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.

Effects of peroxidative stress and age on the concentration of a deoxyguanosine-malondialdehyde adduct in rat DNA.

The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had no effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-alpha-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain >> liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.

Increased formation and degradation of malondialdehyde-modified proteins under conditions of peroxidative stress.

The effect of increased in vivo lipid peroxidation on excretion of the main urinary metabolites of malondialdehyde (MDA) was investigated. peroxidative stress in the form of vitamin E deficiency or the administration of iron nitrilotriacetate or carbon tetrachloride was imposed on rats fed an MDA-free diet. Significant increases were observed in excretion of the lysine-MDA adduct epsilon-propenal lysine, its N-acetyl ester, and free MDA. Under the conditions imposed, the increments in excretion of the lysine adducts reflect increased peroxidative modification of tissue proteins in vivo. These adducts also were found to be the main forms of MDA excreted in human urine. Reacting 14C-bovine serum albumin (BSA) with MDA resulted in its accelerated proteolysis in vitro by soluble enzyme preparations derived from human erythrocytes and rat liver mitochondria. The increments observed were similar to those reported for the hydrolysis of BSA following its exposure to hydroxyl radicals. The results show that lipid peroxidation in vivo results in peroxidative damage to tissue proteins and indicate that such proteins are subject to an accelerated rate of proteolysis.

Identification of a deoxyguanosine-malondialdehyde adduct in rat and human urine.

In an ongoing study, rat and human urine have been examined for the presence of malondialdehyde (MDA) derivatives as indicators of the nature of lipid peroxidative damage caused by this compound in vivo. MDA in urine was found to be present mainly in the form of two lysine adducts, one acetylated and the other unacetylated, reflecting in vivo reactions with tissue proteins. Two minor metabolites were identified as adducts with the phospholipid bases serine and ethanolamine and a third one as an adduct with the nucleic acid base guanine. The identification of an MDA adduct with deoxyguanosine (dG-MDA) among the products of hydrolysis of rat liver DNA suggested the possible occurrence of this compound in urine. In the present study dG-MDA was identified in rat and in human urine, and a high-performance liquid chromatographic method utilizing fluorescence detection was developed for its estimation. The method is sensitive to 1 pmol of dG-MDA and requires a minimum of 1 mL of rat urine or 5 mL of human urine. Its rate of excretion by five-week-old rats (28.54 +/- 2.28 nmol/kg/24 h) (mean +/- SEM) was higher than that for nine-week-old rats (6.29 +/- 1.02) and much higher than that for adult humans (0.40 +/- 0.05). The results indicate that, as reported for 8-hydroxy-deoxyguanosine, dG-MDA excretion is related to metabolic rate. Excretion of dG-MDA by the rat, like the excretion of total MDA, declines during growth on a body weight basis at a rate similar to the decrease in resting energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials.

A comparative evaluation was made of the conventional spectrophotometric procedure and three published high performance liquid chromatographic (HPLC) procedures for the determination of malondialdehyde (MDA) as the thiobarbituric acid (TBA) derivative when applied to liver, fish meal, serum, and urine. Except for urine, spectrophotometric analysis overestimated MDA content. Purification of the TBA-MDA complex obtained from liver and fish meal on reverse phase cartridges was found to entail a loss of complex bound to residual peptides in the trichloracetic acid (TCA) extract. Mincing as opposed to homogenizing liver samples led to a doubling of values for MDA content. Hexanal was a major TBA reactant, in addition to MDA, in all the samples. Acid hydrolysis and heat were necessary for the release of MDA bound to the amino groups of proteins and other amino compounds. Methods for free MDA have limited application to biological materials except short term in vitro preparations such as peroxidizing microsomes, in which free MDA accumulates. On the basis of these and other observations, a modified HPLC procedure for the determination of MDA as the TBA-MDA complex is proposed.

Isolation of a malondialdehyde-deoxyguanosine adduct from rat liver DNA.

