Metabolic studies in a patient with severe carnitine palmitoyltransferase type II deficiency.

Here we report on a patient with severe ("non-classic") carnitine palmitoyltransferase type II (CPT II) deficiency. Hypoglycemia prompted by an infectious episode and associated with non-ketotic dicarboxylic aciduria orientated diagnosis towards beta-oxidation deficiency disorders. Blood carnitine levels revealed a secondary carnitine deficiency that was responsive to oral L-carnitine supplementation. Blood acylcarnitine profiles were abnormal and included acetyl (C2:0), butyryl/isobutyryl (C4:0), isovaleryl/2-methylbutyryl (C5:0), hexanoyl (C6:0), myristoyl (C14:0), palmitoyl (C16:0), hexadecenoyl (C16:1), oleyl (C18:1) and stearoyl (C18:0) carnitine. In urine, excess excretion of dicarboxylylcarnitines, mainly dodecanedioylcarnitine, was noticed. Upon carnitine supplementation, C8 to C12 fatty acylcarnitines, with decanoylcarnitine as well as C10 to C14 dicarboxylylcarnitines being prominent, were observed in urine. Biochemical measurements disclosed a severe reduction of mitochondrial CPT II activity (7% of normal values). Correlations of metabolic findings in the patient and physiological roles of CPT II are briefly discussed.

Magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice: a nutritional model for discriminatory screening of anticonvulsant drugs and original assessment of neuroprotection properties.

A great many animal models for audiogenic seizures have been described. The extent to which these models may provide insight into neuroscience fields such as abnormal locomotor behavior (wild running), seizures and anticonvulsants, and neuroinsults and neuroprotectors is examined here by our study of magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice. MDDASs were induced in all of the eight tested adult murine strains and are presented as a sequence of four successive components (latency, wild running, convulsion, and recovery phase periods). Compared with several classic seizure tests, the nutritional MDDAS model responded to low doses of prototype antiepileptic drugs (AEDs), including phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), valproic acid (VPA), ethosuximide (ESM), and diazepam (DZP). Modulation by AEDs of the four components of MDDAS indicated that this seizure test was discriminatory, distinguishing between phenytoinergic (PHT, CBZ), GABAergic (PB, VPA, DZP), and ethosuximide (ESM) compounds. Suitability of the MDDAS test for evaluation of neuroprotective compounds was also examined: it showed partial (melatonin) and complete (WEB2170, an anti-PAF agent) reduction of recovery phase by non-anticonvulsant doses of test compounds. These neuroprotective responses were compared with neuroprotective potentials determined in a model of neonatal cerebral injury induced by focal injection of ibotenate (a glutamate analog). WEB2170 and melatonin reduced the size of lesions in white matter, but only WEB2170 protected cortical plate against ibotenate-induced lesions. In addition to the original neuroprotective behavior of WEB2170, studies on the neuroprotectors also supported GABAergic anticonvulsant activity of melatonin in the MDDAS test.

Peroxisome-proliferating effects of fenoprofen in mice.

We report on hepatic effects obtained in vivo by treating mice with different doses of fenoprofen, an arylpropionic acid previously shown to inhibit in vitro peroxisomal very long chain fatty acid oxidation. A strong and dose-related induction of peroxisomal palmitoyl-CoA oxidase, and of carnitine acyltransferase and acyl-CoA hydrolase activities was recorded in liver homogenates of mice fed diets supplemented with different contents [0.01, 0.05, 0.1, or 1% (w/w)] of fenoprofen for 6 d. Peroxisomal glycolate oxidase and mitochondrial butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA dehydrogenases were unaffected or increased. Hepatic catalase activity was significantly increased in mice fed the diet with 0.05 and 0.1% fenoprofen but, surprisingly, was not stimulated in mice fed the 1% fenoprofen-containing diet. A time-related but unequal induction of acyl-CoA oxidases and catalase was observed with the 0.1% fenoprofen diet: at 21 d of treatment, the induction of lignoceroyl-CoA and palmitoyl-CoA oxidase activities were five-fold stronger than that of catalase activity. In mice treated with 1% fenoprofen for up to 6 d, only acyl-CoA oxidase activities were found to be significantly increased. Morphometric analysis of the liver peroxisomes in mice treated with 0.1% fenoprofen evidenced an increase in size, volume density, and surface density along with a reduced ratio between perimeter and area of the peroxisomal profiles. No morphological marker for very long chain fatty acid deposition could be detected in livers from fenoprofen-treated animals. Our findings clearly demonstrate that fenoprofen acts as a peroxisome proliferator in the liver of mice and do not support the occurrence of in vivo reduction of very long chain fatty acid oxidation in liver from treated animals.

