Animesh Chandra

Metabolism of the Lipid Peroxidation Product, 4-Hydroxy-trans-2-nonenal, in Isolated Perfused Rat Heart*

The metabolism of 4-hydroxy-trans-2-nonenal (HNE), an α,β-unsaturated aldehyde generated during lipid peroxidation, was studied in isolated perfused rat hearts. High performance liquid chromatography separation of radioactive metabolites recovered from [3H]HNE-treated hearts revealed four major peaks. Based on the retention times of synthesized standards, peak I, which accounted for 20% radioactivity administered to the heart, was identified to be due to glutathione conjugates of HNE. Peaks II and III, containing 2 and 37% radioactivity, were assigned to 1,4-dihydroxy-2-nonene (DHN) and 4-hydroxy-2-nonenoic acid, respectively. Peak IV was due to unmetabolized HNE. The electrospray ionization mass spectrum of peak I revealed two prominent metabolites with m/z values corresponding to [M + H]+ of HNE and DHN conjugates with glutathione. The presence of 4-hydroxy-2-nonenoic acid in peak III was substantiated using gas chromatography-chemical ionization mass spectroscopy. When exposed to sorbinil, an inhibitor of aldose reductase, no GS-DHN was recovered in the coronary effluent, and treatment with cyanamide, an inhibitor of aldehyde dehydrogenase, attenuated 4-hydroxy-2-nonenoic acid formation. These results show that the major metabolic transformations of HNE in rat heart involve conjugation with glutathione and oxidation to 4-hydroxy-2-nonenoic acid. Further metabolism of the GS-HNE conjugate involves aldose reductase-mediated reduction, a reaction catalyzed in vitro by homogenous cardiac aldose reductase.

A SYNTHESIS OF 4-HYDROXY-2-TRANS-NONENAL AND 4-(3H) 4-HYDROXY-2-TRANS-NONENAL

Abstract4-Hydroxy-2-trans-nonenal, the most abundant and toxic unsaturated aldehyde generated during membrane lipid peroxidation, was synthesized starting from fumaraldehyde dimethyl acetal. In the first step of the synthesis, the fumaraldehyde dimethyl acetal was partially hydrolyzed using amberlyst catalyst to obtain the monoacetal. The 4-hydroxy-2-trans-nonenal was synthesized by the Grignard reaction of the fumaraldehyde monoacetal with 1-bromopentane. 4-Hydroxy-2-trans-nonenal, obtained as its dimethylacetal, was oxidized to its corresponding 4-keto derivative using pyridinium chlorochromate buffered with sodium acetate as the oxidizing agent. 4-(3H) 4-Hydroxy-2-trans-nonenal was obtained in one step by the sodium borotriteride reduction of the 4-keto derivative.

Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Active site modification of aldose reductase by nitric oxide donors

Nitric oxide (NO) donors sodium nitrosoprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonemine (SIN-1) caused a time- and concentration-dependent loss of catalytic activity of recombinant human placental aldose reductase. Modification of the enzyme was prevented by NADPH and NADP and reversed partially by dithiothreitol (DTT) and sodium borohydride. The protection by NADPH was lost in the presence of both substrates (NADPH and glyceraldehyde), indicating that the enzyme becomes sensitive to inhibition by SNP during catalysis. Site-directed mutant form of the enzyme, in which active site cys-298 was substituted with serine (C298S) was not inactivated by NO donors, whereas, ARC80S and ARC303 were as sensitive as the wild type enzyme, indicating that inactivation of aldose reductase is due to modification of the active site at cys298. These results suggest that NO may be an endogenous regulator of aldose reductase, and consequently the polyol pathway of glucose metabolism; which has been implicated in the pathogenesis of secondary diabetic complications.

Attenuation of galactose cataract by low levels of dietary curcumin

Abstract Our previous studies have shown that curcumin, a dietary antioxidant present in turmeric, attenuates 4-hydroxynonenal induced lens opacification in organ culture (Awasthi et al , Amer. J. Clin. Nutr. 1996;64:761–766). Oxidative stress has been implicated in the mechanism of galactosemic cataract formation. Therefore, the present studies were designed to examine whether dietary curcumin can attenuate galactosemic cataract in rats in-vivo . 8 week old Sprague-Dawley rats were randomized into four groups and fed with either AIN-76 diet or that containing 30% galactose with or without curcumin 0.0025% (ww) in the diet. Progression of cataract formation was monitored by slit lamp biomicroscopy at days 1, 22 and 29. The eyes were removed out at day 29 and the lenses were dissected out. Images of the isolated lenses were acquired using a digital imager. The lens epithelium was dissected and lens epithelial cells were examined for apoptosis. Lenses of the animals fed a diet containing neither galactose nor curcumin and those fed only a curcumin containing diet remained transparent. The transparency of lenses from rats without or with curcumin was identical as assessed by measuring the average intensity of transmitted light (AITL) (241 ± 4, n=12). Lenses from galactose fed animals without curcumin were partially opacified with an AITL value of 77 ± 9 % of the controls, (n=10, p

