1174 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80 previously treated with antibiotics, or when the stool isolates themselves were incubated with antibiotics. Duodenal intubation demonstrated small bowel bacterial overgrowth associated with high concentrations of toxic methylamines. Consequently, CRF is accompanied by accumulation of Crn-degrading bacteria in the gut <strong>and</strong>/or by induction of creatininase activity in these bacteria. 3) A good deal of convincing evidence has been obtained for two oxidative Crn degradation pathways, FIG. 18. Oxidative Crn degradation pathways favored in uremia <strong>and</strong>/or in inflamed skin tissue: 1) Crn; 2) creatol; 3) creatone A; 4) creatone B; 5) methylguanidine; 6) tautomer of Crn; 7) 1-methylhydantoin; 8) 5-hydroxy-1-methylhydantoin; 9) methylparabanic acid; 10) N 5 - methyloxaluric acid; 11) methylurea. with the first leading to the formation of MG <strong>and</strong> the second to methylurea (Fig. 18). MG is not to be regarded as a metabolic end product, but may be degraded further (1145). Both in vivo <strong>and</strong> in vitro studies have shown that Crn is converted to MG, with creatol, creatone A, <strong>and</strong> creatone B representing consecutive intermediates in this pathway (25, 271, 437, 687, 756). ROS, <strong>and</strong> in particular the hydroxyl radical, strongly stimulate the formation of
July 2000 CREATINE AND CREATININE METABOLISM 1175 MG out of Crn (see Ref. 25). Accordingly, MG production was increased in vivo by hyperbaric oxygen therapy (979) <strong>and</strong> inhibited by superoxide dismutase (677), magnesium lithospermate B, as well as by tannin-containing rhubarb <strong>and</strong> green tea extracts that are thought to reduce the level of ROS (see Refs. 1141, 1142). Because N,N�-dimethylthiourea, an efficient hydroxyl radical scavenger, had no effect in normal rats on the conversion of creatol to MG, whereas it inhibited production of creatol <strong>and</strong> MG out of Crn in a dose-dependent manner (272, 1146), it may be assumed that ROS selectively affect the conversion of Crn to creatol. In vitro experiments contradict this view, in as far as the conversion of creatol to MG was stimulated by Fenton’s reagent, which provides ROS (686). Considerable uncertainty still exists on whether the individual steps of the pathway are enzyme catalyzed or not. In vitro studies revealed that the whole reaction cascade may proceed nonenzymatically (271, 294, 686). On the other h<strong>and</strong>, it might be anticipated that individual steps are catalyzed by bacteria in the intestinal tract (437). However, no difference in MG production <strong>and</strong> urinary excretion was observed between control <strong>and</strong> germfree or antibiotic-treated rats (756). Nagase et al. (684) provided evidence that rat liver, kidney, lung, muscle, red blood cells, <strong>and</strong> the gut flora synthesize MG. Control experiments on isolated rat hepatocytes revealed that nonenzymatic production can account for only a small proportion of MG synthesis from Crn in this tissue. Accordingly, rat liver peroxisomal enzymes were shown to catalyze the conversion of Crn to MG (978), <strong>and</strong> rat liver (microsomal) L-gulono-�-lactone oxidase was identified to catalyze the reaction creatol � O 2 3 creatone A � H 2O 2 (271). Although the K m of this flavoprotein is 5 �M for L-gulono-�-lactone, but 12.8 mM for creatol, the V max values for both substrates are similar. In rat kidney lacking L-gulono-�-lactone oxidase, another flavoprotein, long-chain L-2-hydroxy acid oxidase, was shown to oxidize creatol to produce MG (see Ref. 746). Even though oxygen is consumed in this reaction, no H 2O 2 is liberated. In accordance with the lack of L-gulono- �-lactone oxidase in both guinea pigs <strong>and</strong> primates including humans, evidence for nonenzymatic production of MG by human liver microsomes has been obtained (271). An