Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 μmol/l to more than 60 μmol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients. Received: 3 November 1998 / Revised: 3 May 1999 / Accepted: 3 May 1999     

Trans-inhibition of glutamate transport prevents excitatory amino acid-induced glycolysis in astrocytes

Previous studies have demonstrated that activation of glutamate transporters promotes glycolysis in astrocytes. Current evidence indicates that compounds such as threo-beta-hydroxyaspartate (THA) are both competitive inhibitors and substrates for glutamate transporters. In this study, we have analyzed the effect of THA on excitatory amino acid (EAA) transport and on EAA-induced glycolysis in mouse primary astrocyte cultures. In agreement with previous studies in rat astrocytes, THA competitively inhibited 3H-D-aspartate (3H-D-Asp) uptake with an IC50 of 319 microM (Ki = 36.6 microM). In contrast, it did not prevent D-aspartate-induced 3H-2-deoxyglucose (2DG) uptake in these conditions. Preexposure of cells to THA for at least 15 min revealed another form of glutamate transport inhibition. This effect was concentration-dependent with an apparent IC50 of 47.7 microM and showed kinetic characteristics consistent with a mechanism of trans-inhibition. Preincubation with THA also inhibited D-aspartate-induced 3H-2DG uptake in a concentration-dependent manner with an apparent IC50 of 59.8 microM. Comparison with other transportable analogues reveals that they share with THA the ability to cause trans-inhibition of glutamate transport and to prevent glutamate-stimulated glycolysis; THA, however, is unique in that it has no effect alone on glucose utilization after preexposure. These data indicate that trans-inhibition of glutamate transport may be a mechanism by which certain glutamate transport inhibitors can prevent the stimulation of aerobic glycolysis by glutamate in astrocytes.     

Focal cerebral ischaemia induces a decrease in activity and a shift in ouabain affinity of Na+, K+-ATPase isoforms without modifications in mRNA and protein expression

In a mouse model of focal cerebral ischaemia, we observed after 1 h of ischaemia, that the total Na+, K+-ATPase activity was decreased by 39.4%, and then did not vary significantly up to 6 h post-occlusion. In the sham group, the dose-response curves for ouabain disclosed three inhibitory sites of low (LA), high (HA) and very high (VHA) affinity. In ischaemic animals, we detected the presence of only two inhibitory sites for ouabain. After 1 h of permanent occlusion, the first site exhibited a low affinity while the second site presented an affinity intermediate between those of HA and VHA sites, which evolved after 3 h and 6 h of occlusion towards that of the VHA site. The presence of only two ouabain sites for Na+, K+-ATPase after ischaemia could result from a change in ouabain affinity of both HA and VHA sites (alpha2 and alpha3 isoforms, respectively) to form a unique component. Irrespective of the duration of ischaemia, the smaller activity of this second site accounted entirely for the loss in total activity. Surprisingly, no modifications in protein and mRNA expression of any alpha or beta isoforms of the enzyme were observed, thus suggesting that ischaemia could induce intrinsic modifications of the Na+, K+-ATPase.     

Rôle du foie dans le métabolisme des nutriments en nutrition artificielle

The liver is a prominent organ in nutritional homeostasis. Due to unique metabolic properties, it plays a main role in the metabolism of the three macronutrients ‘as well as the micronutrients’ (vitamins and minerals) storage. Although it represents only 2.5% of the body mass, it consumes 20% of total resting energy expenditure and a similar percentage of the amino acid mixture absorbed via the gut during and after a meal. Due to a peculiar vascularization (portal vein, the entire gastrointestinal venous flux is directed towards the liver with all hydrosoluble nutrients, only water-unsoluble lipids being excluded from this obligatory ‘first-pass mechanism’). Since it is the location for glycogen storage, VLDL synthesis and ketogenesis, the liver is crucial in the fed-to-fasted metabolic alternation. While fat is not physiologically stored in the liver, it is a very important organ in lipid metabolism. Except immunoglobulins, all plasma proteins are synthetised by the liver together with the constitutive proteins, explaining that it is a very powerful organ for protein synthesis. Finally, due to a very active amino acid metabolism, the liver can reshape the amino acid-mixture coming from the gut in the absorptive state. Such a phenomenon has a major implication in the nutritional physiology of amino acid metabolism according to the route: enteral or parenteral. Indeed, in the latter case the remodelling by the liver does not occurs.     

