Fluorometrical Analysis of Guanidino Compounds in Human Cerebrospinal Fluid

Abstract: Guanidino compounds in CSF of 57 human subjects were determined fluorometrically after reaction with phenanthrenequinone in alkali solution, using HPLC. Creatinine (65.2 ± 13.4 nmol/ml), arginine (24.7 ± 6.4 nmol/ml), and homoarginine (0.7 ± 0.3 nmol/ml) were found in all subjects. Trace amounts of guanidinosuccinic acid and guanidinoacetic acid were detected in some of the subjects. Brain guanidino compounds, taurocyamine, N -acetylarginine, and methylguanidine were not detected in CSF.     

Comparative Study of Guanidino Compounds in Serum and Brain of Mouse, Rat, Rabbit, and Man

Abstract: The levels of 12 guanidino compounds were determined in the serum and brain of mouse, rat, rabbit, and man using cation-exchange column chromatography with the fluorescence ninhydrin detection method. A comparative study of these compounds was made in the four groups studied, for serum and brain. In rabbit and man the different guanidino compounds were also determined in various brain regions. This study provides basic analytical data that could facilitate the interpretation of further biochemical and neurochemical studies dealing with guanidino compounds that are identified as being toxins in hyperargininemia and uremia.     

Biochemistry and neurotoxicology of guanidino compounds. History and recent advances

Guanidino compounds are known to have important biological roles, such as the participation of arginine in ureagenesis, and of creatine in muscular contraction. On the other hand, the high toxicity of guanidino compounds, such as methylguanidine and guanidine, has been under study for quite a long time in the biochemical as well as clinical fields. In this review, the author summarizes the experimental results of neurophysiological and neurochemical studies on guanidino compound-induced seizures, conducted by his colleagues since 1966, and introduces several topics arising from their recent investigations on guanidino compounds and seizure mechanism, i.e., (1) alpha-guanidino-glutaric acid in the cobalt epileptic focus and its convulsive activity; (2) guanidino-ethanesulfonic acid and epilepsy; (3) delta-guanidinovaleric acid, and endogenous and specific GABA receptor antagonist; and (4) guanidino compounds as radical generators.     

Fluorometric determination of guanidino compounds by new postcolumn derivatization system using reversed-phase ion-pair high-performance liquid chromatography

A new postcolumn derivatization system using 1,2-naphthoquinone-4-sulfonate as fluorogenic reagent for the fluorometric determination of guanidino compounds is described. The guanidino compounds were separated by reversed-phase ion-pair high-performance liquid chromatography with an isocratic mobile phase containing the fluorogenic reagent and octane-sulfonate as the counterion. Fluorophors were derived from a condensation of guanidino compounds with the fluorogenic reagent in an alkaline solution. The chromatographic system using the mobile phase containing the fluorogenic reagent was simplified because only two pumps were required to deliver the mobile phase and the alkaline solution. Separation of guanidino compounds was completed within 25 min using a Nucleosil C8 column (5 microns, 15 cm X 4.6 mm i.d.). This method was applied to serum obtained from patients on hemodialysis therapy.     

Blood drugs and other analytical challenges (Methodological Surveys in Biochemistry,Vol. 7) : Edited by E. Reid,Ellis Horwood (Wiley), Chichester, 1978, X + 355 pp., price £ 19.50, ISBN 0-85312-124-9

A high-performance liquid chromatographic procedure has been developed for the separation and fluorometric detection of guanidino compounds in physiologic fluids. All guanidino compounds were separated on a 17 × 0.46 cm cation-exchange column using a stepwise pH gradient. The chromatographic system was designed to enable the use of the specific reagent 9,10-phenanthrenequinone as a means of monitoring the guanidino compounds of physiologic fluids. This new analytical method is so sensitive that it enables the analysis at the picomole level. Our automatic guanidino-compound analyzer was successfully applied to the quantitative determination of all guanidino compounds in physiologic fluids from normal controls and uremic patients.     