analogous reaction cascade results in the formation of methylurea out of Crn. 1-Methylhydantoin <strong>and</strong> 5-hydroxy-1-methylhydantoin were identified in the urine of uremic patients <strong>and</strong> rats <strong>and</strong> were shown to be derived from Crn (396, 686). Furthermore, upon oral administration of 1-methylhydantoin to rats, 5-hydroxy-1-methylhydantoin, methylparabanic acid, N 5 -methyloxaluric acid, <strong>and</strong> methylurea were detected in the urine (395). Because 1-methylhydantoin <strong>and</strong> 5-hydroxy-1-methylhydantoin were also identified in rabbit skin inflamed by vaccinia virus inoculation, but not in normal skin (394), the first step of this pathway, the conversion of Crn to 1-methyl- hydantoin (Fig. 18, 1 3 7) may depend on bacterial Crn deaminase rather than on a nonenzymatic mechanism (Fig. 18, 1 3 6 3 7). Even though the two oxidative Crn degradation pathways (Fig. 18, 1 3 5 <strong>and</strong> 7 3 11) seem to proceed via an identical reaction sequence <strong>and</strong> may both be stimulated by ROS, the urinary excretion of creatol (2) is threefold increased in uremic rats compared with normal controls, whereas that of 5-hydroxy-1-methylhydantoin (8) is fourto fivefold lower (396). The reason for this apparent inconsistency is unknown. 4) Some further Crn degradation pathways were proposed but have not been characterized in detail so far. Guanidinoacetate seems to be converted to guanidine in vivo (638, 684, 980). In normal rabbits, partial conversion of Crn to GBA was observed, whereas in a rabbit with decreased Crn clearance, GPA <strong>and</strong> Arg were suggested to be derived from Crn (83). Furthermore, in the presence of ROS in vitro, Crn was oxidized to glycocyamidine (688). Finally, Orita et al. (736), using 15 N-labeled Arg, provided evidence for two distinct pathways for MG formation, with Crn acting as an intermediate in one of these pathways but not in the other. Several groups have shown, however, that this is actually not the case or that, at least, the direct formation of MG out of Arg is quantitatively irrelevant (294, 638, 684, 756, 1144). As far as the serum <strong>and</strong> tissue concentrations as well as the urinary excretion rates of the potential Crn degradation products are concerned, they consistently indicate that the production of MG <strong>and</strong> creatol is increased in uremia (for references, see Refs. 21, 168, 169, 396, 479, 554, 598, 687, 722, 747, 1143). In CRF rats relative to controls, the concentration of MG was increased 3- to 18-fold in serum, blood cells, liver, muscle, colon, <strong>and</strong> kidney (735; see also Ref. 39). In brain, on the other h<strong>and</strong>, MG concentration was increased only twofold, indicating limited permeability of the blood-brain barrier for MG. The same conclusion can be drawn from experiments on the effects of intraperitoneal injection of guanidino compounds into rats, which suggested a low permeability of the blood-brain barrier for MG, Crn, <strong>and</strong> guanidinosuccinic acid (GSA) (1144, 1145, 1147). Guanidine, 4-GBA, <strong>and</strong> GPA may be increased in serum <strong>and</strong> cerebrospinal fluid of uremic patients (163, 165, 168, 757) as well as in serum, heart, skeletal muscle, brain, liver, kidney, <strong>and</strong> intestine of rats <strong>and</strong> mice with acute or chronic renal failure (10, 39, 554). Crn <strong>and</strong> its degradation products are likely to be of critical importance with regard to uremic toxicity. Creatol <strong>and</strong> MG, in contrast to Crn, (further) deteriorate renal function when administered to normal rats or to rats with CRF (1147–1149). Mongrel dogs chronically intoxicated with MG, at concentrations similar to those in plasma of uremic patients, displayed many functional <strong>and</strong> pathological changes characteristic of uremia such as increased
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