Effects of cholecalciferol and calcium on experimental hepatic osteodystrophy in rats

To investigate cholecalciferol (vitamin D) metabolism disorders in hepatic osteodystrophy (HOD) and the effects of vitamin D, its metabolites, and calcium (Ca) on HOD, an experimental HOD model in rats was developed using carbon tetrachloride. In the serum level of 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol, and 24R,25-dihydroxycholecalciferol, there were no significant differences between normal and control cirrhotic rats. Vitamin D supplementation significantly inhibited the atrophy of intestinal villi, reduction of bone calcium content, elevation of bone resorption, reduction of osteoid volume, and reduction of bone volume. Ca supplementation significantly increased the serum free Ca index and inhibited the elevation of bone resorption, the reduction of bone ash and Ca content, and the reduction of bone volume. This experimental study demonstrates that: (1) no marked vitamin D hydroxylation disorder was found in HOD; (2) vitamin D supplementation was effective in inhibiting HOD; and (3) sufficient Ca supplementation was also effective in inhibiting HOD.A portion of this work was presented at the 13th Annual Meeting of the Japanese Society for Bone and Mineral Research, July 1995, Fukuoka, Japan.     

Metabolic and neuroanatomical correlates of barrel-rolling and oculoclonic convulsions induced by intraventricular endothelin-1: a novel peptidergic signaling mechanism in visuovestibular and oculomotor regulation?

The neuroactive peptide endothelin-1 has receptors distributed abundantly among subdivisions and nuclei of the visuovestibular and oculomotor systems. In previous work, we and others described the convulsive manifestations resulting from central injection of this neuropeptide, including nystagmus, oculoclonus, exophthalmos, tonic hindlimb extension, and a generalized repetitive motor disturbance called barrel-rolling. We applied the quantitative, autoradiographic [¹⁴C]deoxy-glucose method to examine the hypothesis that visuovestibular and oculomotor structures would become metabolically stimulated when endothelin was introduced into the brain via the ventricular system in conscious rats. Since previous work had demonstrated that hypermetabolic responses to endothelin in other neural systems were inhibited by an antagonist of neuronal calcium L-type channels, nimodipine, we further tested whether the increased function of vestibulooculomotor nuclei whose metabolic activity was sensitive to endothelin could be altered following nimodipine pretreatment via the ventricle. A single unilateral injection of endothelin (9 pmol in 3 l saline) into a lateral ventricle provoked significantly increased rates of glucose metabolism in 22 of 39 individual anatomical structures of the visuovestibular and oculomotor systems. Among those affected were the superficial stratum of the caudal superior colliculus (+25%), the optic tract bilaterally (+ 35 to 43%), the oculomotor cranial nerve nuclei (III, IV, VI; range of +21 to 47%), and the medial terminal nucleus of the accessory optic tract which harbors dense fields of endothelin binding sites (bilateral increase of +70 to 96%). Several other nuclei involved in the proprioceptive and visuovestibular disturbance caused by endothelin displayed increased metabolic activity, including the cuneate, gracile, sensory trigeminal, and prepositus hypoglossal nuclei, the vestibular subnuclear system, and the cerebellar flocculus. Identification of hypermetabolic responsivity to endothelin in these structures provides further information on the anatomical substrates mediating the behavioral phenomenology of endothelin-induced motor convulsions which involve the paroxysmal participation of the extraocular muscles and motor control systems producing barrel-rolling convulsions. Nimodipine pretreatment inhibited both the convulsive activity and the cerebral hypermetabolic responses to intraventricular endothelin. The results indicate that the neural systems sensitive to intraventricular endothelin become functionally active via a calcium-mediated process that may involve the neuropeptide as an intrinsic signaling molecule.     

A soluble androgen receptor in the cytoplasm of the male mastomys prostate

Summary A steroid receptor protein was isolated from the cytoplasmic fraction of Mastomys prostate. Following in vivo and in vitro labelling of the tissue with tritiated testosterone or dihydrotestosterone, samples were analysed by gel exclusion chromatography or sucrose density gradient centrifugation. A steroid receptor complex was isolated on Sephadex G-200. Analysis of the steroids associated with this complex showed that the major part of the bound radioactivity was 5 -dihydrotestosterone. The binding was inhibited by unlabelled testosterone and could not be demonstrated in the liver cytosol. Using sucrose desity gradient centrifugation, the dihydrotestosterone receptor complex sedimented at 5.6 s together with heavier aggregates. In the presence of 0.4 M KCl a single complex was sedimented at 4. 6 s. The results demonstrate a receptor protein in the cytosol of the Mastomys prostate which binds to dihydrotestosterone and is comparable to that of rat prostate.     

