Andrée G. Roberge

Characteristics of Dihydroxyphenylalanine/5‐Hydroxytryptophan Decarboxylase Activity in Brain and Liver of Cat

Abstract: Dihydroxyphenylalanine/5‐hydroxytryptophan (DOPA/5‐HTP) decarboxylase activity varied widely in different parts of the CNS, being highest in the neostriatum and lowest in the frontal cortex. The addition of 2.5 μm‐pyridoxal 5′‐phosphate (PLP), the coenzyme, increased enzyme activity in brainstem and liver, while higher concentrations led to a decrease in activity. In brainstem, the addition of 1000 μm PLP shows activity similar to that obtained without exogenous PLP. The effects of different monoamine oxidase (MAO) inhibitors on decarboxylase activity were demonstrated. Iproniazid phosphate and harmaline significantly decreased the decarboxylation in liver and brainstem, while pargyline inhibited only liver decarboxylation. Some decarboxylase inhibitors such as RO4–4602 and α‐methyl DOPA, as well as piribedil, a dopaminergic receptors agonist, were added in vitro to measure their action on decarboxylase with or without exogenous PLP or with double concentrations of substrate (5‐HTP). Piribedil (5000 μm) affected the enzymic reaction and triggered a higher inhibition in liver. Inhibition in brainstem needed less RO4–4602 (50 μm) than in liver (300 μm). Addition of PLP did not reverse this inhibition, while doubling the concentration of 5‐HTP nullified the inhibitory effect in liver only. Inhibition induced by α‐methyl DOPA (5 μm) was easily reversed by doubling the concentration of substrate. However, the presence of exogenous PLP restored the enzymic activity in liver only. We conclude from this work thus that the enzyme can decarboxylate its substrate without exogenous PLP, that MAO inhibitors might inhibit decarboxylase activity, and that decarboxylase inhibitors react differently when brain and liver are used as enzymic source. PLP seems to act as a protective agent on the active site of the enzyme in the brainstem and preferentially with the substrate in the liver.

Determination of mono-, di- and polyamines in foods using a single-column amino acid auto-analyzer

A fully automated, rapid and sensitive method was developed to analyze fourteen different biogenic amines in food. Using a Technicon C4 ion-exchange resin column (20 m X 0.5 cm), adapted to an automatic Technicon TSM amino acid analyzer, the following amines were separated and quantified: adrenaline, noradrenaline, 1,3-diaminopropane, putrescine, cadaverine, histamine, spermidine, dopamine, spermine, agmatine, tyramine, serotonin, phenethylamine and tryptamine. Five buffers were required to elute the amines using a gradient of pH from 5.6 to 12.7; the column temperature was maintained at 65 degrees C. The method was also assayed on ground beef, cheese and wine samples. Amines from cheese and ground beef samples were extracted with 0.6 M perchloric acid. No extraction of wine samples was necessary.

Effect of chronically administered L-Dopa on Dopa/5HTP decarboxylase and tyrosine and tryptophan hydroxylases in cat brain

The activity of tyrosine and tryptophan hydroxylases and of Dopa/ 5-HTP decarboxylase was measured in different structures of the brain of cats administered L-Dopa (100 mg/kg/day,per os) during several consecutive days. The activity of tyrosine hydroxylase which is unchanged after 4 and 7 days of treatment, respectively, is significantly decreased after 21 days of L-Dopa. The activity of tryptophan hydroxylase is normal after 4 days of L-Dopa but it is significantly decreased after 7 and 21 days of L-Dopa, respectively. The activity of decarboxylase is normal after 4 days of L-Dopa but it is significantly increased after 7 and 21 days of L-Dopa, respectively.