Effect of vitamin E on antioxidant enzymes, lipid peroxidation products and glomerulosclerosis in the rat remnant kidney.

In rats with five-sixth nephrectomy (remnant kidney), glomerulosclerosis was significantly reduced by dietary administration of vitamin E (alpha-tocopherol) during 11 and 16 weeks after reduction of nephron number. The activity of catalase and the production of H2O2 in remnant kidney cortex homogenate were not influenced by the vitamin E diet; however, the activities of glutathione peroxidase and superoxide dismutase were significantly increased (up to 140 and 180%, respectively, after 16 weeks). Lipid peroxidation, evaluated by malonaldehyde and 4-hydroxynonenal concentrations, was decreased in cortex homogenates and in urine. Though the extent of the effect of vitamin E on antioxidant enzyme levels and lipid peroxidation is small, the important reduction of glomerulosclerosis is in favor of dietary supplementation with vitamin E.

Metabolic studies in twin brothers with 2-methylacetoacetyl-CoA thiolase deficiency.

We report clinical and biological investigations in two patients (twin brothers) with 2-methylacetoacetyl-CoA thiolase deficiency. Main clinical features included important staturo-ponderal delay, frequent infectious rhinopharyngitis episodes and an acute metabolic acidosis at the age of 4 years, this metabolic decompensation being adequately halted by bicarbonate supplementation. Since that age, patients developed rather favorably, however, with persistence of the staturo-ponderal delay. Organicaciduria typical of 2-methylacetoacetyl-CoA thiolase deficiency was recorded consisting of excessive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-methylglutaconate, adipate and 2-methylacetoacetate. Blood carnitine levels were altered in patients with increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Determination of acylcarnitine profiles showed that patients excreted excessive amounts of several acylcarnitines in urine including propionyl, butyryl, isobutyryl, isovaleryl, 2-methylbutyryl and tiglyl-carnitine, the latter acylcarnitine being prominent with, in one of the patients, occurrence of a previously undescribed isomer of this carnitine ester, possibly 2-ethylacrylyl-carnitine. Excretion of these acylcarnitines in urine was increased in response to L-carnitine although, as a whole, this therapy resulted in a less important stimulation of esterified carnitine removal in urine from patients than in the case of supplemented controls. Biochemical investigations on cultured skin fibroblasts confirmed 2-methylacetoacetyl-CoA thiolase deficiency. Through the present report on this rare disease in two siblings, we would like to underline that acylcarnitines can be used in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency, a view supported by acylcarnitine profiles further determined in another patient with proven oxothiolase deficiency, adding this pathology to the list of beta-oxidation disorders that may be screened successfully through determination of acylcarnitine profiles in body fluids.

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

Peroxisomes in mice fed a diet supplemented with low doses of fish oil.

The influence of low dietary doses (0.1 and 0.8% w/w) of a commercial fish oil preparation on peroxisomes in normal mice was studied and compared to the known strong inductive effects of high (10%) fish oil diets. Low fish oil doses were chosen to supply the mice with a concentration of docosahexaenoic acid, which was beneficial to patients with a peroxisomal disease. Peroxisomes were evaluated by cytochemical, morphometric, and enzymological techniques. The 0.1% fish oil diet had no effect on peroxisomes in liver, heart, and kidney even after prolonged treatment. The 0.8% diet did not change the peroxisomal number nor the catalase (EC 1.11.1.6) activity in the liver. Hepatic peroxisomal beta-oxidation, however, was increased by 50% after 14 d. This was accompanied by reduced peroxisomal size. The 0.8% diet also caused a small increase (+25%) in myocardial catalase activity. No effect was observed in kidneys. Our results indicate that in mice a low (< 0.8%) dietary fish oil dose has no or only a slight effect on hepatic peroxisomal beta-oxidation. This may be of particular interest to patients with a peroxisomal fatty acid beta-oxidation defect and who display a severe deficiency of docosahexaenoic acid--diets supplemented with low fish oil doses will improve the docosahexaenoic acid level without adding a strong load to the disturbed fatty acid metabolism.

Comparative anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6-dimethylphenyl)phthalimide and prototype antiepileptic drugs in mice and rats.

