Ilse Possemiers

Guanidino compounds in serum, urine, liver, kidney, and brain of man and some ureotelic animals

Guanidino compound levels were quantitatively determined in serum, urine, liver, kidney, and brain of man and of some ureotelic animals. The guanidino compounds were separated over a cation exchange resin, using sodium citrate buffers, and detected with the fluorescence ninhydrin method. Species-specific differences in the levels of some guanidino compounds in the studied ureotelic animals are shown. alpha-Keto-delta-guanidinovaleric acid is a naturally occurring guanidino compound in ureotelic animals, and is not restricted to the pathobiochemistry of hyperargininemic patients. The fasting serum levels observed in beagles are the same as those found in hyperargininemic patients. In serum, liver, and kidney, the homoarginine, beta-guanidinopropionic acid, and gamma-guanidinobutyric acid levels are the highest in rats. The last two compounds have the highest levels of the studied guanidino compounds, with the exception of creatinine, in kidney. Specific high levels of gamma-guanidinobutyric acid and argininic acid are found in brain of rabbits.

Guanidino compound levels in brain regions of non-dialyzed uremic patients

Guanidino compounds have been suggested to contribute to the complex neurological complications associated with uremia. Several of them have previously been reported to accumulate in physiological fluids of renal insufficient subjects. We report on guanidino compound levels in 28 brain regions in control and uremic brains. In all brain regions studied, in controls as well as in uremic patients, concentrations of alpha-keto-delta-guanidinovaleric acid, alpha-N-acetylarginine and beta-guanidinopropionic acid remained below detection limits. Creatine, guanidinoacetic acid, argininic acid, gamma-guanidinobutyric acid, arginine and homoarginine were not increased in uremic patients. Argininic acid and homoarginine were detectable in some brain regions only. Creatine concentrations varied from 2500 +/- 2100 nmol/g tissue in hypophysis to 10500 +/- 1200 nmol/g tissue in cerebellar cortex. Even more pronounced regional differences were found for gamma-guanidinobutyric acid with the lowest concentration in the caudate nucleus (0.6 +/- 0.3 nmol/g tissue) and highest in substantia nigra, pallidum and cerebellar dentate nucleus (8.3 +/- 2.8 nmol/g tissue). The guanidinosuccinic acid levels were below detection limit in controls in the majority of brain regions. Taking into account the detection limit of guanidinosuccinic acid for a certain amount of tissue applied to the analytical system, important increases (approx. up to > 100 fold) were observed in all brain regions of uremic patients. Accumulation of guanidinosuccinic acid increased with increasing degree of renal failure with levels up to 65 nmol/g tissue in the hypophysis. Creatinine concentrations were also found to be increased in uremic brain regions but increases seemed to be less strictly related to serum urea levels. Guanidine and methylguanidine were found only occasionally in brain regions of controls while respectively 100- and 30-fold increases were found in brain regions of uremic subjects. Levels of guanidinosuccinic acid and creatinine in uremic brain were comparable to those previously observed in brain of experimental animals displaying convulsions following intraperitoneal injection of the respective compounds. Our findings further establish guanidino compounds as probable uremic toxins contributing to the neurological complications in uremia.

Guanidino Compound Analysis as a Complementary Diagnostic Parameter for Hyperargininemia: Follow-Up of Guanidino Compound Levels during Therapy

ABSTRACT: The aim of this collaborative study was to investigate whether guanidino compound analyses in the biologic fluids can be used as a complementary diagnostic parameter for hyperargininemia. Guanidino compounds were determined in the biologic fluids of all known living hyperargininemic patients using a cation exchange Chromatographie system with a fluorescence detection method. The serum arginine, homoarginine, α-keto-δ-guanidino-valeric acid, argininic acid, and N-α-acetylarginine levels of all the hyperargininemic patients are higher than the normal range. Similar increases were seen for the urinary excretion of α-keto-δ-guanidinovaleric acid and argininic acid. Untreated hyperargininemic patients have the highest guanidino compound levels in cerebrospinal fluid. However, even under therapy, the arginine, homoarginine, α-keto-δ-guanidinovaleric acid, and argininic acid levels in cerebrospinal fluid are still increased. Protein restriction alone is not sufficient to normalize the hyperargininemia, but protein restriction together with supplementation of essential amino acids with or without sodium benzoate decreases further the arginine levels. However, whereas the argininemia can be normalized, the catabolites of arginine are still increased. We conclude that the urinary amino acid levels may remain normal in hyperargininemia, whereas consistent increases of the guanidino compounds are observed. Thus, guanidino compound analyses can be used as a complementary biochemical diagnostic parameter for hyperargininemia. Although the argininemia can be normalized by therapy, the levels of the catabolites of arginine are still elevated.