Effect of ammonium acetate-induced hyperammonemia on metabolism of guanidino compounds.

Guanidino compounds are synthesized from arginine in various tissues such as liver, kidney, brain, and skeletal muscle. Guanidino compounds such as arginine and creatine play an important role in nitrogen metabolism, whereas other guanidino compounds such as guanidinosuccinic acid and alpha-N-acetylarginine are known toxins. In order to understand the changes in the metabolism of guanidino compounds during ammonia toxicity, we investigated the effect of hyperammonemia induced by an ammonium acetate injection on the levels of guanidino compounds in plasma, liver, kidney, and brain of rats. Control animals were injected with an equal volume of saline. Blood and tissues were removed 1 h following ammonium acetate or saline injection and guanidino compounds were analyzed by high-performance liquid chromatography. Plasma and kidney levels of guanidinosuccinic acid were significantly elevated in rats challenged with ammonium acetate. Brain alpha-N-acetylarginine levels were also significantly higher in rats injected with ammonium acetate as compared to those in controls. Our results suggest that guanidinosuccinic acid and alpha-N-acetylarginine may play an important role in hyperammonemia.     

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography—fluorescence detection method

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These guanidino compounds are separated on a 6 × 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.     

Effect of guanidine dderivatives on choline acetylase and acetylcholinesterase of the rabbit brain]

The effects of 14 guanidino compounds on the activity of rabbit brain choline acetyltransferase (CAT) and acetylcholine esterase (ACE) have been studied, comparatively with that of pentylentetrazol. No guanidino compound examined nor pentylentetrazol had an effect on CAT activity. 4 of them showed a moderate inhibitory effect on ACE, of the same order of magnitude as that of pentylentetrazol, but significantly lower than that of the specific inhibitors eserine and mytelase.     

Guanidino compound metabolism in arginine-free diet induced hyperammonemia.

Guanidino compounds, intermediates of arginine metabolism, are altered in many pathological conditions especially those involving the urea cycle. Arginine and creatine play an important role in nitrogen metabolism whereas other guanidino compounds such as guanidinosuccinic acid and N-acetylarginine are toxins. Our objective was to investigate the relationship between guanidino compounds and hyperammonemia. Young and adult ferrets were fed a single meal of either an arginine-containing diet (ACD) or an arginine-free diet (AFD). Guanidino compounds were determined by HPLC in the plasma, liver, kidney and brain 3 h after feeding the specified diet. Only young ferrets fed AFD developed hyperammonemia. Plasma and kidney arginine was decreased whereas guanidinosuccinic acid was increased in young ferrets fed AFD. Hepatic creatine and kidney and brain guanidinoacetic acid were significantly decreased in this group. These results indicate that AFD-induced hyperammonemia produced decreased methylation activity in the liver and transamidination activity in kidney. Elevated guanidinosuccinate levels coupled with deficient hepatic creatine synthesis may play a role in the pathophysiology of hyperammonemia.     

Effect of Urease-Induced Hyperammonemia on Metabolism of Guanidino Compounds

We previously reported that guanidino compounds produced by the catabolism of arginine play an important role in the pathophysiology of acute hyperammonemia. In order to understand the metabolism of guanidino compounds during sustained hyperammonemia, we investigated the effect of intraperitoneal urease injection (800 IU/kg) on the levels of guanidino compounds in blood, liver, kidney, and brain of rats. Control rats received an equal volume of saline. Eight hours following injection, rats were sacrificed and blood and tissues were removed. Ammonia and urea were determined by enzymatic and colorimetric assays, respectively. Guanidino compounds were analyzed by high-performance liquid chromatography. Blood and tissue ammonia were significantly increased and urea decreased in urease-treated animals. Blood and kidney arginine levels were significantly decreased although hepatic arginine was increased following urease injection. Elevated hepatic arginine may be due to the rapid conversion of urea to ammonia by urease and the development of a futile urea cycle. Catabolites produced by the transamidination of arginine were significantly decreased in the blood, liver, kidney, and brain of urease-treated rats, whereas acetylation of hepatic arginine to α-N-acetylarginine was increased. Blood and tissue guanidinosuccinic acid levels were not elevated during urease induced hyperammonemia, supporting the hypothesis that urea is a precursor for the synthesis of guanidinosuccinic acid.     

