Effect of different dietary triglycerides on 7alpha-hydroxylation of cholesterol and other mixed-function oxidations

The effect of a diet containing triglycerides of different fatty acid composition on hepatic 7alpha-hydroxylation of cholesterol was studied. 7alpha-Hydroxylation of exogenous as well as endogenous cholesterol was significantly lower in the liver of rats fed trilinolein and triolein than in those fed tripalmitin and trierucin. The concentration of cytochrome P-450 in liver microsomes was significantly lower in the rats fed tripalmitin and trierucin than in those fed triolein and trilinolein. The inhibitory effect of triolein and trilinolein on 7alpha-hydroxylation of cholesterol and the stimulatory effect of these triglycerides on the concentration of cytochrome P-450 was not due to the small amounts of peroxides present in the unsaturated triglycerides. Thus addition of the antioxidant butylated hydroxyanisol did not change the general pattern with respect to 7alpha-hydroxylation and concentration of cytochrome P-450. However, a diet consisting of peroxidized linoleic acid further decreased 7alpha-hydroxylation of cholesterol. The difference between the effect obtained with triolein and trilinolein on the one hand and trierucin and tripalmitin on the other was observed also in experiments with lower concentrations of fat in the diet and in experiments with different lighting conditions and feeding patterns. The inverse relation between cytochrome P-450 and 7alpha-hydroxylation of cholesterol, as well as results obtained with substrates for mixed-function oxidation other than cholesterol suggest that most of the changes observed due to the different diets are specific for 7alpha-hydroxylation of cholesterol. The level of cholesterol 7alpha-hydroxylase activity was found to be better related to the degree of absorption of fat than to total amount of absorbed fat or degree of unsaturation of the fat. The results are discussed in relation to previous knowledge concerning mechanisms regulating biosynthesis of bile acid.     

Effects of Bile Salt-Stimulated Lipase-Treated Triglycerides and Free Fatty Acids on Extracellular Stages of Eimeria tenella

Normal human milk (NHM) has antiprotozoal activity unrelated to immunological components; this activity extends to sporozoites of Eimeria tenella . This activity may be due to free fatty acids (FFA) enzymatically hydrolyzed from tnacyl glycerols by a bile salt-stimulated lipase (BSSL) found in NHM. Sporozoites were therefore incubated in the presence of several saturated and unsaturated FFA. Anticoccidial activity was observed for many unsaturated fatty acids and for some saturated fatty acids. In addition, sporozoites were added to solutions of triglycerides (trilinolein, triolein and trilinolenin) preincubated with BSSL and sodium cholate. which resulted in killing of the parasites. Triglycerides alone showed no anticoccidial activity. These results were duplicated with first generation merozoites. Intracellular stages of E. tenella were affected by FFA only at concentrations that inhibited host cells.     

Effects of bile salt-stimulated lipase-treated triglycerides and free fatty acids on extracellular stages of Eimeria tenella

Normal human milk (NHM) has antiprotozoal activity unrelated to immunological components; this activity extends to sporozoites of Eimeria tenella. This activity may be due to free fatty acids (FFA) enzymatically hydrolyzed from triacyl glycerols by a bile salt-stimulated lipase (BSSL) found in NHM. Sporozoites were therefore incubated in the presence of several saturated and unsaturated FFA. Anticoccidial activity was observed for many unsaturated fatty acids and for some saturated fatty acids. In addition, sporozoites were added to solutions of triglycerides (trilinolein, triolein and trilinolenin) preincubated with BSSL and sodium cholate, which resulted in killing of the parasites. Triglycerides alone showed no anticoccidial activity. These results were duplicated with first generation merozoites. Intracellular stages of E. tenella were affected by FFA only at concentrations that inhibited host cells.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Regulation of the metabolism of lipid emulsion model lipoproteins by a saturated acyl chain at the 2-position of triacylglycerol

Lipid emulsion particles were prepared by sonicating four different lipid mixtures (triacylglycerol (TAG), 70%; phospholipid, 25%; cholesteryl oleate (CO), 3%; and free cholesterol, 2%), then purified by density gradient ultracentrifugation. For three test mixtures, the TAG contained 50, 75, or 100% 1,3-dioleyl-2-stearylglycerol (OSO) with the remainder being triolein (OOO); 100% triolein in the lipid mixture was used as the control. After intravenous injection of the lipid particles into unanesthetized rats, removal of radioactive TAG fatty acid and CO from plasma was measured for 30 min, then liver and spleen uptakes were measured. When emulsions contained 75% or 100% OSO as TAG, the plasma removal rates of CO were, respectively, 60% or 30% of the rate when the TAG was 100% triolein; smaller recoveries of CO were found in the liver. The clearances of TAG fatty acid did not differ significantly and the recoveries of TAG fatty acid in the organs were not affected by the type of emulsion injected. Remnant particles were derived from donor rats in which uptake was blocked by exclusion of liver and other viscera from the circulation before injection of 100% OOO and 100% OSO emulsions. When injected into recipient intact rats, the removal of remnants from plasma was slower for remnants derived 15 min after injection of 100% OSO emulsions than from 100% OOO emulsions, showing that the slower removal of emulsion CO was due to slower remnant uptake from the plasma with OSO emulsions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Disruption of mitochondrial beta -oxidation of unsaturated fatty acids in the 3,2-trans-enoyl-CoA isomerase-deficient mouse

