Naoki Fujitsuka

Production of Methylguanidine from Creatinine via Creatol by Active Oxygen Species: Analyses of the Catabolism in vitro

The chemical oxidative conversion of creatinine (Cr) into methylguanidine (MG) has been followed by 1H-NMR and HPLC. By using active oxygen species generated by Fentons reagent (Fe2+ and H2O2) or the similar reagent (Fe3+ and H2O2), creatol (CTL), a metabolite newly isolated from the urine of uremic patients, and creatones A and B were experimentally detected in the reaction mixture and implicated as successive intermediates in the pathway from CR to MG. An alternative oxidation of Cr to demethylcreatinine (glycocyamidine) was also observed. The importance of CTL in this in vitro oxidation mechanism is discussed.

Production of Methylguanidine from Creatinine in Normal Rats and Rats with Renal Failure

Creatinine (Cr) was administered intraperitoneally to both normal rats and those given adenine, and time-course changes in methylguanidine (MG) production from Cr were compared. In rats with renal failure, the accumulation of MG in the body increased gradually with time after Cr administration. In particular, the MG level in skeletal muscle was markedly high in comparison with that in serum, liver or kidney, and a high concentration of MG was still present 24 h after Cr loading. In contrast, the amount of MG excreted into urine in these rats during 24 h after Cr administration was lower than the corresponding values in normal rats. Thus, the amount of MG per rat, distributed at 24 h after intraperitoneal administration of Cr 300 mg/100 g body weight was calculated. The production of MG from Cr was found to be markedly higher in rats with adenine-induced renal failure (172.09 micrograms/100 g body weight) than in normal rats (70.30 micrograms/100 g body weight). Produced MG was mostly excreted into urine in normal rats, whereas in rats with renal failure as much as 79.1% of produced MG was accumulated in the body.

Studies on the Precursor of Methylguanidine in Rats with Renal Failure

Each of creatinine (Cr), guanidinoacetic acid (GAA) and arginine (Arg) was administered intraperitoneally to rats with renal failure, and the levels of methylguanidine (MG) in the serum, liver, kidney, muscle and urine were determined at certain intervals. The levels of MG in the serum, liver, kidney, muscle and urine after Cr administration increased markedly with time. The amount of total MG at 24 h was estimated to be 114 micrograms/100 g body weight, which accounted for 0.46% of the Cr dose. In contrast, changes in MG levels after administration of GAA or Arg were only slight in comparison with those after Cr administration. Thus, MG was proved to be produced mainly from Cr.

Purification of Methylguanidine Synthase from the Rat Kidney

Methylguanidine (MG)-synthesizing enzyme was purified from rat kidney lysosomes and peroxisomes. The enzyme was a flavoprotein with a molecular weight of about 37,000 and oxidized creatol to produce MG. The present results suggest that the reaction mechanism of this enzyme is different from that of L-gulono-gamma-lactone oxidase (EC 1.1.3.8) isolated from rat liver microsomes.

Variations in the Distribution of Methylguanidine with the Progression of Renal Failure after Methylguanidine Loading

Methylguanidine (MG) was intraperitoneally administered to both normal rats and those given adenine, and MG levels in the serum, liver, kidney, muscle, brain and urine were compared. The accumulation of MG in the body increased with the progression of renal failure, whereas the rate of urinary excretion of MG in rats given adenine for 30 days was lower than the corresponding values in rats given adenine for 10 and 20 days. The velocity of MG elimination from serum and tissues became lower as the period of adenine administration lengthened. In particular, the rate of MG elimination from muscle was markedly low in comparison with that from the serum, liver, kidney or brain, and a high concentration of MG was still present 24 h after MG loading. In addition, the amount of MG obtained by subtracting the total amounts detected in the serum, liver, kidney, muscle, brain and urine from the dose decreased gradually as the period of adenine administration lengthened. The MG-scavenging effect is diminished according to the progress of renal failure.

Determination of Radical Species in the Kidney of Rats with Chronic Renal Failure by the Spin Trapping Method

Determination of Radical Species in the Kidney of Rats with Chronic Renal Failure by the Spin Trapping Method T. Takako Yokozawa N. Naoki Fujitsuka H. Hikokichi Oura A. Akitane Mori H. Hiroshi Kashiwagi Research Institute for Wakan-Yaku and Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, and Institute of Molecular and Cellular Medicine, Okayama University Medical School, Shikata, Okayama, Japan

L-gulono-γ-lactone oxidase is the enzyme responsible for the production of methylguanidine in the rat liver

A methylguanidine-synthesizing enzyme localized in rat liver microsomes produces methylguanidine via the intermediates creatone A and creatone B from the substrate, creatol, a substance produced from creatinine mainly by reaction with hydroxyl radicals. This enzyme has been identified as L-gulono-gamma-lactone oxidase (EC 1.1.3.8). However, no corresponding activity was found in extracts from human livers.

Effects of ginsenoside-Rb2 on adenine nucleotide content of rat hepatic tissue.