Biochemical and behavioural phenotyping of a mouse model for GAMT deficiency

Deficiency of guanidinoacetate N-methyltransferase (GAMT) is the first described creatine (CT) deficiency syndrome in man, biochemically characterized by accumulation of guanidinoacetic acid (GAA) and depletion of CT. Patients exhibit severe developmental and muscular problems. We created a mouse model for GAMT deficiency, which exerts biochemical changes comparable with those found in human GAMT-deficient subjects. CT and creatinine (CTN) levels are significantly decreased and GAA is increased in knockout (KO) mice. In patients, other guanidino compounds (GCs) appear to be altered as well, which may also contribute to the symptomatology. Extensive evaluation of GCs levels in the GAMT mouse model was therefore considered appropriate. Concentrations of 13 GCs in plasma, 24-h urine, brain and muscle of GAMT mice were measured. We also report on the detailed behavioural characterization of this model for GAMT deficiency. Besides an increase of GAA and a decrease of CT and CTN in plasma, 24-h urine, brain and muscle of KO mice, we observed a significant increase of other GCs in brain and muscle that was sometimes reflected in plasma and/or urine. KO mice displayed mild cognitive impairment. In general, it could be concluded that the GAMT mouse model is very useful for biochemical research of GAMT deficiency, but shows only a mild cognitive deficit.

Guanidino compounds in serum and urine of cirrhotic patients

To investigate the metabolic relationship between urea and guanidinosuccinic acid (GSA), we determined the levels of the guanidino compounds, including GSA, and urea in serum and urine of cirrhotic patients. Linear correlation studies between serum urea and GSA levels were performed. Good positive linear correlation coefficients were found in the Child-Turcotte C subgroup (r = .847, P < .001) and in the total subgroup including B and C patients (r = .848; P < .0001). Serum guanidinoacetic acid levels were significantly increased in the Child-Turcotte C subgroup (P < .0001 for men and P < .001 for women). In contrast, GSA levels were significantly (P < .0001) decreased in the three studied subgroups. Similar results were found for urinary GSA excretion levels. Within each subgroup, serum and urinary GSA levels were significantly lower in patients with alcohol-induced cirrhosis than in nonalcoholic cirrhotic patients. Similar results were obtained for urea. The findings in cirrhotic patients clearly demonstrate a metabolic relationship between urea and GSA. They also show that urea and GSA biosynthesis is significantly lower in cirrhotic patients with an alcoholic origin than in cirrhotic patients with a nonalcoholic origin.

The pathobiochemistry of uremia and hyperargininemia further demonstrates a metabolic relationship between urea and guanidinosuccinic acid

To better understand the biosynthesis of guanidinosuccinic acid, we determined urea, arginine, and guanidinosuccinic acid levels in nondialyzed uremic and hyperargininemic patients. These substances were also determined during several years of therapy in one hyperarginiemic patient. Interrelationships of guanidinosuccinic acid levels with their corresponding urea and arginine levels were assessed by linear correlation studies. In uremic patients, a significant positive linear correlation (r = .821, p less than .001) was found between serum urea and guanidinosuccinic acid levels A significant positive linear correlation was also found between serum urea levels and urinary guanidinosuccinic acid levels (r = .828, P less than .001), but not between serum arginine levels and urinary guanidinosuccinic acid levels in hyperargininemic patients. In the intrahyperargininemic patient study, a similar significant positive correlation was found between serum urea levels and the corresponding urinary guanidinosuccinic acid levels (r = .866, P less than .001); the correlation between serum arginine levels and the corresponding urinary guanidinosuccinic acid levels was smaller. The presented analytical findings in uremic and hyperargininemic patients clearly demonstrate a metabolic relationship between urea and guanidinosuccinic acid.

Serum Guanidino Compound Levels and Clearances in Uremic Patients Treated with Continuous Ambulatory Peritoneal Dialysis

Guanidino compounds are increased in uremia and have been implicated as uremic toxins. The serum concentrations of 13 guanidino compounds and the clearances of 10 guanidino compounds were determined in 15 steady-state uremic patients treated with continuous ambulatory peritoneal dialysis. Guanidino compounds were determined using liquid cation-exchange chromatography with a sensitive fluorescence detection method. Standardized dialysis procedures were performed, including an overnight and a 3-hour dwell period. Guanidino compound levels did not significantly differ at the end of an overnight or a 3-hour exchange, indicating a steady-state blood chemistry for these substances in chronic ambulatory peritoneal dialysis. High levels were found for guanidinosuccinic acid, creatinine, guanidine and methylguanidine, while creatine and homoarginine levels were lower than in controls. Guanidinosuccinic acid, creatinine and methylguanidine reached levels associated with toxic effects in vitro. Significantly different clearances were found ranging from 4.02 +/- 1.08 ml/min for arginine to 7.94 +/- 2.76 ml/min for creatine during a 3-hour exchange.

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.