We compared the anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6-dimethylphenyl)phthalimide (ADD 213063) with those of phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), ethosuximide (ESM), valproate (VPA), and felbamate (FBM). Evaluation of anticonvulsant properties performed according to well-established procedures in rats and mice showed that ADD 213063 is most effective in protecting animals against maximal electroshock seizures (MES). This anti-MES activity is achieved with nontoxic doses, with the optimal effect recorded in rats dosed orally with anti-MES ED50 and protective index (PI) values of 25.2 mumol/kg and > 75, respectively. ADD 213063 protects to a lesser extent against seizures induced by subcutaneous (s.c.) picrotoxin and subcutaneous pentylenetetrazol (PTZ) in mice dosed intraperitoneally and orally, respectively. The profile of anticonvulsant action of ADD 213063 closely parallels that of CBZ.

Stroke, hemiparesis and deficient mitochondrial beta-oxidation.

We describe on a 3-year-old child referred for evaluation and therapy of a cerebral vascular accident with residual hemiplegia and partial epilepsy. Metabolic investigations initially showed normal urinary organic acids as well as normal blood and urinary amino acids. Blood carnitine fractions had been pathological and a secondary carnitine deficiency was diagnosed and treated by oral L-carnitine supplementation. During carnitine treatment, abnormal urinary acylcarnitine profiles were noticed with excessive amounts of several carnitine esters including propionylcarnitine, butyryl- and/or isobutyryl-carnitine, isovaleryl- and/or 2-methylbutyryl-carnitine, hexanoylcarnitine and octanoylcarnitine. Subsequently, an urinary organic acid profile suggestive of glutaric aciduria type II was recorded during a clinical decompensation crisis. Morphological and biochemical studies on skeletal muscle and skin fibroblasts were performed and confirmed the existence of a defect of the mitochondrial beta-oxidation pathways with lipidic myopathy, reduced palmitate and octanoate oxidation rates in cultured fibroblasts. Glutaric aciduria type II increases the list of metabolic disorders characterized by hemiplegia and other sequelae of brain ischaemia such as stroke-like episode, seizures, aphasia, ataxia and myoclonia, similar to those seen in MELAS.

Peroxisomes in liver, heart, and kidney of mice fed a commercial fish oil preparation: original data and review on peroxisomal changes induced by high-fat diets.

Male NMRI mice were fed a diet with 10% w/w Beromegan for up to three weeks. Beromegan is a commercial fish (salmon) oil preparation rich in eicosapentaenoic acid and docosahexaenoic acid. Peroxisomal beta-oxidation capacity, catalase activity, and ultrastructural morphometry of the hepatic peroxisomes were investigated. In myocardium and kidney, catalase activity, peroxisomal staining after catalase cytochemistry, peroxisomal morphology, and morphometry (in myocardium) were evaluated. In liver, we found a significant increase in peroxisomal beta-oxidation, catalase activity, and peroxisomal number already after 3 days of dietary treatment. These changes were more pronounced after 3 weeks. Peroxisomal size was not changed. Positive correlations were found between peroxisomal enzyme activities and the number but not the size of the peroxisomes, and between catalase activity and beta-oxidation capacity. The mean peroxisomal diameter per animal was inversely proportional to catalase activity measured in homogenate. In myocardium, catalase activity was increased with duration of fish oil feeding. Peroxisomal staining, number, and size were also increased when compared to controls. In kidney, no alterations were observed. Our results indicate a beneficial effect of a diet supplemented with fish oil on the peroxisomal metabolism in liver and myocardium; it differs from the changes induced by xenobiotic peroxisome proliferation.

Effect of various n-3/n-6 fatty acid ratio contents of high fat diets on rat liver and heart peroxisomal and mitochondrial beta-oxidation.

The present work extends tissue investigations previously performed in rat gastric mucosa on lipid metabolism alterations caused by n-3 and n-6 fatty acid-enriched diets. Liver and heart tissues are here studied and demonstrated to undergo, upon exposure to high fat diets with various n-3/n-6 fatty acid ratio contents, biochemical and morphological changes which may be enumerated as follows: (1) Rat liver peroxisomal prostaglandin E2, fatty acid but not bile acid beta-oxidation rates are enhanced, especially upon the diet with the higher n-3/n-6 fatty acid ratio. Mitochondrial beta-oxidation rates are little or not affected by the high fat diets. (2) Rat liver carnitine acyltransferases are stimulated by the high fat diets, the more rich the n-3 fatty acid content, the more pronounced the stimulatory effect. (3) Rat heart peroxisomal and mitochondrial beta-oxidation rates were increased in animals receiving the n-3 fatty acid-enriched diet. At a low n-3/n-6 fatty acid ratio content of the diet, these oxidizing rate values were in control range. The carnitine acyltransferase activities were increased in rat heart to different extents, depending on the n-3/n-6 fatty acid ratio content of the diet. (4) Ultrastructural examination and morphometric determinations on hepatocytes from rats receiving the diets with the lowest and the highest n-3/n-6 fatty acid ratio contents disclose that in the latter case the numbers and fractional volumes of peroxisomes and mitochondria are significantly higher than in the former case.

