Creatine and Creatinine Metabolism - Physiological Reviews

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1176 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80 catabolism, anemia, a decrease in the circulating platelet count, central and peripheral neuropathy, pruritus, arrhythmias and myocardial degeneration, congestion, and, in later stages of intoxication, anorexia, vomiting, increased salivation, and diarrhea (see Ref. 53). In vitro studies have shown that MG has a variety of potentially toxic effects by inhibiting, for example, mitochondrial oxidative phosphorylation, growth of cultured cells, or Na � -K � -ATPase in the brain (for references, see Refs. 168, 735, 1147) or by affecting membrane fluidity (1138). Furthermore, MG, which is also present in various foods (for references, see Refs. 223, 294, 685), may be nitrosated intragastrically and may thereby become a potent mutagen (223) (see also sect. IXF). Recently, MG was identified as a nonselective reversible inhibitor of NOS isoenzymes (see Refs. 341, 581, 930, 931, 1004, 1114). The three major isoenzymes of NOS, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), were inhibited with IC 50 values of 90–370 �M (931, 1114). By inhibiting eNOS, intravenously administered MG increased mean arterial blood pressure in rats, thus raising the hypothesis that MG may contribute to the hypertension seen in patients with CRF (929, 930). Similarly, by inhibiting NOS in kidney and brain, MG may have an impact on the regulation of glomerular capillary pressure and on neurological functions, respectively. Guanidino compounds including Cr, Crn, guanidine, MG, GAA, GPA, GBA, and GSA were also shown to have a series of other, potentially harmful, side effects (see also sect. VIIIB). Insulin resistance is a common finding in CRF. Accordingly, insulin binding to erythrocyte receptors was shown to be decreased in uremic patients, but increased rapidly upon hemodialysis (814). Binding of insulin to erythrocytes is also depressed by 1 mM concentrations of Crn, Cr, and guanidinoacetate. Plasma of uremic patients as well as �- and �-GPA inhibit the hexose monophosphate shunt in red blood cells (453). GPA, GBA, GAA, and GSA inhibit the phytohemagglutinin-induced stimulation of proliferation of normal human lymphocytes (878). This inhibitory action may possibly explain the depression in total lymphocyte count and function as well as the greatly depressed cell-mediated response to protein antigens in uremia. Finally, guanidine, MG, Crn, Cr, GSA, and GBA have convulsive effects in animals and may thus contribute to the neurological symptomatology in uremia (see sect. IXG). In conclusion, Crn degradation seems to be virtually irrelevant under normal conditions when Crn might even have a beneficial effect by acting as a hydroxyl radical scavenger. At greatly reduced GFR, on the other hand, when the serum concentration of Crn as well as oxidative stress are considerably increased, the formation of toxic Crn degradation products is favored and may contribute significantly to further disease progression. Because Crn degradation is stimulated by ROS and in particular by the hydroxyl radical, the serum concentrations of creatol and MG as well as the creatol/Crn and MG/Crn ratios may not only serve as diagnostic indexes for the degree of CRF, but may also be used as measures of oxidative stress in uremic patients (25, 683, 687, 747, 1146). XI. ANALYTICAL METHODS AND THEIR IMPLICATIONS FOR CLINICAL DIAGNOSIS Ever since the suggestion of Popper and Mandel in 1937 (779) that the clearance of endogenous Crn approximates the GFR, investigation of serum and urinary [Crn] has been popular in clinical medicine. Even though the parallelism between Crn clearance and GFR turned out to be less strict than previously presumed (see sect. IXH), there is still a broad interest in improving analytical methods for measuring [Crn] and [Cr] in biological samples. For many decades, chemical methods prevailed (551, 758, 932, 1071). Crn has been, and still is, measured mostly by the Jaffé reaction (422) in which Crn and picric acid under alkaline conditions form an orange-red-colored complex. Despite considerable effort, the detailed mechanism of color formation could not yet be resolved (see Refs. 551, 932, 958). The major disadvantage of the Jaffé reaction is its lack of specificity due to interference by a variety of metabolites, e.g., ketones and ketoacids, protein, bilirubin, and cephalosporins (e.g., Refs. 71, 443, 635, 926, 932, 958, 1071, 1110). Although a large number of modifications and improvements of the Jaffé reaction were proposed, none has eliminated all interferences. This disadvantage was outscored in the past by the usefulness of the Jaffé reaction in the clinical environment, by the lack of valid alternatives, and, in particular, by the low price of the required chemicals. Cr was frequently determined chemically by the �-naphthol-diacetyl reaction (1117), but this method is rather cumbersome and nonspecific as well. Already in 1937, using crude bacterial extracts, Miller and Dubos managed to estimate enzymatically the Crn content of plasma and urine (see Ref. 55). But it is only in recent years that significant progress has been made toward developing and improving enzymatic Cr and Crn determination methods (see Refs. 758, 862, 926, 932). In keeping with the diversity of Crn degradation pathways (see Fig. 7), a series of alternative reaction sequences was proposed to be specific and accurate. 1) A first method is based on creatininase, creatinase, sarcosine oxidase, and a peroxidase. The hydrogen peroxide liberated in the sarcosine oxidase reaction is used by the peroxidase to produce a colored substance that can be measured spectrophotometrically or fluorimetrically (for references, see Refs. 432, 443, 926, 958, 1110). 2) In the reaction sequence of creatininase, CK, pyruvate kinase, and lactate dehydro-