In previous studies, an adduct of malondialdehyde (MDA) with guanine was identified in rat and human urine. Subsequent detection of an adduct with deoxyguanosine (dG) in urine prompted an investigation of its possible occurrence in DNA. Rat liver DNA was hydrolyzed using nuclease P1 and alkaline P-ase and subjected to deoxyribonucleoside analysis using reverse phase high-pressure liquid chromatography (HPLC) with fluorescence detection. A compound was isolated that could not be separated from a synthetic pyrimidinopurine adduct of MDA and dG (dG-MDA). Partial hydrolysis released guanine (Gua), Gua-MDA, and dG in amounts that, in aggregate, were the molar equivalent of the starting material calculated by fluorescence analysis as dG-MDA. Complete acid hydrolysis of the isolate yielded an equimolar amount of MDA. Analysis of liver DNA isolated from growing rats yielded a value for dG-MDA content of 9.0 +/- 1.6 pmol/100 micrograms DNA (mean +/- SEM, N = 5). This value is approximately 7 times those reported for the 8-hydroxy deoxyguanosine content of rat liver nuclear DNA. This study demonstrates that DNA is modified in vivo by reactions of its guanylate moiety with MDA, and indicates that, at least in the case of rat liver DNA, the prevalence of such modifications is greater than those caused by reactions with hydroxyl radicals.

Calcium glycerophosphate as a source of calcium and phosphorus in total parenteral nutrition solutions.

Calcium glycerophosphate (CaGP) was tested as an alternative to calcium gluconate (CaGluc) and potassium mono- and dibasic phosphate (KPhos) as a source of Ca and P in total parenteral nutrition (TPN) solutions for piglets. Four-day-old piglets were infused for 7 days with a TPN solution that provided either 4.2 mmol Ca and 2.1 mmol P/kg/24 h as CaGluc and KPhos (the maximum quantities that can be provided using these sources), or 15.0 mmol Ca and 15.0 mmol P/kg/24 h as CaGP. Ca and P retentions were more than six times greater (p less than 0.01) in the piglets receiving CaGP (14.5 +/- 0.2 vs 2.2 +/- 0.3 mmol Ca/kg/24 h and 13.3 +/- 0.4 vs 2.4 +/- 0.1 mmol P/kg/24 h) (Mean +/- SEM). The ratio of Ca to fat-free dry weight, an indicator of bone mineralization, was significantly higher (p less than 0.05) in the humerus (174.8 +/- 2.2 vs 147.2 +/- 6.7) and femur (158.3 +/- 4.8 vs 130.1 +/- 7.8) in the CaGP group. This study showed that CaGP is efficiently used as a source of Ca and P in TPN solutions for piglets. The results suggest that the use of CaGP as the source of Ca and P in TPN solutions may prevent the development of the undermineralized bone seen in low-birth weight infants nourished intravenously.

A review of recent studies on the metabolism of exogenous and endogenous malondialdehyde.

1. The generation of malondialdehyde (MDA), a mutagenic product of the oxidative decomposition of highly unsaturated fatty acids in vivo, is increased by exposure to certain environmental oxidants and xenobiotics. 2. This increase is reflected in enhanced excretion of several MDA derivatives in the urine. The main urinary metabolites of MDA have been identified as N-epsilon-(2-propenal)lysine and its N-alpha-acetyl ester. 3. Two minor metabolites have been identified as enaminals formed by reactions with the phospholipid bases serine and ethanolamine. A further MDA metabolite has been identified as a cyclized adduct with guanine. 4. These urinary compounds reflect the turnover of proteins, phospholipids and nucleic acids that have been modified by reactions with MDA in vivo. Monitoring of the urinary adduct with guanine may provide a practicable method of assessing the effect of xenobiotics and other factors on in vivo lipid peroxidation. 5. The proportion of total MDA in the diet, blood, urine and solid tissues that exists in the free state appears to be negligible. 6. Chronic oral administration of the enol Na salt of MDA to animals produced no significant pathology except for dose-dependent lesions of hepatic nuclei. Nuclear abnormalities in cultured rat skin fibroblasts were seen at intracellular concentrations as low as 10(-7) M.

Isolation of a guanine-malondialdehyde adduct from rat and human urine.

A 1:1 adduct of guanine with malondialdehyde (MDA) was isolated from rat and human urine. This compound was shown to be identical to a synthetic adduct prepared according to the procedure of Seto et al. (Bull. Chem. Soc. Jpn. 58, 3431-3435, 1985). The UV, NMR and other characteristics of the compound were consistent with the tricyclic pyrimidinopurine structure proposed by these investigators. Its endogenous origin is indicated by its presence in the urine of rats fed an MDA-free diet, and by the observation that its excretion increased following iron or carbon tetrachloride administration. It may serve as a marker for nucleic acid modification caused by lipid peroxidation in vivo.

Effect of a high protein intake on acid-base balance in adult rats.