Hybrid information provided by the 14C-aminopyrine breath test studies with 14C-monomethylaminoantipyrine in the guinea pig

Summary N-Demethylation of ¹⁴C-aminopyrine (¹⁴C-AP), labelled at the methyl groups of the tertiary amino group, yields H¹⁴CHO and ¹⁴C-monomethylaminoantipyrine (¹⁴C-MMAAP) which also undergoes N-demethylation, however, at a slower rate as measured in hepatic microsomes. As after intraperitoneal application to male guinea pigs of ¹⁴C-AP (75 mg/kg; 50 Ci/kg), exhalation rate of ¹⁴CO₂ declines in a biphasic manner, the hypothesis was tested whether the terminal part might reflect N-demethylation of MMAAP. The application of ¹⁴C-MMAAP (70 mg/kg; 10 Ci/kg), resulted in monophasic curves of ¹⁴CO₂ exhalation rate. Their half lives were, however, longer than terminal half lives obtained after ¹⁴C-AP. Obviously, this terminal phase does not represent ¹⁴CO₂ formation from the metabolite MMAAP only, but ¹⁴C-AP might still contribute to ¹⁴CO₂ production. Confirmation was obtained by HPLC determination of AP and MMAAP in serum after injection of AP. Shortly after injection, high concentrations of AP and low ones of MMAAP were found in blood from the portal vein and systemic circulation. Thus, initial parts of ¹⁴CO₂-exhalation rate curves reflect predominantly AP metabolism whereas later phases provide hybrid information.     

Effects of enkephalins and two enzyme resistant analogues on monoamine synthesis and metabolism in rat brain

Summary Methionine-enkephalin and leucineenkephalin, administered into the lateral ventricle of intact rats, increased the accumulation of DOPA by a naloxone-sensitive mechanism in different brain regions after inhibition of the aromatic l-amino acid decarboxylase. Because of the rapid enzymatic degradation of both enkephalins large doses (500 g) were required to enhance brain catecholamine synthesis. The two enzyme resistant enkephalin analogues d-Ala²-methionine enkephalin amide (DALA (4–256 g) and FK 33-824 (0.003–1 g) also increased the synthesis of DOPA, dose-dependently and by naloxone-sensitive mechanisms, but at much lower dosage level. The enkephalins markedly enhanced the brain tyrosine concentration but this effect was not antagonized by naloxone, probably because the enzymatic cleavage releases tyrosine from the administered peptides. In contrast, neither DALA nor FK 33-824 increased the brain tyrosine concentration. The formation of 5-HTP and the brain tryptophan concentration were also increased by the enkephalins, although these effects were not blocked by naloxone. The enkephalin analogues, however, enhanced the formation of 5-HTP and the brain tryptophan concentration by naloxonesensitive mechanisms. All four peptides accelerated the disappearance of dopamine, noradrenaline and 5-hydroxytryptamine after inhibition of monoamine synthesis. The results suggest that endogenous enkephalins, through the activation of opiate receptors, are involved in the short-term regulation of central monoaminergic systems.Preliminary data were presented at the Nobel Symposium 42, Principles for the Central Regulation of the Endocrine System, Stockholm, June 8–10, 1978, and at the 4th International Catecholamine Symposium, Asilomar Conference Center, Pacific Grove, California, September 17–22, 1978     

Cerebral energy metabolism in patients with brain lesions at normo- and hyperbaric oxygen pressures

Summary The object of this study was to ascertain the oxygen tolerance limit and the oxygenation state of the injured brain in man. While breathing air, oxygen and hyperbaric oxygen at pressures of 1.5 and 2.0 atmospheres absolute (ATA), the cerebral arteriovenous differences (AVD) for O₂, glucose, lactate, pyruvate and blood gas pressures and pH values were measured. The balance of the cerebral glucose metabolism was calculated. The results showed that the injured brain did not tolerate the exposure to an oxygen pressure of 2.0 ATA for 10 to 15 min, but exposure to 1.5 ATA for 35–40 min was tolerated and had a favorable effect on the glucose or energy metabolism of the brain as well as on the clinical course. There was a distinctly increased cerebral glycolysis while breathing air indicating insufficient oxygen delivery to the brain. The change from breathing air to oxygen resulted in a distinct inhibition of cerebral glycolysis, which indicated improved cerebral oxygenation and energy production and gave evidence for a Pasteur effect regulating the glucose metabolism of the injured brain in man. At an inspiratory oxygen pressure of 1.5 ATA we had a nearly balanced cerebral glucose metabolism indicating an adequate cerebral oxygenation and energy formation. Further increase in inspiratory oxygen pressure to 2.0 ATA (performed only in group A) increased cerebral glycolysis considerably. This was assumed to be due to cerebral oxygen poisoning resulting in disturbed oxidative energy formation. Following this alteration an extreme reduction of the cerebral glucose uptake appeared, probably due to a disturbance of the specific glucose transport system. These metabolic alterations were not accompanied by seizures or any other clinical neurological manifestation. In group B, exposed to 1.5 ATA, such alterations of the cerebral glucose metabolism did not appear. A nearly balanced cerebral glucose metabolism was found at inspiratory oxygen pressures of 1.0 and particularly of 1.5 ATA, indicating an improved oxygenation and energy production of the affected brain. Finally, a renewed increase of the cerebral glycolysis occurred following the change from breathing oxygen to air. This again indicated an insufficient oxygen delivery to the affected brain.

