Possible pathogenic mechanism of propofol infusion syndrome involves coenzyme q.

BACKGROUND: Propofol is a short-acting intravenous anesthetic agent. In rare conditions, a life-threatening complication known as propofol infusion syndrome can occur. The pathophysiologic mechanism is still unknown. Some studies suggested that propofol acts as uncoupling agent, others suggested that it inhibits complex I or complex IV, or causes increased oxidation of cytochrome c and cytochrome aa3, or inhibits mitochondrial fatty acid metabolism. Although the exact site of interaction is not known, most hypotheses point to the direction of the mitochondria. METHODS: Eight rats were ventilated and sedated with propofol up to 20 h. Sequential biopsy specimens were taken from liver and skeletal muscle and used for determination of respiratory chain activities and propofol concentration. Activities were also measured in skeletal muscle from a patient who died of propofol infusion syndrome. RESULTS: In rats, authors detected a decrease in complex II+III activity starting at low tissue concentration of propofol (20 to 25 µM), further declining at higher concentrations. Before starting anesthesia, the complex II+III/citrate synthase activity ratio in liver was 0.46 (0.25) and in skeletal muscle 0.23 (0.05) (mean [SD]). After 20 h of anesthesia, the ratios declined to 0.17 (0.03) and 0.12 (0.02), respectively. When measured individually, the activities of complexes II and III remained normal. Skeletal muscle from one patient taken in the acute phase of propofol infusion syndrome also shows a selective decrease in complex II+III activity (z-score: -2.96). CONCLUSION: Propofol impedes the electron flow through the respiratory chain and coenzyme Q is the main site of interaction with propofol.

Valproic acid induces antioxidant effects in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a fatal, axonal demyelinating, neurometabolic disease. It results from the functional loss of a member of the peroxisomal ATP-binding cassette transporter subfamily D (ABCD1), which is involved in the metabolism of very long-chain fatty acids (VLCFA). Oxidative damage of proteins caused by excess of the hexacosanoic acid, the most prevalent VLCFA accumulating in X-ALD, is an early event in the neurodegenerative cascade. We demonstrate here that valproic acid (VPA), a widely used anti-epileptic drug with histone deacetylase inhibitor properties, induced the expression of the functionally overlapping ABCD2 peroxisomal transporter. VPA corrected the oxidative damage and decreased the levels of monounsaturated VLCFA (C26:1 n-9), but not saturated VLCFA. Overexpression of ABCD2 alone prevented oxidative lesions to proteins in a mouse model of X-ALD. A 6-month pilot trial of VPA in X-ALD patients resulted in reversion of the oxidative damage of proteins in peripheral blood mononuclear cells. Thus, we propose VPA as a promising novel therapeutic approach that warrants further clinical investigation in X-ALD.

Toxicity of peroxisomal C27-bile acid intermediates.

Peroxisomes play an important role in bile acid biosynthesis because the last steps of the synthesis pathway are performed by the beta-oxidation system located inside peroxisomes. As a consequence, C(27)-bile acid intermediates accumulate in several peroxisomal disorders. It has been suggested that C(27)-bile acids are especially toxic and contribute to the liver disease associated with peroxisomal disorders. For this reason, we investigated the toxicity of C(27)-bile acids and the underlying mechanisms. We studied the effects of conjugated and unconjugated C(27)-bile acids on cell viability, mitochondrial respiratory chain function and production of oxygen radicals in the rat hepatoma cell line McA-RH7777. Cell viability decreased progressively after incubation with increasing concentrations of different bile acids with dihydroxycholestanoic acid (DHCA) being clearly the most cytotoxic bile acid. In addition, the different bile acids caused a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate and glutamate. Finally, there was a dose-dependent stimulation of ROS generation in the presence of C(27)-bile acids. In conclusion, our studies showed that C(27)-bile acids are more cytotoxic than mature C(24)-bile acids. In addition, C(27)-bile acids are potent inhibitors of oxidative phosphorylation and enhance mitochondrial ROS production by inhibiting the respiratory chain.

A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis.

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders.

Characterization of the human omega-oxidation pathway for omega-hydroxy-very-long-chain fatty acids.