Effect of L-Dopa on delayed response and visual discrimination in cats and its relation to brain chemistry

SummaryThe effect of L-DOPA (30 mg/kg, per os) on the performance of a delayed response and a visual discrimination task was investigated in low-performing cats, with a naturally occurring DA brain deficiency and in experimentally naive cats with a normal DA brain content.Delayed response performance was significantly improved in low-performing cats 4 to 8 hours after administration of L-DOPA. In experimentally naive cats, delayed response was improved in 30% of experiments only. L-DOPA had no or a negative effect on the performance of the visual discrimination task. The effect of chronic administration of L-DOPA was also investigated. Biochemical assays show that improved performance coincides with increased DA content in striata.Results are discussed in relation to observations in human patients with Parkinsons disease and to short term memory mechanisms.

Chlordiazepoxide, go-nogo successive discrimination and brain biogenic amines in cats.

Chlordiazepoxide (CDP; 0.4 mg/kg/day, per os) was administered to cats during either the acquisition (CDP 21-22 days) of a go-nogo successive discrimination task (SD) or the performance (CDP 10 days) of the previously learned SD task. Endogenous levels of serotonin, 5-hydroxyindoleacetic acid, noradrenaline and dopamine were assayed in 12 brain areas, in trained as well as in untrained cats. This study has shown that (1) CDP strongly impaired the acquisition but not performance of the SD task, revealing a dissociation of the effects of CDP on these two stages of training; (2) the CDP administration, as well as the SD training, produced regional changes in brain levels of biogenic amines, suggesting the involvement of particular monoaminergic neurons in the behavioral effects of CDP and in operant behavior; and (3) in particular brain areas, interactions were observed between the effects of the SD training and those of the CDP administration on monoamines, indicating that the behavioral state may interfere with the neurochemical effects of CDP.

THE EFFECTS OF A RIBOFLAVIN SUPPLEMENTATION ON THE NUTRITIONAL STATUS AND PERFORMANCE OF ELITE SWIMMERS

Abstract The present experiment was undertaken to verify if a riboflavin supplementation is required for the maintainance of a normal status for this vitamin in athletes on training and to determine its effect on performance. Fourteen elite swimmers having a food intake respecting Canadian dietary recommendations, participated in the present study. From this sample, two subgroups were formed. The first group was submitted to a 60 mg/day riboflavin supplementation during 16 to 20 days while the second received a placebo during the same period of time. Before the nutritional supplementation, a blood sample was obtained to determine the erythrocyte gluthatione reductase (EGR) enzyme activity and the amount of free flavine adenine dinucleotide (FAD). Athletic performance was assessed by a swimming test consisting of six 50-meter free style bouts. A treadmill test was also performed to determine maximal oxygen consumption (max VO 2 ) and ventilatory anaerobic threshold (VANT). All subjects were characterized by a normal riboflavin status before the experiment. The supplementation did not change EGR activity while free FAD tended to increase after the protocol. This latter effect, which did not attain statistical significance (p>0.05), was believed to reflect a delay in the elimination of the vitamin excess. Riboflavin supplements had also no influence on swimming performance, max VO 2 , and VANT. This observation was reinforced by the fact that no correlation was noted between EGR activity and performance indices. In conclusion, the present results indicate that athletes submitted to a large amount of training can maintain a normal riboflavin status without any supplementation. It also appears that a riboflavin supplementation does not affect the biochemical indices of this vitamin and the performance level in non-deficient athletes.

Behavioral and biochemical effects of α-methyltyrosine in cats

A single dose of orally administered DL-α-methyltyrosine methylester hydrochloride (AMT) significantly increased the number of errors made by cats performing a spatial delayed response (DR) task. The same doses of AMT had no such effect on simultaneous visual discrimination (VD) performance. Response latencies were increased in all cats, whichever task performed. In an attempt to correlate the behavioral and biochemical effects of AMT, dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were assayed in 5 brain structures, 4 to 48 h after 100 to 175 mg/kg. DA was reduced significantly with all doses of AMT used, NA with doses of 125 mg/kg and more. 5-HT increased significantly after 100 and 125 mg/kg but remained unchanged after higher doses. Due to the simultaneous action of AMT on DA, NA and 5-HT, the respective roles of these amines are difficult to dissociate. In contradistinction, previous findings suggest that brain DA be responsible for the impairment of DR performance.

