Vitamin E and probucol reduce urinary lipophilic aldehydes and renal enlargement in streptozotocin-induced diabetic rats.

Diabetes mellitus is characterized by complications affecting several organs, including the kidney. Lipid peroxidation increases in diabetes and has been implicated in the pathogenesis of diabetic complications. In this study, we examined the ability of two antioxidants, vitamin E and probucol, to reduce lipid peroxidation in vivo and renal hypertrophy, an early stage of diabetic nephropathy, in rats. Animals were divided into four groups: non-diabetic, diabetic, diabetic treated with vitamin E, and diabetic treated with probucol. Animals were given antioxidants by intraperitoneal injection after induction of diabetes by streptozotocin injection. After 7 wk, lipid peroxidation in vivo was measured by analyzing urinary excretion of lipophilic aldehydes and related carbonyl compounds (LACC) as 2,4-dinitrophenylhydrazones by high-performance liquid chromatography. A number of urinary lipophilic nonpolar and polar aldehydes and related carbonyl compounds were identified, almost all of which increased in diabetes. Antioxidant treatment resulted in significantly decreased excretion of urinary LACC excretion. Antioxidant treatment of diabetic rats reduced renal hypertrophy. There was a high correlation between kidney weight and urinary LACC. Since LACC are accepted markers of lipid peroxidation, these results indicate that antioxidants can reduce the elevated lipid peroxidation of diabetes and may slow the onset of diabetic nephropathy.

Urinary aldehydes as indicators of lipid peroxidation in vivo.

The excretion of malondialdehyde (MDA), lipophilic aldehydes and related carbonyl compounds in rat and human urine was investigated. MDA was found to be excreted mainly in the form of two adducts with lysine, indicating that its predominant reaction in vivo is with the lysine residues of proteins. Adducts with the phospholipid bases serine and ethanolamine and the nucleic acid bases guanine and deoxyguanosine also were found. Except for the adduct with deoxyguanosine (dG-MDA), the excretion of these compounds increased with peroxidative stress imposed in the form of vitamin E deficiency or the administration of iron or carbon tetrachloride. Marked differences in the concentration of dG-MDA in different tissues were correlated with their content of fatty acids having three or more double bonds, the putative source of MDA. Fourteen nonpolar and eleven polar lipophilic aldehydes and other carbonyl compounds were identified as their 2,4-diphenylhydrazine derivatives in rat urine. The excretion of five nonpolar and nine polar compounds was increased under conditions of peroxidative stress. The profile of lipophilic aldehydes obtained for human urine resembled that for rat urine. Except for a reported 4-hydroxynon-2-enal conjugate with mercapturic acid, the conjugated forms of the lipophilic aldehydes excreted in urine remain unidentified. Aldehyde excretion is influenced by numerous factors that affect the formation of lipid peroxides in vivo such as energy status, physical activity and environmental temperature, as well as by wide variations in the intake of peroxides in the diet. Consequently, urinalysis for aldehydic products of lipid peroxidation is an unreliable indicator of the general state of peroxidative stress in vivo.

Response of urinary lipophilic aldehydes and related carbonyl compounds to factors that stimulate lipid peroxidation in vivo.

Peroxidation of lipids results in the formation of a number of aldehydic and other carbonyl-containing secondary degradation products. The effect of peroxidative stimuli mediated by vitamin E deficiency, a diet high in polyunsaturated fatty acids (containing cod liver oil), and carbon tetrachloride administration on urinary excretion of a number of lipophilic aldehydes and related carbonyl compounds was examined in rats. These secondary lipid peroxidation products were measured as 2,4-dinitrophenylhydrazine derivatives. All three treatments increased urinary excretion of secondary lipid peroxidation products, although the pattern of excretion of these products varied somewhat among the treatments. Significant increases were found in butanal, hexanal, octanal, butan-2-one, pentan-2-one, hex-2-enal, hepta-2,4-dienal, 4-hydroxyhex-2-enal, 4-hydroxyoct-2-enal, 4-hydroxynon-2-enal, and a number of unidentified carbonyl compounds. These results suggest that urinary excretion of these lipophilic secondary lipid peroxidation products is a useful and noninvasive marker of whole-body lipid peroxidation.

Lipophilic aldehydes and related carbonyl compounds in rat and human urine.

