5-15% left shift in oxygen isotherms. After discontinuing the administration of carbamyl phosphate, the disappearance of modified hemoglobin with a return to normal oxygen binding values follows the gradual replacement of old cells by new cells in the circulation. When the level of in vivo carbamylation in dog blood is greater than 0.16, it is similar to the levels of in vitro carbamylation of hemoglobin SS which result in the interference with sickling of erythrocytes at low levelsof oxygen.

Administration, Oral↗
PubMed1975

Enzymatic determination of carbamyl phosphate in blood.

A specific assay for determination of carbamyl phosphate (carbamyl P) in blood has been developed using the enzymatic conversion of carbamyl P to citrulline by ornithine carbamyl transferase (OCT). This assay, for evaluation of bioavailability of carbamyl P, is necessary for study of the in vivo and in vitro effects of carbamyl P as an antisickling agent. The recovery of carbamyl P as citrulline was 88 to 92 per cent. Carbamyl P in whole dog blood in vitro decomposed with first-order kinetics with a t-1/2 of 42 minutes, the same as the t-1/2 reported for decomposition of carbamyl P to cyanate in buffers at physiologic temperature and pH. When carbamyl P (190 mg. per kilogram) was injected intraperitoneally into mice, it appeared in the blood, with a maximum concentration of 0.2 mM observed between 1 and 5 minutes after injection, followed by a rapid decrease within 10 minutes. The clearance of carbamyl P from dog blood after intravenous injection was first-order, with a t-1/2 of approximately 2.2 minutes. The rapid disappearance of carbamyl P in vivo may possibly by explained by the action of tissue acyl phosphatases which have been reported to hydrolyze carbamyl P.

Animals↗