Monique Fontaine

Comparison of seizure reduction and serum fatty acid levels after receiving the ketogenic and modified Atkins diet

The ketogenic diet (KD) and the modified Atkins diet are effective therapies for intractable epilepsy. We compared retrospectively the KD and modified Atkins diet in 27 children and also assessed serum long chain fatty acid profiles. After 3 months, using an intent-to-treat analysis, the KD was more successful, with >50% seizure reduction in 11/17 (65%) vs. 2/10 (20%) with the modified Atkins diet, p=0.03. After 6 months, however, the difference was no longer significant: 7/17 (41%) vs. 2/10 (20%) (p=0.24). We observed a preventive effect of both diets on the occurrence of status epilepticus. After 1 and 3 months of either diet, responders experienced a significant decrease in serum arachidonic acid concentration compared to non-responders. The KD and modified Atkins diet led to seizure reduction in this small pilot series, with slightly better results after 3 months with the KD, but not after 6 months. The decrease of serum arachidonic acid levels might be involved in the anticonvulsive effects of KD or modified Atkins diet.

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

Purification of complement components by hydrophobic affinity chromatography on phenyl—Sepharose: Purification of human C5

Human complement components C5 and C3 were purified with 41% and 20% yields, respectively, by euglobulin precipitation, DEAE—Sephacel ion‐exchange chromatography and gel filtration. Phenyl—Sepharose chromatography allowed the complete separation of C3 and C5. C3 bound loosely on the resin whereas C5 bound firmly and was eluted with 50% glycerin solution. Gel filtration on Sephacryl S‐300 allowed the depletion of C4bp and H that contaminated C5 preparations. Homogeneity of C5 and C3 preparations was demonstrated by SDS—PAGE and immunochemical analysis. C5 and C3 consisted of two chains (α, 110000; β, 75000) linked by disulfide bridges.

A hemolytic assay for clinical investigation of human C2.

The heat lability of early acting components of human complement was studied in detail. Kinetic disappearance of individual components was monitored by hemolytic assay. C2 and factor B were the most heat labile components. We took advantage of the difference in heat stability between C2 and C1 to develop a hemolytic assay for human C2.

Stroke, hemiparesis and deficient mitochondrial β-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 octanoyl-carnitine. Subsequently, an urinary organic acid profile suggestive of glutaric aciduria type 11 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 β-oxidadation pathways with lipidic myopathy, reduced palmitate and octanoate oxidation rates in cultured fibroblasts. Glutaric aciduria type 11 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.

Mitochondrial dysfunction and lipid homeostasis.

This review is aimed at illustrating that mitochondrial dysfunction and altered lipid homeostasis may concur in a variety of pathogenesis states, being either contributive or consecutive to primary disease events. Underlying mechanisms for this concurrence are far from being the exhaustive elements taking place in disease development. They may however complicate, contribute or cause the disease. In the first part of the review, physiological roles of mitochondria in coordinating lipid metabolism and in controlling reactive oxygen species (ROS), ATP and calcium levels are briefly presented. In a second part, clues for how mitochondria-driven alterations in lipid metabolism may induce toxicity are discussed. In the third part, it is illustrated how mitochondrial dysfunction and lipid homeostasis disruption may be associated (i) to complicate type 1 diabetes (pancreatic β-cell mitochondrial dysfunction in ATP yield induces reduced insulin secretion and hence disruption of glucose and lipid metabolism), (ii) to contribute to type 2 diabetes and other insulin resistant states (mitochondrial impairment may induce adipocyte dysfunction with subsequent increase in circulating free fatty acids and their abnormal deposit in non adipose tissues (pancreatic β-cells, skeletal muscle and liver) which results in lipotoxicity and mitochondrial dysfunction), (iii) to offer new clues in our understanding of how the brain controls feeding supply and energy expenditure, (iv) to promote cancer development notably via fatty acid oxidation/synthesis imbalance (in favor of synthesis) further strengthened in some cancers by a lipogenetic benefit induced by a HER2/fatty acid synthase cross-talk, and (v) to favor cardiovascular disorders by impacting heart function and arterial wall integrity.

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.