Effect of hydrazine on urea cycle enzymes in vitro and in vivo

Abstract The effect of hydrazine on urea cycle enzymes was studied in adult male rats. Addition of different concentrations of hydrazine to homogenates of rat liver produced a gradual inhibition of citrulline and urea synthesis. A daily injection of subconvulsive doses (32 mg/kg) of this toxic substance for 4 days provoked an increase of citrulline and urea contents in different tissues. The activities of urea cycle enzymes were not affected by such a treatment except for argininosuccinase where an increase was noted. The activity of ornithine-ketoacid transaminase was greatly inhibited. The results suggest that the inhibition of ornithine-ketoacid transaminase provokes an accumulation of ornithine. The presence of a high concentration of this amino acid together with an increased ammonia production stimulates urea synthesis. Under these physiological conditions, the condensation reaction of citrulline with aspartic acid may become ratelimiting in the operation of the urea cycle with the resultant accumulation of citrulline.

Effects of dietary protein intake and immobilization on plasma dopamine-β-hydroxylase activity in cats

Abstract Dopamine-β-hydroxylase (DBH) was measured in the plasma in normal cats and in cats immobilized for 2 h after being fed for 14 days semi-purified diets containing different dietary proteins. The food intake (dry weight) for all animals fed semi-purified diet was significantly decreased compared to that of Purina fed cats. Body weight of the cats, however, was maintained constant thoughout the experiment except for the decrease observed in lentil-fed cats. Lowered DBH activity was found in the plasma of cats fed Purina, skim milk, tofu, egg white, or lentils, compared to casein, diets. Fish, beef and casein-fed cats had similar DBH activity. A 2 h immobilization period increased the plasma DBH activity in all groups except in casein and legume fed cats. These results suggest that dietary protein composition affects plasma DBH activity.

DIFFERENTIATION IN BRAIN AND LIVER DOPA/SHTP DECARBOXYLASE ACTIVITY AFTER L-DOPA ADMINISTRATION WITH OR WITHOUT PYRIDOXINE IN CATS

Abstract— Pyridoxine (50mg/kg, per os) given for 7 consecutive days did not modify the content of dopamine, noradrenaline, and serotonin in the neostriatum of the brain 3, 6 and 18 h after the last dose, but significantly increased DOPA/5HTP decarboxylase activity in both the neostriatum and liver. The administration of l‐DOPA and pyridoxine (100 and 50mg/kg, per os, respectively) together for 7 days increased DOPA/5HTP decarboxylase activity in the brain to the same extent as did l‐DOPA and pyridoxine given individually. Liver DOPA/5HTP decarboxylase activity remained normal when both drugs were administered together. However it decreased significantly after l‐DOPA administration for 7 days but not after pyridoxine treatment. In cats under treatment with l‐DOPA for 7 days, actinomycin D given for the final 3 days prevented the increased DOPA/5HTP decarboxylase activity induced by l‐DOPA in the neostriatum and mesencephalon but had no effect on the enzymatic activity in the liver. These findings indicate that differences exist between brain and liver DOPA/SHTP decarboxylase activity in uivo. In addition, denatured supernatant from livers of animals treated with l‐DOPA contained a dialysable compound which inhibits DOPA/SHTP decarboxylase activity in the supernatant from livers of untreated cats. In animals who received pyridoxine along with l‐DOPA, no such inhibitor was found. These results may explain the mechanism by which l‐DOPA exerts its beneficial effects and why pyridoxine administered with l‐DOPA reduces the therapeutic effectiveness of l‐DOPA in Parkinsons disease. These findings are consistent with the possibility that a tetrahydro‐isoquinoline derivative formed in vivo in the liver after l‐DOPA therapy for 7 days might affect DOPA/5HTP decarboxylase activity in the liver but not in brain. A tetrahydroisoquinoline derivative did not appear to be formed when l‐DOPA and pyridoxine were administrated together suggesting that pyridoxine protected the enzyme and favored a more rapid degradation of l‐DOPA peripherally with less l‐DOPA available for the CNS.