Rat and human urine samples were analyzed for lipophilic aldehydes and other carbonyl products of lipid peroxidation. The following compounds were identified as their 2,4-dinitrophenyl hydrazones by cochromatography with pure standards using three solvent systems: butanal, butan-2-one, pentan-2-one, hex-2-enal, hexanal, hepta-2,4-dienal, hept-2-enal, octanal, non-2-enal, deca-2,4-dienal, 4-hydroxyhex-2-enal, and 4-hydroxynon-2-enal. In general, fasted rats excreted less of these compounds than fed rats, indicating they were partially of dietary origin or that the endogenous compounds were excreted in a form not susceptible to hydrazone formation. The compounds excreted in human urine were similar to those excreted in rat urine but were present in lower concentrations. Identification of the conjugated forms of the lipophilic aldehydes and related carbonyl compounds excreted in urine may be a source of information about their reactions in vivo.

The effect of superoxide anion in the production of seven major cholesterol oxidation products in aptoric and protic conditions.

The reaction of cholesterol with superoxide anion was investigated in aprotic (dry) and in protic (aqueous) conditions. Superoxide anion was produced by electro-chemical reduction of molecular oxygen in a solution of 0.1 M tetrabutylammonium bromide in acetonitrile. The cholesterol and cholesterol oxidation products, 7-ketocholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol and 25-hydroxycholesterol were measured by high performance liquid chromatography using a mu-Porasil normal phase column and a UV detector. Cholesterol triol, 5 alpha and 5 beta cholesterol epoxides were measured by capillary gas chromatography. None of these cholesterol oxidation products was detected from the reaction of cholesterol with superoxide anion in aprotic conditions. This indicates that cholesterol was not oxidized by direct attack of superoxide anion. When 4% water (v/v) was added to the aprotic reaction mixture, to induce the dismutation of superoxide anions and the production of hydroxy free radicals, it was found that cholesterol was not oxidized by the active oxygen species thus produced. The effects of hydrogen peroxide, on the reaction of cholesterol with superoxide anion in the protic condition was also investigated. When hydrogen peroxide was added to superoxide anion in aqueous solution, only 7-ketocholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol were formed from cholesterol.

Vitamin C nutriture has little short-term effect on vitamin E concentrations in healthy women.

To determine whether the postulated sparing effect of vitamin E by ascorbic acid (AA) is important for human nutrition, we studied vitamin E status in 20 healthy pre-menopausal women (age 20-43 y) with high or low vitamin C intakes for 6 wk in a live-in metabolic unit. The experimental diet contained no fruits and vegetables and provided 5 mg/d of AA (Recommended Dietary Allowance = 60 mg/d), 3 mg/d of alpha-tocopherol (RDA = 10 mg/d) and 42 g/d of tocopherol-stripped safflower oil to increase the vitamin E requirement. Half of the subjects revived a daily AA supplement of 495 mg (high AA group). A biochemical ascorbate deficiency was attained in the low AA group as indicated by plasma AA concentrations that reached the lower limit of normal by study d 15. Oral doses (20 mg) of hexadeuterated RRR-alpha-tocopherol acetate (d6-alphaT) were given daily to all subjects on d 15-21. Measures of vitamin E status included d6-alphaT and unlabeled alpha-tocopherol concentrations in plasma, platelets, buccal cells and adipose. The levels of unlabeled alpha-tocopherol decreased over time in plasma and platelets and were unchanged for buccal cells and adipose, but were not significantly affected by AA intake. Likewise, the rise and fall of plasma and platelet d6-alpha T, and measures of lipid peroxidation, were not affected by AA intake. Although vitamin C nutriture did not significantly affect vitamin E status within the 6-wk time period of this experiment, further study of this question is warranted, because some of the present results indicate a trend toward sparing of tissue tocopherol by high AA intake.

The influence of vitamin E and selenium on lipid peroxidation and aldehyde dehydrogenase activity in rat liver and tissue.

