Shoji Ohba

FORMATION OF GUANIDINOSUCCINIC ACID, A STABLE NITRIC OXIDE MIMIC, FROM ARGININOSUCCINIC ACID AND NITRIC OXIDE-DERIVED FREE RADICALS

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking actions such as vasodilatation and activation of the N-methyl-D-aspartate (NMDA) receptor. We have reported that GSA is the product of argininosuccinate (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We tested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incubated with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 degrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in turn, generated peroxynitrite which was then converted to the hydroxyl radical. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA was incubated with 1 mM SIN-1, the amount of GSA produced depended on the incubation time and the concentration of ASA. Among the tested SIN-1 concentrations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, completely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited GSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the presence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic. Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed back mechanism.

Pharmacokinetics of Tamsulosin Hydrochloride in Patients with Renal Impairment: Effects of α1‐Acid Glycoprotein

The pharmacokinetics of tamsulosin hydrochloride in patients with renal impairment were compared with those in healthy volunteers, and the factors that influenced plasma levels of tamsulosin were elucidated. A single oral dose of 0.2 mg of tamsulosin was given and blood and urine samples were obtained for 36 hours after administration. Unbound plasma concentration of tamsulosin was measured by a combination of equilibrium dialysis and liquid chromatography tandem mass spectrometry methods to examine the effect of protein binding on the pharmacokinetics of tamsulosin. Mean values for maximum concentration (Cmax) and area under the concentration—time curve (AUC) of total drug (Cmax,t and AUC1 in patients with renal impairment were 73% and 211% greater, respectively, than those in healthy volunteers. Mean Cmax and AUC of unbound drug (Cmax,u and AUCu), however, were almost the same in the two groups. A high correlation was found between α1‐acid glycoprotein (α1‐AGP) concentration and AUCt, but no correlation was found between α1‐AGP concentration and AUCu,0–36 or between creatinine clearance (ClCR) and AUCu,0–36. These results show that in patients with renal impairment, the pharmacokinetics of tamsulosin are affected by the change in protein binding that is associated with alteration of plasma α1‐AGP concentration, but are not largely affected by the decrease in the renal excretion. Although total tamsulosin levels increased as plasma protein binding increased, unbound tamsulosin levels (which are directly associated with the pharmacologic effects) remained unchanged in these patients.

Biosynthesis of Guanidinoacetic Acid in Isolated Rat Hepatocytes

Guanidinoacetic acid (GAA), a precursor of creatine, is formed from arginine and glycine by transamidinationl. GAA formation in rat is not detected in liver homogenates2 or in isolated perfused livers3. Therefore, creatine synthesis in rat liver is regulated by the activity of transamidinase in the kidney4. However, recent improvements in GAA analysis reveal that the blood level is 1/4 of the value6 reported previously3. Moreover, Natelson et al. reported that isolated rat hepatocytes had GAA synthetic activity5. The details of which are not clear. Therefore, GAA synthesis in isolated rat hepatocytes was investigated quantitatively to evaluate the role of its synthesis in liver. In addition, some regulatory mechanisms in this synthesis were also investigated.

Is Guanidine a Marker of Peroxidation in Uremics

There are many reports concerning the toxicity of oxygen in various disease states1–4 Recently, attention has been called to the unfavorable effects of active oxygen in renal diseases5–9. We have reported that the hydroxyl radical plays an important role in vitro10 and in isolated rat hepatocytes11 in the peroxidation of creatinine (CRN) to methylguanidine (MG), a guanidino compound known to be a potent uremic toxin. In this study, we investigate the correlation between some markers of the peroxidative state and the concentration of various guanidino compounds in the sera of patients undergoing regular hemodialysis.

Effect of Active Oxygen on Guanidine Synthesis in Vitro

Recently there have been many reports describing the toxicity of oxygen in various disease states 1–4. Some investigators have reported that patients with chronic renal failure are in a stronger peroxidative state than normal persons5–9. We have reported that methylguanidine (MG) is a peroxidative product of creatinine (CRN) and that the hydroxyl radical plays an important role in MG synthesis both in vitro and in isolated rat hepatoeytes. We also suggest that MG is a useful indicator of peroxidation in uremic serum10–13.

Elastase-type activity in human radial artery in patients with chronic renal failure.

To elucidate the nature of the abnormality of elastin metabolism in arteriosclerosis, we determined the elastase-type activity in the human radial artery of patients with chronic renal failure due to glomerular disease and diabetes. Elastase-type activity was determined by HPLC analysis of the hydrolyzed products of succinyl-trialanine-4-nitroanilide, a sensitive synthetic substrate for elastase. Three kinds of hydrolyzed products, (L-Ala)2-NA, L-Ala-NA and NA, were found after incubation of the substrate with human radial artery in the presence of amastatin (an inhibitor of aminopeptidases). We assumed the activity that liberates NA to be an elastase-type activity because purified human aorta elastase liberates NA from the substrate. The pH optima of the human artery and rat aorta activities were 6.0 and 6.8, respectively. The elastase activity in human radial artery and rat aorta was inhibited by diisopropyl phosphofluoridate, a serine protease inhibitor, and by elastatinal, an elastase inhibitor. The elastase-type activity in the radial artery of patients with chronic renal failure was significantly lower than that of the control group, and the decrease was especially marked in the patients with juvenile onset diabetes. These results suggest that the elastin metabolism is abnormal in the radial artery in diseases that tend to cause atherosclerosis.

The Effect of Lactulose on the Metabolism of Guanidino Compounds in Chronic Renal Failure

Serum concentrations of urea and creatinine increase in patients with decreased renal function. Urea has been demonstrated to undergo enterohepatic circulation1,2,3,4. Creatinine has also been thought to have an enterohepatic circulation in patients with decreased renal function5,6,7. Serum concentrations of some guanidino compounds, especailly guanidinosuccinic acid (GSA) and methylguanidine (MG) increase in uremic states8,9,10,11. We reported that urea stimulated the synthesis of GSA in isolated rat hepatocytes12,13,14. Cohen proposed that MG might be produced from creatinine15.

Biosynthesis of Guanidinosuccinic Acid in Isolated Rat Hepatocytes: Evaluation of Guanidine Cycle and Acidosis

Guanidinosuccinic acid (GSA), a guanidine derivative, is implicated as a uremic toxin1 To clarify the synthetic pathway of GSA and the mechanism of its increased synthesis in renal failure, we investigated GSA synthesis in isolated rat hepatocytes, in vitro and obtained the following results2. 1) GSA synthesis increased as urea concentration rose. 2) Ornithine and arginine3 which stimulated urea synthesis inhibited GSA synthesis. 3) D,LNorvaline which is an inhibitor4 of urea cycle enzymes: arginase, argininosuccinate synthetase and argininosuccinate lyase, inhibited GSA synthesis. These results support the theory that GSA is formed from urea via the guanidine cycle which consists of microsomal enzymes and urea cycle enzymes5. In this study, the guanidine cycle which is proposed as a synthetic pathway for GSA was examined in isolated rat hepatocytes. In addition, the effects of some basic conditions on GSA synthesis in isolated rat hepatocytes were investigated.