Effects of dietary n-6/n-3 ratios on lipid and prostaglandin E2 metabolism in rat gastric mucosa.

The effects of increased dietary n-3 polyunsaturated fatty acids on gastric mucosal lipid metabolism were studied in rats fed for 8 weeks with different combinations of fish and corn oils. Lipid composition, ex vivo prostaglandin E2 (PGE2) production and enzymatic activities involved in phospholipid metabolism and peroxisomal oxidative catabolism of fatty acids and PGE2 were examined. With dietary n-6/n-3 compositional ratios ranging between 75 and 3.3 it was observed that: (i) the arachidonic acid-to-eicosapentaenoic acid ratio (AA/EPA) fell from infinity to 3.1 and 5.1 in phosphatidylcholines (PC) and phosphatidylethanolamines (PE), respectively; (ii) ex vivo production of PGE2 was lowered by a factor of about 2; and (iii) gastric phospholipase A2 activity was enhanced by 32%. With dietary n-6/n-3 ratio lower than 3.3, stimulation of PGE2-CoA oxidase activity was observed whilst the PGE2 level remained constant. These data suggest that the fish oil-induced decrease in ex vivo PGE2 production is more closely related to a decrease in the membrane AA level than to an enhanced oxidative catabolism of PGE2.

Effects of dietary corn oil and salmon oil on the oxidation of fatty acids and prostaglandin E2 in rat gastric mucosa.

The investigations previously carried out by Grataroli and colleagues (1) to elucidate the relationships between dietary fatty acids, lipid composition, prostaglandin E2 production and phospholipase A2 activity in the rat gastric mucosa are, here, extended. In the present investigations, fatty acid and prostaglandin E2 catabolizing enzymes were assayed in gastric mucosa from rats fed either a low fat diet (corn oil: 4.4% w/w) (referred as control group), a corn oil-enriched diet (17%) or a salmon oil-enriched diet (12.5%) supplemented with corn oil (4.5%) (referred as groups of treated animals) for eight weeks. Peroxisomal fatty acyl-CoA beta-oxidation was induced in the treated animals whereas the activities of catalase and mitochondrial tyramine oxidase were increased and normal, respectively. Mitochondrial acyl-CoA dehydrogenations occurred at higher rates and carnitine acyltransferase activities were enhanced. In addition, the induction of peroxisomal but not mitochondrial prostaglandoyl-E2-CoA beta-oxidation could be demonstrated. Induction of peroxisomal oxidation of fatty acids and prostaglandins is suggested to contribute to the decrease of prostaglandin E2 production in the treated animals, especially those receiving the salmon oil diet, that the above mentioned authors originally reported.

Peroxisomal proliferation in heart and liver of mice receiving chlorpromazine, ethyl 2(5(4-chlorophenyl)pentyl) oxiran-2-carboxylic acid or high fat diet: a biochemical and morphometrical comparative study.

Chlorpromazine and related drugs including trifluoperazine, clopenthixol, and fluphenazine are in vitro inhibitors of mitochondrial carnitine palmitoyltransferase and cytochrome c oxidase and of peroxisomal carnitine octanoyltransferase from mouse heart and liver. By contrast with 0.1% ethyl 2(5(4-chlorophenyl)pentyl) oxiran-2-carboxylic acid or 0.1% clofibrate-containing diets, the treatment of mice with 0.1% chlorpromazine-containing diet fails to induce peroxisomal proliferation in liver and heart. An 0.5% chlorpromazine-containing diet did induce peroxisomal proliferation. Inhibition of peroxisomal beta-oxidation presumably via the reduction of carnitine octanoyltransferase by chlorpromazine elicits the appearance in liver of lamellar structures resembling those seen in human peroxisomal disorders and induces accumulation of very long-chain fatty acids in plasma. The peroxisomal proliferation induced by administration of high dose chlorpromazine is ascribed to its ability to depress mitochondrial fatty acid oxidation by impairing cytochrome c oxidase and carnitine palmitoyltransferase activities.