Malondialdehyde (MDA) production and cytosolic aldehyde dehydrogenase (ALDH) response were examined in rat liver tissues after feeding different levels of dietary vitamin E and/or selenium and polyunsaturated fat for 12-38 wk. MDA production was significantly increased by vitamin E deficiency or by high levels of polyunsaturated fat intake, but not by selenium deficiency. The activity of cytosolic ALDH increased upon increased production of MDA after 12-16 wk of feeding the lipid peroxidation-inducing diets. However, ALDH activity was suppressed after 38 wk of feeding the vitamin E-deficient diet. The results indicate that the hepatic cytosolic ALDH may be involved in the metabolism of MDA during a relatively short-term increase in in vivo lipid peroxidation, but that ALDH activity becomes suppressed after more severe in vivo lipid peroxidation has been produced. Hepatic and plasma alpha-tocopherol levels and lipid peroxidation products were measured for the various dietary groups.

Diabetes increases excretion of urinary malonaldehyde conjugates in rats.

The effect of streptozotocin-induced diabetes on the urinary excretion of thiobarbituric acid test-positive materials was examined. In diabetic rats, urinary excretion of thiobarbituric acid reactive substances was increased 5-fold over that in nondiabetic animals. High-performance liquid chromatography of urine samples revealed that five of the six fractions previously found to be increased in vitamin E deficiency [Lee, H.-S., Shoeman, D.W., and Csallany, A.S. (1992) Lipids 27, 124-128] were also significantly increased in streptozotocin-induced diabetes. The data suggest that a high level of oxidative stress is induced by uncontrolled diabetes in rats.

alpha-Tocopherol oxidation mediated by superoxide anion (O2-). I. Reactions in aprotic and protic conditions.

The reaction of alpha-tocopherol (alpha-T) with superoxide anion (O2-) in both dry acetonitrile and in aqueous acetonitrile solution is described. The O2- was generated by the electrochemical reduction of molecular oxygen in acetonitrile, using tetrabutylammonium bromide as an electrolyte. alpha-T was reacted with O2- either in dry acetonitrile or in a 10% aqueous acetonitrile solution. In dry acetonitrile, alpha-T was oxidized to a very unstable primary intermediate, which was further oxidized to a secondary, more stable intermediate. The formation of the secondary intermediate depended upon the presence of molecular oxygen. This intermediate readily converted into two compounds in equimolar amounts (designated A and B). The primary, very unstable intermediate was readily reduced again to alpha-T by treatment with LiAlH4 or ascorbic acid. However, the secondary intermediate or the stable oxidation products could not be reduced to alpha-T. In the 10% aqueous acetonitrile, alpha-T was oxidized to alpha-tocopheryl quinone, alpha-tocopherol dimer and alpha-tocopherol dihydroxy dimer, and an unknown compound. In the aqueous medium, no intermediates were formed by the action of O2-. The results of this study indicate that the reaction of alpha-T with O2- under aprotic conditions is different from that observed under protic conditions.

alpha-Tocopherol oxidation mediated by superoxide anion (O2-). II. Identification of the stable alpha-tocopherol oxidation products.

The present paper describes the identification of two stable end products of alpha-tocopherol oxidation that were previously detected among the products of the reaction of alpha-tocopherol with superoxide anion (O2-) under aprotic conditions. One compound, previously designated compound A, was identified as trans-7-hydroxy-trans-8,8a-epoxy-alpha-tocopherone, and the other, designated compound B, was identified as cis-7-hydroxy-cis-8,8a-epoxy-alpha-tocopherone. It was also observed that under protic conditions (10% water in acetonitrile) the reaction of alpha-tocopherol with O2- did not produce compounds A and B, but rather alpha-tocopheryl quinone, alpha-tocopherol dimer, alpha-tocopherol dihydroxy dimer, and the previously designated compound C. Compound C was identified in the present study as alpha-tocopheryl-quinone-2,3-epoxide.

Urinary response to in vivo lipid peroxidation induced by vitamin E deficiency.

Experiments were carried out to measure the urinary excretion of free and conjugated malonaldehyde (MDA) and other thiobarbituric acid reactive substances (TBARS) in vitamin E deficient and vitamin E supplemented rats. From both dietary groups, six TBA positive fractions were isolated, in addition to that containing free MDA, by high-performance liquid chromatography (HPLC) on a TSK-GEL G-1000PW column. Three of the fractions isolated were found to be significantly increased in vitamin E deficiency. After acid hydrolysis, only one of the above compounds produced free MDA which indicated the presence of derivatized MDA. Only this fraction exhibited fluorescence at excitation 370 nm and emission 450 nm. The five other fractions formed 2,4-dinitrophenylhydrazones (2,4-DNPH), indicating the presence of carbonyl groups, but the derivatized MDA fraction did not. No significant differences were found in free MDA levels between the vitamin E deficient and the vitamin E supplemented groups.