CoA esters of valproic acid and related metabolites are oxidized in peroxisomes through a pathway distinct from peroxisomal fatty and bile acyl-CoA β-oxidation

In rat liver homogenates fortified with the appropriate cofactors (ATP and CoA), valproic acid induced H2O2 production rates by far lower than those recorded on the straight medium‐chain fatty acid n‐octanoic acid. Using directly the CoA esters of these carboxylic acids as substrates for the rat liver H2O2‐generating enzyme activities, valproyl‐CoA, and n‐octanoyl‐CoA were found to induce similar oxidation rates. In the rat liver homogenates, cyanide‐insensitive valproyl‐CoA and octanoyl‐CoA oxidations occurred at rates similar to those of valproyl‐CoA and octanoyl‐CoA oxidase(s), respectively. Studies on fractions obtained from rat liver postnuclear supernatants by isopycnic centrifugation on a linear sucrose density gradient disclose that the density distribution of valproyl‐CoA oxidase superimposes to those of catalase, fatty acyl‐CoA oxidase and cyanide‐insensitive fatty acyl‐CoA oxidation, three peroxisomal marker activities. By contrast, the cyanide‐insensitive valproyl‐CoA oxidation does not adopt the typical peroxisomal distribution of these activities but rather exhibits a mitochondrial localization with, however, a minor peroxisomal component. Interestingly enough, the comparative study of rat tissue distribution, inducibility by clofibrate and sensitivity to deoxycholate indicated that valproyl‐CoA oxidase is an enzyme distinct from fatty acyl‐CoA oxidase and bile acyl‐CoA oxidase. Taken as a whole, the results presented here support the occurrence of a peroxisomal oxidation of the CoA ester of valproic acid and its Δ4‐enoic derivate which might be characterized by two major features: initiation by an acyl‐CoA oxidase distinct from fatty and bile acyl‐CoA oxidases, and inability to complete the β‐oxidation cycle which would not proceed, at significant rates, further than the β‐hydroxyacyl‐CoA dehydrogenation step in peroxisomes.

Deuterated palmitate-driven acylcarnitine formation by whole-blood samples for a rapid diagnostic exploration of mitochondrial fatty acid oxidation disorders

BACKGROUND The biochemical diagnosis of mitochondrial fatty acid oxidation defects (FAOD) currently rests on enzyme assays. A dynamic ex vivo exploration consisting of incubations of whole-blood samples with stable-labeled palmitate and determining leukocyte capacities to produce deuterated acylcarnitines was developed on healthy controls (n=52) and patients with very-long- (VLCADD) (n=2), medium- (MCADD) (n=6), or short- (SCADD) (n=1) chain acyl-CoA dehydrogenase deficiencies. METHODS Incubations were optimized with L-carnitine and [16-(2)H(3), 15-(2)H(2)]-palmitate at 37 degrees C for various time periods on MCADD and control whole-blood samples. Labeled acylcarnitines were quantified by electrospray-ionization tandem mass spectrometry after thawing, extraction and derivatization to their butyl esters and the method was applied to patients with defects mentioned above. RESULTS The production of acylcarnitines was linear until 6 h of incubation and optimal on 50 to 200 nmol deuterated substrate. A good discrimination between MCADD patient and control data was found, with median C8/C4 acylcarnitine production rate ratios of 81.0 (5th-95th percentile range: 16.6-209.9) and 0.21 (5th-95th percentile range: 0.06-0.79), respectively. The method also discriminated from controls the VLCADD and SCADD patients. Preliminary studies on a healthy control indicated that the storage at 4 degrees C does little or not alter capacities of whole-blood samples to generate labeled acylcarnitines over a period of 48 h. CONCLUSION The rapid management afforded by the method, its abilities to characterize patients and to work on whole-blood samples after a stay of 24-48 h at 4 degrees C make it promising for the diagnostic exploration of FAOD.

Structure-function relations in the third component of human complement (C3)—I. Hydrophobic sites

Charge shift electrophoresis and crossed hydrophobic interaction immuno-electrophoresis were used to demonstrate the presence of hydrophobic sites in the human C3 molecule. C3b and C3d were true amphiphilic proteins that could bind to hydrophobic surfaces. To the contrary, native C3, that presented the characteristics of amphiphilic proteins upon charge shift electrophoresis, did not bind to hydrophobic surfaces. These results suggested that the hydrophobic sites were located in the internal part of the C3 molecule and they were exposed in the external part when C3 was activated. The action of chaotropes on C3 was studied in detail and showed that the hydrophobic sites protected the thiolester bond (present in the labile site) from hydrolysis by water and thereby preserved the biological properties of native C3.