Very-long-chain fatty acids (VLCFAs) have long been known to be degraded exclusively in peroxisomes via beta-oxidation. A defect in peroxisomal beta-oxidation results in elevated levels of VLCFAs and is associated with the most frequent inherited disorder of the central nervous system white matter, X-linked adrenoleukodystrophy. Recently, we demonstrated that VLCFAs can also undergo omega-oxidation, which may provide an alternative route for the breakdown of VLCFAs. The omega-oxidation of VLCFA is initiated by CYP4F2 and CYP4F3B, which produce omega-hydroxy-VLCFAs. In this article, we characterized the enzymes involved in the formation of very-long-chain dicarboxylic acids from omega-hydroxy-VLCFAs. We demonstrate that very-long-chain dicarboxylic acids are produced via two independent pathways. The first is mediated by an as yet unidentified, microsomal NAD(+)-dependent alcohol dehydrogenase and fatty aldehyde dehydrogenase, which is encoded by the ALDH3A2 gene and is deficient in patients with Sjögren-Larsson syndrome. The second pathway involves the NADPH-dependent hydroxylation of omega-hydroxy-VLCFAs by CYP4F2, CYP4F3B, or CYP4F3A. Enzyme kinetic studies show that oxidation of omega-hydroxy-VLCFAs occurs predominantly via the NAD(+)-dependent route. Overall, our data demonstrate that in humans all enzymes are present for the complete conversion of VLCFAs to their corresponding very-long-chain dicarboxylic acids.

Characterization of the final step in the conversion of phytol into phytanic acid.

Phytol is a branched-chain fatty alcohol that is a naturally occurring precursor of phytanic acid, a fatty acid involved in the pathogenesis of Refsum disease. The conversion of phytol into phytanic acid is generally believed to take place via three enzymatic steps that involve 1) oxidation to its aldehyde, 2) further oxidation to phytenic acid, and 3) reduction of the double bond at the 2,3 position, yielding phytanic acid. Our recent investigations of this mechanism have elucidated the enzymatic steps leading to phytenic acid production, but the final step of the pathway has not been investigated so far. In this study, we describe the characterization of phytenic acid reduction in rat liver. NADPH-dependent conversion of phytenic acid into phytanic acid was detected, although at a slow rate. However, it was shown that phytenic acid can be activated to its CoA ester and that reduction of phytenoyl-CoA is much more efficient than that of phytenic acid. Furthermore, in rat hepatocytes cultured in the presence of phytol, phytenoyl-CoA could be detected, showing that it is a bona fide intermediate of phytol degradation. Subcellular fractionation experiments revealed that phytenoyl-CoA reductase activity is present in peroxisomes and mitochondria. With these findings, we have accomplished the full elucidation of the mechanism by which phytol is converted into phytanic acid.

Identification of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid.

Phytol is a branched chain fatty alcohol, which is abundantly present in nature as part of the chlorophyll molecule. In its free form, phytol is metabolized to phytanic acid, which accumulates in patients suffering from a variety of peroxisomal disorders, including Refsum disease. The breakdown of phytol to phytanic acid takes place in three steps, in which first, the alcohol is converted to the aldehyde, second the aldehyde is converted to phytenic acid, and finally the double bond is reduced to yield phytanic acid. By culturing fibroblasts in the presence of phytol, increases in the levels of phytenic and phytanic acid were detected. Interestingly, fibroblasts derived from patients affected by Sjögren Larsson syndrome (SLS), known to be deficient in microsomal fatty aldehyde dehydrogenase (FALDH) were found to be deficient in this. In addition, fibroblast homogenates of these patients, incubated with phytol in the presence of NAD+ did not produce any phytenic acid. This indicates that FALDH is involved in the breakdown of phytol.

Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of long-chain dicarboxylic acids.

Dicarboxylic acids (DCAs) are omega-oxidation products of monocarboxylic acids. After activation by a dicarboxylyl-CoA synthetase, the dicarboxylyl-CoA esters are shortened via beta-oxidation. Although it has been studied extensively where this beta-oxidation process takes place, the intracellular site of DCA oxidation has remained controversial. Making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this paper that peroxisomes, and not mitochondria, are involved in the beta-oxidation of C16DCA. Additional studies in fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, together with direct enzyme measurements with human recombinant l-bifunctional protein (LBP) and DBP expressed in a fox2 deletion mutant of Saccharomyces cerevisiae, show that the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP, and sterol carrier protein X, possibly together with the classic 3-ketoacyl-CoA thiolase. This is the first indication of a specific function for LBP, which has remained elusive until now.

Studies on the metabolic fate of n-3 polyunsaturated fatty acids.

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.

A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency.

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.

Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.

Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.

An inherited metabolic disorder presenting as ethylene glycol intoxication in a young adult.

Despite the abundance of reports emerging in the literature on metabolic disorders, some disorders remain undiagnosed or misdiagnosed, not only in clinical pathology but also in forensic pathology. The authors report a patient who had recurrent episodes characterized by nausea, vomiting, and signs of dehydration necessitating admission to the hospital. At each admission, he was found to have lactic acidosis. On the first admission, glycolic acid was detected in his blood and he was diagnosed as having ethylene glycol intoxication. Only at the third admission, 2 years after the first, was the possibility of an underlying metabolic disorder considered. Laboratory investigations showed a deficiency of complex I in the mitochondrial oxidative phosphorylation. Possible medicolegal implications are discussed.