Identification of vitamin E-dependent water soluble fluorescent compounds in mouse tissues.

The present paper describes the identification of two vitamin E-dependent, water soluble fluorescent compounds in mouse tissues. Ultraviolet and fluorescent spectroscopy, derivatization with 1-dimethylamino-naphthalene-5-sulfonyl chloride (dansyl chloride) and cochromatography using high performance liquid chromatography (HPLC) were utilized for the identification of the unknown compounds. The water soluble fluorescent compounds in mouse tissues were identified as tyrosine and tryptophan. The compounds were previously found to increase significantly in vitamin E deficiency in various tissues.

HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues.

A fast, sensitive, high performance liquid chromatographic method was developed for the quantitation of cholesterol and four of its major oxidation products: 3 beta-hydroxycholest-5-en-7-one (7-ketocholesterol), cholest-5-ene-3 beta, 7 alpha-diol (7 alpha-hydroxycholesterol), cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol), and cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol). In this procedure 2:1 chloroform:methanol (v/v) extracts of tissue homogenate were combined, dried over anhydrous Na2SO4, filtered, evaporated to dryness under N2 and dissolved with a mobile phase of either 97:3 or 93:7 hexane:isopropanol (v/v). After membrane filtration and without further purification, aliquots were directly injected onto a 10-microns pore size, 30 X 0.39 cm mu-Porasil normal phase column. The separation of cholesterol and its oxidation products was monitored by a UV detector at 206 and 233 nm. This method was successfully applied to pork muscle as well as mouse liver tissues and was able to detect cholesterol oxidation products (COP) in the ppm range. The identity of the COP was confirmed by mass spectroscopy.

Malondialdehyde-containing proteins and their relationship to vitamin E.

A high molecular weight (Sephadex G-15 void volume), water-soluble, fluorescent material that was found to increase significantly in the mouse liver in response to vitamin E deficiency was separated into six proteins by high performance liquid chromatography (HPLC) using a TSK G2000 SW column. One of these proteins increased significantly in concentration due to vitamin E deficiency and had a molecular weight of 20,000 daltons. This protein was found to contain malondialdehyde, an end product of lipid peroxidation, attached to it presumably in a Schiff-base type structure with amino groups. This appears to be the first report in the literature of direct evidence that malondialdehyde is attached to protein in vivo.

Separation of alpha-tocopherol and its oxidation products by high performance liquid chromatography.

A very sensitive high performance liquid chromatographic (HPLC) method was developed for the separation of alpha-tocopherol (alpha-T) and its five oxidation products: alpha-tocopheryl quinone (TQ), dimer (D), dihydroxy dimer (DHD), trimer (T) and 9-methoxy-alpha-tocopherone commonly called alpha-tocopheroxide (TO). The separation was achieved on a normal-phase silica-based column (Ultrasphere-Si), using a mobile phase of hexane/chloroform/isopropanol (95:4.5:0.5, v/v/v) at a flow rate of 0.4 ml/min, and the eluants were monitored simultaneously at their maximum absorptions using a variable-wavelength UV detector. The minimum detection limit is 0.01 microgram for alpha-T, TQ and TO, 0.05 microgram for DHD and D, and 0.1 microgram for T/injection. This normal-phase method has the combined advantages of being very sensitive, fast and capable of separating all six compounds at the same time.

Measurement of free and bound malondialdehyde in vitamin E-deficient and -supplemented rat liver tissues.

The quantity of free malondialdehyde (MDA) in liver tissues of rats fed vitamin E-deficient or -supplemented diets for 43 wk was measured by a newly developed high performance liquid chromatographic (HPLC) method. Bound MDA was quantified by the same HPLC method after alkaline hydrolysis of tissue homogenates. Tissues from vitamin E-deficient animals showed levels of free MDA about 15 times higher but levels of bound MDA less than 2 times higher than the vitamin E-supplemented animals. Free MDA is the major form in vitamin E-deficient tissues, but bound MDA is predominant in vitamin E-supplemented tissues. Conventional thiobarbituric acid (TBA) test results revealed that the content of TBA-reactive substances expressed in MDA equivalents was much higher than the actual free MDA levels in all groups. Results indicate that free MDA level measured by HPLC is a more sensitive index than the TBA value for lipid peroxidation. Some other TBA-reactive substances seem to exist in liver tissue regardless of the dietary treatment.