The pseudodisaccharides: a novel class of group I intron splicing inhibitors.

Lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes. We report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group I introns, thus identifying pseudodisaccharides as a novel class of group I intron splicing inhibitors. Lysinomicin inhibited the self-splicing of the sunY intron of phage T4 with a Ki of 8.5 microM (+/- 5 microM) and was active against other group I introns. Inhibition was found to be competitive with the substrate guanosine, unlike aminoglycoside antibiotics, which act non-competitively to inhibit the splicing of group I introns. Competitive inhibitors of group I intron splicing known to date all contain a guanidino group that was thought to be required for inhibition; lysinomicin lacks a guanidino group.     

A serine endopeptidase from cucumber leaves is inhibited by L-arginine, guanidino compounds and divalent cations.

An endopeptidase was purified and characterized from green leaves of cucumber (Cucumis sativus L. suyo). The purified enzyme, a basic amino acid-specific endopeptidase with a pI of 5.0, was a monomeric protein of 80 kDa whose pH optimum was 9.5. Inhibitor analysis suggested that it was a serine endopeptidase and contained sulfhydryl groups essential for catalytic activity. Analysis of internal amino acid sequences of the endopeptidase showed no significant similarity to other proteins. Its activity was inhibited by L-Arg and guanidino compounds having high hydrophobicity, as well as divalent cations such as Mg2+ and Ca2+. The K(i) values of L-Arg and Mg2+, which are also likely in vivo inhibitors, were 3.5 and 10 mM, respectively. Inhibition by L-Arg and Mg2+ was additive, and more than 70% of the activity was reversibly inhibited under their physiologically significant concentrations. These results suggest that the enzyme is possibly regulated by L-Arg and/or guanidino compounds, and by divalent cations in vivo.     

First detection and quantification of N-monomethylarginine,a structural isomer of N-monomethylarginine,in humans using MS

The l-arginine metabolites methylated at the guanidino moiety, such as N^G-monomethyl-l-arginine (LNMMA), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), and symmetric N^G,N^G'-dimethyl-l-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, N^δ-monomethyl-l-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D₆-SDMA was used as internal standard. The calibration ranges were 25–1000 nmol/L for LNMMA and 5–350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found.     

Isolation and identification of some guanidino compounds in the urine of patients with hyperargininaemia by liquid chromatography,thin-layer chromatography and gas chromatography-mass spectrometry

Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.     

Use of substituted (benzylidineamino)guanidines in the study of guanidino group specific ADP-ribosyltransferase

A number of substituted (benzylidineamino)guanidines with different substitutents in the benzene nucleus are synthesized by coupling substituted benzaldehydes with aminoguanidine, and these compounds are tested as substrates for cholera toxin catalyzed ADP-ribosylation. A spectrophotometric assay method for the measurement of ADP-ribosyltransferase activity is developed, making use of the absorption characteristics of some of these compounds and the difference in the ionic character of the free compounds and the ADP-ribosylated products. The kinetic parameters for the ADP-ribosylation of these compounds are evaluated. A correlation between log kcat or log (kcat/Km) and the Hammett substituent constant sigma is observed. This correlation suggests the importance of substrate electronic effects on the enzymatic reaction. The reactivity of these compounds as acceptors of ADP-ribosyl groups in the reaction catalyzed by cholera toxin increases with increasing electron-donating power of the substituents in the benzene function. The effect is primarily on the catalytic rate constant, kcat, not on the binding constant, Km. The results are consistent with an SN2 reaction mechanism in which the deprotonated guanidino group makes a nucleophilic attack on the C-1 carbon of the ribose moiety.     

