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.     

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.     

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.     

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.     

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.     

Serum protein binding characteristics of cyproterone acetate, gestodene, levonorgestrel and norethisterone in rat, rabbit, dog, monkey and man

Protein binding characteristics including percentage of total binding, total binding capacity (pmol/mg protein), degree of specific binding, competition with dihydrotestosterone (DHT) and estradiol (E2) binding sites and dissociation constants (Kd) of low and high affinity binding sites were investigated for the progestins cyproterone acetate (CPA), gestodene (G), norethisterone (NET) and levonorgestrel (LN) in serum or plasma pools from man and four laboratory animal species (rat, rabbit, dog and monkey). Serum pools from animals were constructed from samples obtained either prior to or 1 day after pretreatment with ethinyl estradiol (EE2) (5 micrograms/kg/day for 7 days). Human plasma pools differed by SHBG levels (normal/induced). All serum pools were characterized by protein content and distribution. Equilibrium dialysis or dextran-coated charcoal (DCC) methods were used to separate bound and free steroids labelled with tritium. All progestins were highly (greater than 80%) bound to proteins in all undiluted samples. Total binding capacity was highest in rat and lowest in monkey. Human plasma showed a capacity of 1.5-2.1 microgram steroid/ml. In man, monkey and rabbit LN and G were specifically bound to the same degree as DHT, whereas NET binding was 50% lower. Specific binding of CPA to dog serum was 2-3 times higher than for other steroids. Two (high and low affinity) binding sites were found for LN, G and NET in man, monkey and rabbit and in dog for LN. Kd values for high affinity binding ranged from 3.5 (G in man) to 23 (NET in man) x 10(-9)M. Kd values of low affinity binding varied from 0.5 (CPA in dog) to 4 (NET in man) x 10(-6)M. E2 and DHT competition experiments confirmed the concept of SHBG as a carrier protein of 19-nor-progestins and DHT and its occurrence in man, monkey and rabbit. A sex hormone binding protein (SBP) in the dog seems to be responsible for the relatively high specific binding of CPA. SHBG is inducible by means of EE2 in man and monkey, but not in rabbit. EE2 may induce SBP in the dog. Comparison of in vitro Kds (high affinity binding) and in vivo metabolic clearance rates showed the same rankings for LN, G and NET in man, monkey and rabbit.     

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.     

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.     

Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism.

The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...     

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.     

Identification of Motilin mRNA in the Brain of Man and Rabbit. Conservation of Polymorphism of the Motilin Gene Across Species

Depoortere, I., P. De Clercq, M. Svoboda, L. Bare and T. L. Peeters. Identification of motlin mRNA in the brain of man and rabbit. Conservation of polymorphism of the motlin gene across species. Peptides 18(10) 1497–1503, 1997.—The data regarding the identity of motilin-like immunoreactivity in the central nervous system are controversial. The aim of the present study was to clarify whether motilin mRNA is present in the brain of rabbit and man. Total RNA, prepared from several regions of the rabbit brain, was subjected to RT-PCR aimed at amplifying a 294 bp cDNA fragment of the rabbit motilin precursor. The amplified product was subcloned and sequenced. The sequence showed 7 differences compared to the one reported for the duodenal precursor     

Studies on adenosine triphosphate transphosphorylases. XVII. A physicochemical comparison of the ATP-creatine transphosphorylase (creatine kinase) isozymes from man,calf, and rabbit

All of the creatine kinase isozymes from human, calf, and rabbit brain and muscle are composed of two noncovalently linked polypeptide chains, based upon sedimentation equilibrium analyses in the presence and absence of disruptive agents. The brain-type isozymes of man, calf, and rabbit proved to be slightly heavier than the muscle types. Various physicochemical properties of the isozymes are recorded. Each group of isozymes, i.e., the muscle, hybrid (muscle-brain), and brain isozymes from man, calf, and rabbit, showed similar electrophoretic behavior, although isoelectric points were not precisely identical for the muscle and hybrid types. Theoretical titration curves constructed from amino acid compositions of the calf isozymes showed reasonable agreement between their calculated and measuredpI ₀ values (isoelectric point extrapolated to zero ionic strength). The three native muscle isozymes and brain isozymes all contain two reactive sulfhydryl groups per mole or one per polypeptide chain of their two-chain proteins, which may be titrated with 5,5′-dithiobis (2-nitrobenzoic acid); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of ten- SH groups per mole of each brain-type and eight- SH groups per mole of muscle-type isozyme in the case of man, calf, and rabbit. A comparison of their amino acid compositions and tryptic peptide maps shows that there is only a slightly greater degree of homology between the individual isozymes of the same type (muscle type or brain type) than between the muscle- and brain-type isozymes of the same species.     

Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits

Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 +/- 0.1 and 3.9 +/- 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 +/- 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to a value of 2.1 +/- 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inter-tissue and inter-species comparison of butyrylcholinesterases

Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.     

All three isoforms of the voltage-dependent anion channel (VDAC1, VDAC2, and VDAC3) are present in mitochondria from bovine, rabbit, and rat brain

All three isoforms of the voltage-dependent anion channel (VDAC) were detected by immunoblot analysis of mitochondria isolated from rat, rabbit, and bovine brain. All three isoforms were associated with mitochondria after fractionation of rat brain extracts on sucrose density gradients. No VDAC isoforms were detected in non-mitochondrial fractions. Relative levels of the mRNAs coding the VDAC isoforms in rat, rabbit, and bovine brain were determined by RT-PCR. In all three species, the mRNA for VDAC2 was predominant. Relative to the mRNA for VDAC3, mRNAs for both VDAC1 and VDAC2 were more highly expressed in bovine brain than in rat brain. These results are consistent with the possibility that differences in relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of Type A:Type B hexokinase binding sites on brain mitochondria.     

The kinetics of lithium in the rat serum, brain and liver]

The kinetics of lithium in the serum, liver and brain of rats is described. The serum levels resembled those of man, whereas considerable quantitative differences were observed when comparing specific kinetic parameters. The brain level increased with the increasing doses, approaching the corresponding serum level. Concentration differences between different brain areas could be observed only after repeated administrations. Striatum, cortex and hippocampus showed significantly higher levels than the thalamus. The liver content remained low with increasing doses, and was below the brain level.     

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.     

KINETICS OF CHOLINE ACETYLTRANSFERASES (EC 2.3.1.6) FROM HUMAN AND OTHER MAMMALIAN CENTRAL AND PERIPHERAL NERVOUS TISSUES

Abstract— Choline acetyltransferase (EC 2.3.1.6) was partially purified from human caudate nucleus and putamen, human sciatic nerve, rabbit and rat brain, and rabbit sciatic nerve. Kinetic constants were determined under the same conditions for all six extracts. Extrapolated K_m values were between 6.6 and 18 μM for acetyl-CoA and between 0.4 and 1.2 mM for choline. Product inhibition patterns indicated that ChAc from both central and peripheral nervous tissues of man and the rabbit obeys a Theorell-Chance mechanism. Kinetic parameters suggest a possible influence on ACh synthesis of the in vivo concentration ratio, CoA/acetyl-CoA.     

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.     

Bitterness Reduction of Citrus Fruits by β-Cyclodextrin

The levels of free amino acid, ammonia nitrogen and guanidino compounds were examined in renal failure rats induced by adenine. Among the essential amino acids in the serum, the marked reduction of lysine, valine, leucine and isoleucine was confirmed in the adenine-fed group as compared with the control group. Tyrosine and ornithine were also significantly reduced in the adenine-fed rats, while glycine, arginine and aspartic acid were significantly elevated. The urinary excretion of leucine, isoleucine and non-essential amino acids (glutamic acid, histidine, aspartic acid, citrulline, tyrosine, ornithine) was found to be high. On the other hand, adenine administered orally caused hyperammonemia. Furthermore, the results of the present study show that intake of adenine increased extraordinarily the level of guanidinosuccinic acid and methylguanidine in the serum, while the value of serum guanidinosuccinic acid and methylguanidine in rats fed on a control diet was not detectable.