Abstract— In comparison with non‐diabetic rats, the tissue ATP content of diabetic rats is lower and the AMP content higher. However, daily intraperitoneal administration of ginsenoside‐Rb2 for several days resulted in an increase of tissue ATP content and a decrease of AMP content, characterized by an increase in the adenine nucleotide content, as well as improvement of energy charge. Thus, ginsenoside‐Rb2 administration caused a change in the pattern of metabolism, activating the ATP supply system.

Contribution of hydroxyl radical to the production of methylguanidine from creatinine.

Dr. Takako Yokozawa, Department of Applied Biochemistry, Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-01 (Japan) Dear Sir, Methylguanidine (MG) is a probable uremic toxin demonstrated to have a strong association with the induction of uremia. The idea that creatinine (Cr) is a precursor of MG is currently dominant. Giovannetti et al. [1], Perez and Faluotico [2], Gonella et al. [3], Orita et al. [4] and Nagase et al. [5] have reported a positive correlation between the blood Cr level and the degree of MG production, indicating that Cr is very probably a precursor of MG. Our previous study using normal rats and rats with renal failure also demonstrated that MG production increased in proportion to an increase in the level of Cr in the blood [6]. We also examined temporal changes in MG production after Cr loading, and found that the MG produced was excreted rapidly into the urine in normal rats [6]. On the other hand, the production of MG from Cr seems to be increased by factors which promote MG synthesis and inhibit MG degradation. Recently, Aoyagi et al. [7] observed an increased in vitro production of MG from Cr in the presence of active oxygen. Furthermore, in an experiment using isolated rat hepatocytes, they also demonstrated the inhibition of MG production by dimethyl sulfoxide (DMSO), a scavenger of hydroxyl radical, suggesting the involvement of active oxygen in MG production [7]. Data indicating histological damage due to active oxygen in patients with renal failure have been reported by Fillit et al. [8], Giardini et al. [9], Kuroda et al. [10] and Flament et al. [11]. On the basis of these reports, it is considered that, in a state of renal failure, active oxygen increases in quantity and reacts with Cr, resulting in enhanced MG production. However, it remains unclear to what extent active oxygen contributes to the in vivo production of MG from Cr. In this connection, the involvement of hydroxyl radical was investigated using radical scavengers under conditions of increased MG production after Cr loading. Table 1. Effect of hydroxyl radical scavengers on MG production from Cr Scavenger Dose MG μg/3h DMTU 0 13.07 ± 0.80 mg/kgB.W. 20 8.47 + 0.29* 100 6.86 ± 0.28* 500 6.34 ± 0.40* DMSO 0 13.81 ± 0.92

Progressively Decreasing Incidence of Membranoproliferative Glomerulonephritis in Spanish Adult Population

Spanish Society of Nephrology, Hospital Ramón y Cajal, Carretera de Colmenar, Km. 9,100, E-28034 Madrid (Spain) Dear Sir, 35η Several recent reports have called attention to the progressively decreasing incidence of membranoproliferative glomerulonephritis (MPGN) in the last decade [1–7]. In order to test this observation, we reviewed 8,545 renal biopsies in adult patients with primary glomerulonephritis (GN) examined at 33 nephrology units in Spain from January 1970 to December 1986. The study was entirely retrospective. The diagnosis was established on the basis of a kidney biopsy studied by light microscopy and immunofluorescence. Patients less than 14 years of age were excluded. MPGN was classified as type I (subendothelial deposits) or type II (dense intram-embranous deposits). A variance analysis was made to test the ‘null hypothesis’, that is to say two variables are independent. A marked reduction in the annual incidence of MPGN, expressed as a percentage of the total number of GN, was observed after 1976 (fig. 1). Thus, we compared the mean annual incidence of the various types of GN during three periods: period I (1970–1976), period II (1977–1981) and period III (1982–1986). As shown in table 1, in spite of the increased total number of patients submitted to biopsy, the incidence of MPGN (26.4,14.7 and 11.9% for the three periods, respectively) was significantly lower for each of the periods when compared with the preceding period (p < 0.001 for periods I vs. II; p < O.Ol for II vs. III). This reduced incidence was observed only in patients with type I MPGN (table 2), whereas the incidence of type II MPGN and its contribution to the total number of patients with primary GN (1.8,1.9 and 2% for the three periods, respectively) did not significatively vary (data from 23 hospitals). In addition, except for idiopathic nephrotic syndrome whose frequency remained unchanged, we observed variations in the incidence of three other primary GN. The incidence of crescentic GN between period I (4.8%) and period III (7.3%; p < O.Ol), membranous nephropathy in period I (8.1%) versus period III (11.6%; p < 0.01) and IgA 30–25–20–15–10–5Year: 1970 -71 -72–73–74–75–76 -77–78–79–80–81 -82–83 -84–85–86 6–7–8-12–15–17–19– 23–27–27–32–33–33–33–33–33–33 Number of hospitals Fig. 1. Annual incidence of MPGN in the Spanish adult population expressed as the percentage of the total number of GN throughout the 17-year study period. nephropathy (10.9, 19.9 and 22.3% for the three periods, respectively, p < 0.01) was found to increase significantly with time (table 1). The incidence of crescentic GN in period III was similar to that of the Milan study [4] and the increased frequency has also been previously reported [3].