A rapid and specific enzymatic method for the estimation of L-Arginine.

Arginine can be estimated spectrophotometrically by the oxidation of NADH using octopine dehydrogenase. The method is highly specific for this amino acid and is applicable to the estimation of arginine in the presence of mono- and disubstituted guanidino compounds.     

Measurement of beta-guanidinopropionate and phosphorylated beta-guanidinopropionate in tissues.

The Sakaguchi color reaction for monosubstituted guanidino compounds was applied to the measurement of β-guanidinopropionate and phosphorylated β-guanidinopropionate. The phosphorylated derivative was measured as an increase in β-guanidinopropionate following incubation with 0.1n HCl in a boiling-water bath for 10 min. After feeding rats 1% of β-guanidinopropionic acid in their diet for 69 days, skeletal muscle, heart, liver, kidney, and spleen contained 5–10 μmoles of a monosubstituted guanidino compound per gram wet weight of tissue. No β-guanidinopropionate was detected in brain or testes. Phosphorylated β-guanidinopropionate was found only in skeletal muscle (27 μmoles/g) and in heart (7 μmoles/g). Creatine hydrate (2%) added to the diet containing β-guanidinopropionic acid inhibited the accumulation of phosphorylated β-guanidinopropionate in the heart and partially inhibited its accumulation in skeletal muscle.     

Synthesis of guanidino analogues of PMPDAP and their immunobiological activity

A series of novel 9-, 7- and 3-substituted 2- or 6-guanidinopurines as analogues of potent antiviral and immunobiologically active compound enantiomers of PMPDAP was synthesized and evaluated for their biological activity. Compounds containing the combination of guanidino and amino group at the purine moiety enhanced the interferon-gamma-triggered NO production in murine macrophages and stimulated the secretion of cytokines and chemokines in both murine macrophages and human peripheral blood mononuclear cells. The most active compounds are 27 and 54. None of the compounds tested exhibited any significant cytostatic effect or antiviral effect.     

Cerebrocellular Swelling in the Presence of Uraemic Guanidino Compounds: Ameliorative Effects of Taurine

Cell volumes (equilibrium non-inulin spaces) have been measured in slices of rat cerebral cortex incubated in the presence of uraemic guanidino compounds. Of 5 guanidino compounds tested, all but one caused significant cell swelling. This was most pronounced for guanidinosuccinic acid (GSA, 40 μmol/l)(+22%) and guanidine hydrochloride (G, 3 μmol/l)(+13%). Swelling was reduced by taurine in a dose-dependent manner, being completely abolished at 20 mmol/l. Swelling was also abolished by the antioxidants ascorbic acid (0.4 mmol/l) and butylated hydroxytoluene (0.5 mmol/l), the free radical scavenger N-acetyl-l-cysteine (10 mmol/l) and the lipid peroxidase inhibitor desmethyl tirilazad (100 μmol/l). The remission of swelling by 20 mmol/l taurine was reduced by 50% by the taurine transport inhibitor guanidinoethylsulphonate (GES, 1 mmol/l). This figure was not significantly altered when the concentration of GES was increased to 10 mmol/l. It was also reduced by 45% by the GABA_a receptor antagonist bicuculline (100 μmol/l). It was completely abolished when both GES and bicuculline were present. It is suggested that guanidino compounds result in cells undergoing oxidative-nitrosative stress, and that taurine protects against the resultant cell swelling by 2 mechanisms One (intracellular) requires taurine transport and depends on its role as an antioxidant, with lipid peroxidation being probably a significant factor. The other (extracellular) is associated with activation of GABA_a receptors. Special issue dedicated to Simo S. Oja     

Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans.

Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used, long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, 16 healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20 g/day for the first week (loading phase) and 5 g/day for 19 subsequent wk (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10, and 20 wk of supplementation, and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained approximately 30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), alpha-keto-delta-guanidinovaleric acid (+45%), and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on l-arginine:glycine amidinotransferase. Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.