Adult rats are able to maintain Ca balance under protein loads that produce Ca loss in adult humans. This species difference was investigated by determining the relationship between protein intake, endogenous acid production (EAP), net acid excretion (NAE), and urinary Ca in adult rats for comparison with a similar study on adult humans. Diets containing 10, 30, and 50% casein were fed in conjunction with proportionate increments in the sulfur amino acid (SAA) methionine (0.6, 1.8, and 3.0%). Urine volume, Ca, sulfate, organic anions, TA (titratable acidity as acid phosphates), and ammonium increased progressively with increases in protein intake, and pH decreased. When protein intake was increased at a constant level of SAA, no increase in urinary Ca, sulfate, and TA or decrease in pH was observed. Both SAA and non-SAA enhanced ammonium excretion but only non-SAA enhanced organic anion excretion, an indicator of incomplete oxidation of organic acids. SAA were responsible for 89 and 91% of the increase in EAP and Ca excretion, respectively, caused by increasing protein intake from 10-30% of the diet. In a comparison experiment, human adults on a high protein intake exhibited a much smaller increase in acid excretion as ammonium, a greater increase as TA, no change in organic anion excretion, and no increase in EAP from non-SAA. The importance of these species differences in acid-base response to a high protein intake in the greater ability of rats to maintain Ca balance on high protein intakes is unclear; however, the smaller fraction of endogenous Ca excreted in the urine of rats is probably an important factor.

Sources of protein-induced endogenous acid production and excretion by human adults.

The origin of the increase in endogenous acid production and excretion associated with the calciuretic action of high protein intakes was investigated in human adults. Eight subjects, 4 males and 4 females, aged 25-36 years, were fed a low protein diet (50 g/day) and a high protein diet (120 g/day in males and 106 g/day in females) for 7 days each. The high protein diet was formulated by supplementing the low protein diet with a mixture of four purified proteins. Increased protein intake was associated with increases in urinary Ca, sulfate, titratable acidity (acid phosphates) and ammonium, and decreases in urinary pH and bicarbonate. There was no increase in organic anion excretion. The increases in endogenous acid production (EAP) and net acid excretion (NAE) were entirely attributable to the oxidation of excess sulfur amino acids (SAA), which yields 2 moles of hydrogen ions per mole of amino acid catabolized. The results differ in this respect from those reported for studies on the effect of SAA loading, which indicate that non-SAA make a major contribution to the increase in EAP seen under these conditions.

Identification of N-(2-propenal)ethanolamine as a urinary metabolite of malondialdehyde.

N-(2-propenal)ethanolamine was isolated from rat and human urine using anion exchange, cation exchange, size exclusion and high performance liquid chromatography. Acid hydrolysis of the isolate yielded malondialdehyde (MDA) and ethanolamine (E) in a 1:1 molar ratio. A 1:1 E-MDA adduct was synthesized and found to be chromatographically inseparable from the urinary metabolite. Its NMR and UV spectra and lack of fluorescence were consistent with those of an enaminal formed by a Schiff's base reaction. The identification in urine of an adduct of MDA with ethanolamine, and the previous identification of an adduct with serine, constitutes direct evidence for the oxidative decomposition in vivo of polyunsaturated fatty acids present in the relevant phospholipids. The absence in urine of MDA adducts with other alpha-amino compounds (at least in comparable amounts) indicates that the ethanolamine and serine derivatives are formed in situ and not as a result of reactions with MDA generated in enzymatic processes.

Effect of Mechanical Deboner Head Pressure on Lipid Oxidation in Poultry Meat.

The oxidation rates of mechanically deboned poultry meat (MDPM) from whole breasts of roasters obtained under different deboner head pressures (40, 75, 120 and 150 lb/in2) were determined. Mechanical deboning significantly (P<0.05) increased fat, ash, calcium and iron content, and reduced moisture and protein levels, compared to hand deboning. The highest head pressure resulted in significantly lower fat and higher iron content than the other treatments and produced the slowest rate of oxidation. The lowest head pressure (40 lb/in2) resulted in the highest oxidation rate. Fatty acid analysis indicated that 150 lb/in2 caused a loss of linoleic acids with a resulting increase in the proportion of C16 fatty acids. Commercial MDPM samples prepared from broiler backs & necks showed significantly (p<0.05) higher oxidation rates than fowl frames and all of the mechanically deboned roaster meat samples except those deboned at 40 lb/in2.