Copper inhibition of the thiobarbituric acid reaction in Bedlington terriers.

The effect of copper on thiobarbituric acid (TBA) reaction values, an index of lipid peroxidation, was examined in Bedlington Terriers, healthy dogs, and rats. High hepatic concentrations of copper appeared to lower TBA values in the inherited, chronic, progressive hepatic degeneration of Bedlington Terriers, a disease associated with copper toxicosis. The suspected TBA inhibition was confirmed when Cu2+ was added to homogenates of healthy dog or rat liver or a malondialdehyde standard. The amount of copper added approximated that in diseased Bedlington Terriers. Because of the interference by copper, the TBA test was judged to be an inappropriate test for the evaluation of lipid peroxidation in samples containing high copper concentrations such as those in diseased Bedlington Terriers.

Organic solvent-soluble lipofuscin pigments and glutathione peroxidase in mouse brain and heart: effects of age and vitamin E.

The effect of age and dietary supplementation of vitamin E or N,N'-diphenyl-p-phenylenediamine (DPPD) on organic solvent-soluble lipofuscin pigments (OLP) and glutathione peroxidase (GSH-Px) activity in mouse heart and brain was investigated. Four groups of 32 female weanling mice were fed a basal diet containing either 0, 30 or 300 ppm RRR-alpha-tocopheryl acetate (d-alpha-tocopheryl acetate) or 30 ppm DPPD from 2 to 18 mo of age. Neither GSH-Px activity nor dietary supplementation of vitamin E or DPPD had an effect on OLP concentrations in the brain or heart. OLP levels were two- to fourfold higher at 12 mo of age in the heart and were lower at 18 mo of age in the brain than at 2 or 9 mo of age. GSH-Px activity increased with age in the heart tissue of vitamin E-deficient and DPPD-supplemented mice only. No change in GSH-Px activity was observed in the brain due to diet or increasing age. These results suggested that OLP concentrations were not affected by dietary supplementation of vitamin E or DPPD but were affected by age-related factors in the mouse brain and heart.

Organic solvent soluble lipofuscin pigments and glutathione peroxidase in weanling and old rats.

Tissues from weanling and 19-month-old Sprague Dawley rats were spectrophotofluorometrically analyzed for organic solvent soluble lipofuscin pigments (OLP) and assayed for glutathione peroxidase (GSH-Px) activity. Significantly lower levels of OLP and higher levels of GSH-Px activity were found in the kidney, heart and testis at 19 months of age than were found in weanling rats. No age-related differences in OLP were observed in the brain, lung or liver. The results appeared to indicate an inverse relationship between OLP and GSH-Px activity in some tissues, but the magnitude of the changes observed coupled with conflicting observations in the liver suggested that GSH-Px activity and OLP concentrations were not directly related.

Ozone-related fluorescent compounds in mouse liver and lung.

Groups of ten female, weanling mice were fed a basal, vitamin E-deficient diet or a basal diet supplemented with RRR-alpha-tocopheryl acetate for 14 months. During the last month one group from each dietary regimen was exposed for 30-60 min/day to 1.5 ppm ozone (25 hr total ozone exposure) and the remaining groups to control ambient air. The liver and lung tissues were homogenized and extracted with 2:1 chloroform:methanol and water. The water-soluble and organic solvent-soluble fluorescent materials were separated on Sephadex G-25 and LH-20 columns, respectively. Excitation and emission wavelengths for the eluting fractions were determined by continuous emission scans from 250 to 600 nm for each excitation wavelength between 250 and 500 nm (in increments of 25 nm). Ozone exposure did not effect the concentration of any of the fluorescent materials examined in the lung, but it resulted in a significant increase in two of four water-soluble compounds in the liver with excitation wavelength maxima/emission wavelength maxima of 270 nm/310 nm and 275 nm/350 nm (smaller molecular weight material) suggesting in vivo lipid oxidation.