Cardiac Metabolism of Enals

Recent evidence suggests that in addition to their well studied toxic effects, reactive oxygen species (ROS) are also essential mediators of cell growth, differentiation and apoptosis (Sen and Packer, 1996; Lander, 1997). Nonetheless, the mechanisms by which ROS alter cell function remain obscure. Since free radicals derived from oxygen are generally short-lived and react predominantly at their site of generation, it is likely that their metabolic as well as the toxic effects are mediated, in part, by their metastable products, particularly those derived from the peroxidation of membrane lipids. Peroxidation of unsaturated fatty acids generates a variety of metastable compounds of which aldehydes are the most abundant end-products (Esterbauer et al., 1991; Witz, 1983). In comparison only minor amounts of ketones, epoxides, hydrocarbons, alcohols and acids are formed (Grosch, 1987). Therefore, in cellular systems, aldehyde burden may be an important consequence of lipid peroxidation.

REGULATION OF ALDOSE REDUCTASE BY ALDEHYDES AND NITRIC OXIDE

Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. It was initially discovered in the seminal vesicles, where it is believed to catalyze the NADPH-mediated reduction of glucose to sorbitol-which in turn is metabolized to fructose required for spermatogenesis (for review see Bhatnagar and Srivastava, 1992). The conversion of glucose to fructose via sorbitol, or the so called polyol pathway was later identified in several other tissues and AR was found to be universally expressed in most tissues examined and in species ranging from yeast to man. Based on the observations that high concentration of polyols accumulate during sugar cataractogenesis, it was suggested that during hyperglyce- mia, the flux of glucose via the polyol pathway is increased and that due to the low permeability of the membrane to polyols, sugar alcohols accumulate in diabetic tissues leading to marked hydration, cell swelling and lysis (Kinoshita and Nishimura, 1988).

Structure-Function Studies of FR-1

The aldo-keto reductase (AKR) gene superfamily represents a collection of proteins expressed in a wide variety of plants, animals, yeast, and procaryotic organisms. Most AKRs were originally identified as enzymes capable of catalyzing the NADPH-dependent reduction of carbonyl groups contained in a broad range of substrates (Bachur, 1976). However, recent genetic studies mediated by genome and expression sequencing approaches have identified several new members of the AKR superfamily. Many of these new proteins are characterized by high sequence homology to AKR enzymes although little or no information is available about their potential catalytic activities. One such new protein, designated FR-1* was identified as the product of a gene upregulated in serum-starved mouse fibroblasts following treatment with fibroblast growth factor I (FGF-I) (Donohue et al., 1994). High amino acid sequence identity (~70%) was observed between FR-1 and aldose reductase as well as other AKRs. Many amino acid residues known to contribute to the catalytic mechanism in other AKR enzymes including aldose reductase (AKRlB I), aldehyde reductase (AKRIAI) and 3a.-hydroxysteroid dehydrogenase (AKR1C9) are conserved in FR-l. These residues include Tyr-48, His-ll 0, Lys-77 and Asp-43 (numbering is that of aldose reductase) (Barski et al. , 1995; Pawlowski & Penning, 1994; Schlegel et al., 1998; Tarle et al. , 1993). The present study was undertaken to evaluate whether FR -1 is a catalyst of carbonyl reduction and to measure the affinity of FR-1 for various ligands such as nucleotide cofactors, carbonyl substrates and aldose reductase inhibitors. Our studies show that FR-1 catalyzes the NADPH-dependent reduction of substrates representative of diverse structural classes of aliphatic and aromatic aldehydes. Both saturated and unsaturated aldehydes were excellent substrates. Unlike aldose reductase and aldehyde reductase, FR-1 catalyzed the reduction of simple ketones such as acetone and butanone; however virtually no catalytic activity could be detected using steroid and aldose substrates. FR-1 was inhibited by various aldose reductase inhibitors in a manner similar to human aldose reductase. Besides being an excellent substrate, 4-hydroxy-2-nonenal (HNE) inactivated the enzyme through a mechanism involving Michael addition to Cys-298.