July 2000 CREATINE AND CREATININE METABOLISM 1177 genase, degradation of Crn (or Cr) is coupled stoichiometrically to the oxidation of NADH to NAD � , which can be measured spectrophotometrically (65, 80, 926, 1110). 3) Some Crn deaminase-based methods involve quantitation of the liberated ammonia either in a bromophenol blue indicator reaction (for references, see Ref. 758) or via glutamate dehydrogenase. In the latter case, Crn degradation is coupled stoichiometrically to NADPH oxidation (258, 333). These Crn deaminase-based methods have the advantage of comprising only two steps which may limit the risk of interferences. On the other hand, they involve quantitation of a volatile substance, ammonia, which might have a negative impact on the accuracy of the methods. 4) The last procedure involves five steps and, thus, is the most intricate one: Crn deaminase, 1-methylhydantoin amidohydrolase, N-carbamoylsarcosine amidohydrolase, sarcosine oxidase, and a peroxidase (715, 893). The second part of this sequence is, evidently, identical to that of method 1. Even though enzymatic methods are deemed to be quite specific, they are still not free from interferences. For example, peroxidase detection systems suffer from bilirubin or ascorbate interference, thus necessitating incorporation of potassium ferrocyanide, bilirubin oxidase, and/or ascorbate oxidase in the assay (e.g., Refs. 70, 432, 715, 862, 926, 932, 1094). On the other hand, cytosine derivatives such as the antibiotic 5-fluorocytosine may act as substrates of Crn deaminase (see sect. VIIG), a problem to be circumvented by the use of a more specific Crn deaminase either obtained from a natural source (298) or engineered biotechnologically. Further disadvantages of the enzymatic methods are the potentially lower storage stability and the (still) much higher price relative to the Jaffé reaction. It goes without saying that the enzymatic methods also have distinct advantages. Although it was not possible to eliminate the interference problem for the Jaffébased methods despite considerable effort over several decades, comparative studies have shown that already now, enzymatic methods produce less outliers and, in general, display a better performance than the Jaffé methods (see Refs. 70, 71, 258, 333, 443, 958, 1110). Even more importantly, molecular engineering offers great promise for further improving the properties of the enzymatic methods. For instance, mutagenesis approaches gave rise to more stable creatinase and sarcosine oxidase mutants (498, 709, 869, 871), and it is expected that more elaborate molecular design principles will allow us in the future to engineer enzymatic Crn and Cr determination methods virtually devoid of interference problems. HPLC (see, e.g., Refs. 43, 111, 1137, 1158), capillary electrophoresis (186, 1015), and biosensors with immobilized enzymes (582) represent alternative approaches for Cr, PCr, and Crn determination that may be less prone to interference problems than the Jaffé methods. Improved analytical tools for Crn and Cr determination are likely to find wide application not only in the diagnosis and monitoring of kidney function, but also in investigations and clinical evaluation of energy metabolism in various bodily tissues. Moreover, they may be instrumental in monitoring the efficacy of Cr supplementation in sports physiology (see sect. XI) and in the treatment of diseases responsive to Cr therapy. Even though, for instance, the nonspecific Sakaguchi reaction has been suggested to be useful for the screening of GAMT deficiency (867), more specific enzymatic tests for the quantitation of guanidinoacetate and/or Cr are likely to facilitate and improve the accuracy of such screenings. XII. CREATINE SUPPLEMENTATION IN SPORTS PHYSIOLOGY With increasing commercialization of the sports business, improving muscle performance by any means has become a critical issue. The efficacy of carbohydrate loading is widely accepted. Carbohydrates can be consumed in sufficient amounts via natural foods. On the other hand, stimulating muscle growth by, e.g., androgens is banned, since unphysiologically high concentrations of these hormones are required. Over the last few years, Cr supplementation as an ergogenic aid has boomed tremendously (for reviews, see Refs. 46, 103, 128, 305, 441, 674, 829, 1106). The success of British sprinters and hurdlers at the beginning of the 1990s has been associated with Cr supplementation (see Ref. 1106). Nowadays, there is widespread enthusiasm about the performance-boosting effects of Cr, which is seemingly used by many top athletes in explosive sports disciplines. Cr supplementation is popular, for example, among bodybuilders, wrestlers, tennis players, cyclists, mountain bikers, rowers, skijumpers, or cross-country skiers as well as among ski, rugby, handball, basketball, football, and ice hockey teams. It is not the principal goal of this section to share the widespread enthusiasm, but to critically discuss the scientific data published so far on Cr supplementation and its effects on muscle performance. During high-intensity exercise, ATP hydrolysis is initially buffered by PCr via the CK reaction. Whereas PCr is available instantaneously for ATP regeneration, glycolysis is induced with a delay of a few seconds, and stimulation of mitochondrial oxidative phosphorylation is delayed even further. On the other hand, the PCr stores in muscle are limited so that during high-intensity exercise, PCr is depleted within �10 s. Therefore, if it were possible to increase the muscle stores of PCr and thereby to delay PCr depletion, this might favorably affect muscle performance. The normal muscle concentration of total Cr is �125 mmol � (kg dry mass) �1 (46). As early as in the 1920s, oral
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