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Zusammenfassung Ziel dieser Arbeit war es, die Wirkung verschiedener inspiratorischer O₂-Drucke auf den zerebralen Glukose- bzw. Energiestoffwechsel zu untersuchen. Dabei sollte insbesondere die Sauerstoff-Toleranzgrenze und der Zustand der Oxygenierung des geschädigten Hirns bestimmt werden. Unter Luft-, Sauerstoff und hyperbarer Sauerstoffatmung, d. h. bei Drucken von 1,5 und 2,0 Atmosphären, wurden die arterio-hirnvenösen Differenzen (AVD) für O₂, Glukose, Laktat, Pyruvat sowie die Blutgasdrucke und die pH-Werte gemessen. Die Bilanz des zerebralen Glukosestoffwechsels wurde bestimmt. Die Ergebnisse zeigten vor allem, daß das geschädigte Hirn eine Sauerstoffbelastung von 2,0 Atmosphären mit einer Expositionszeit von 10 bis 15 min nicht toleriert. Dagegen wurde eine Sauerstoffbelastung von 1,5 Atmosphären mit einer Expositionszeit von 35–40 min vertragen und hatte einen günstigen Einfluß auf den zerebralen Glukose- bzw. Energiestoffwechsel sowie auf den Krankheitsverlauf von traumatischen oder ischämischen Hirngewebsveränderungen. Während der Luftatmung fand sich eine erhebliche Steigerung der zerebralen Glykolyse, was auf eine mangelhafte O₂-Versorgung des Hirngewebes hinwies. Der Wechsel von Luft- auf Sauerstoffatmung führte zu einer deutlichen Hemmung der zerebralen Glykolyse. Dies zeigte eine Besserung der zerebralen Sauerstoffversorgung und Energieproduktion an und wies auf einen Pasteur Effekt bei der Regulation des Glukosestoffwechsels des geschädigten Hirns hin. Bei einem inspiratorischen Sauerstoffdruck von 1,5 Atmosphären war eine praktisch ausgeglichene Bilanz des zerebralen Glukosestoffwechsels nachweisbar, was für eine ausreichende Sauerstoffversorgung und Energiebildung des Hirns sprach. Der weitere Anstieg des inspiratorischen Sauerstoffdruckes auf 2,0 Atmosphären, der nur in Gruppe A durchgeführt wurde, bewirkte jedoch eine erhebliche Steigerung der zerebralen Glykolyse. Es ist anzunehmen, daß diese Stoffwechseländerung durch eine zerebrale Sauerstoffvergiftung hervorgerufen wurde, die vor allem zu einer Störung der oxydativen Energiegewinnung führte. Anschließend trat eine extreme Reduzierung der zerebralen Glukoseaufnahme auf, die am ehesten durch eine Störung des spezifischen Glukosetransportsystems des Hirns bedingt war. Diese Stoffwechselstörungen gingen nicht mit epileptischen Anfällen oder sonstigen klinisch-neurologischen Veränderungen einher. Bei den Patienten der Gruppe B, die nur mit einem Sauerstoffdruck von 1,5 Atmosphären belastet wurden, traten derartige Veränderungen des zerebralen Glukosestoffwechsels nicht auf. Eine praktisch ausgeglichene Bilanz des zerebralen Glukosestoffwechsels wurde bei inspiratorischen Sauerstoffdrucken von 1,0 und vor allem von 1,5 Atmosphären nachgewiesen und zeigte eine Besserung der Sauerstoffversorgung und Energiebildung des geschädigten Hirns an. Schließlich beobachteten wir nach dem Wechsel von Sauerstoff- auf Luftatmung einen erneuten Anstieg der zerebralen Glykose, was wiederum auf eine insuffiziente Sauerstoffversorgung des Hirns hinwies.