Cellular energy metabolism is largely sustained by mitochondrial beta-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci(-/-) mouse). ECI is the link in mitochondrial beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial beta-oxidation of unsaturated fatty acids is interrupted in eci(-/-)mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci(-/-) mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci(-/-) mice together with peroxisomal beta- and microsomal omega-oxidation enzymes. Combined peroxisomal beta- and microsomal omega-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci(-/-) mouse might be a model of a yet undefined inborn mitochondrial beta-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the beta-oxidation spiral of saturated fatty acids.     

Metabolic origins of urinary unsaturated dicarboxylic acids

Previously, we [Jin, S.-J., & Tserng, K.-Y. (1989) J. Lipid Res. 30, 1611-1619] reported the structures of urinary octenedioic acids occurring in patients with dicarboxylic aciduria. We proposed that these unsaturated octenedioic acids were derived from the oxidation of oleic and linoleic acids. By comparison with synthetic decenedioic acids, we have further identified the higher homologues of unsaturated dicarboxylic acids in urine as cis-5-decenedioic (c5DC10), cis-4-decenedioic (c4DC10), cis-3-decenedioic (cDC10), trans-4-decenedioic, trans-3-decenedioic, cis-5-dodecenedioic (c5DC12), cis-3-dodecenedioic (c3DC12), and trans-3-dodecenedioic acids. The presence of these isomeric decenedioic and dodecenedioic acids in urine is consistent with the proposed metabolic origins. In vitro studies using synthetic unsaturated fatty acids and rat liver homogenates support the proposed metabolic origins of these acids. The following metabolic sequences are proposed for metabolites derived from oleic acid: (route A) cis-5-tetradecenoic acid----cis-5-tetradecenedioic acid----c5DC12----c5DC10----suberic (DC8)----adipic (DC6); (route B) cis-3-dodecenoic acid----c3DC12----c3DC10----c3DC8 (cis-3-octenedioic)----DC6. A similar route is derived from linoleic acid: cis-4-decenoic acid----c4DC10----c4DC8 (cis-4-octenedioic)----DC6. The presence of a double bond at position 3, 4, or 5 of fatty acid appears to be rate limiting for further beta-oxidation; therefore, metabolic products with cis-3, cis-4, or cis-5 structure accumulate. Urinary DC8 and DC6 are derived partially from the metabolic degradation of these unsaturated dicarboxylic acids.     

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Structure and polymorphism of 18-carbon fatty acyl triacylglycerols: effect of unsaturation and substitution in the 2-position

The polymorphic behavior of symmetric diacid triacylglycerols (TGs), 1,3-dioleoyl-2-stearoyl (OSO), 2-elaidoyl (OEO), and 2-vaccinoyl (OVO) glycerols were studied by differential scanning colorimetry (DSC) and X-ray diffraction and compared with the corresponding monoacid TGs triolein (OOO), tristearin (SSS), trielaidin (EEE), and trivaccinin (VVV). The monoacid TGs formed a bilayered structure in all the polymorphic forms. On quenching from the melt, the diacid TGs OEO and OVO formed a bilayered (D = 45 A) beta'-phase with the exception of OSO, which formed a hexagonally packed bilayered (D = 52 A) alpha-phase. At -7 degrees C, the alpha-phase of OSO quickly transformed to a bilayered (D = 45 A) beta'-phase. Incubation at the beta'-phase melting temperature transformed OVO, OEO, and OSO into a trilayered (D = 65 A) beta-phase, where the 1,3-dioleoyl chains are segregated from the vaccinoyl, elaidoyl, or stearoyl chains into alternating layers. In summary, when all the acyl chains in a TG are the same (saturated, cis or trans unsaturated), the stable beta-phase packs into a bilayered structure. However, when the 1- and 3-acyl chains are cis unsaturated (bent) and the 2-acyl chain is either saturated or trans-unsaturated (straight), a bilayered beta'-phase can form, but transforms to a stable trilayered beta-phase, where the 2-acyl chains form a layer between two different layers of 1,3-oleoyl chains.     

The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats

The conversion of radioactive C6-C16-monocarboxylic acids to urinary adipic, suberic, sebacic and 3-hydroxybutyric acids was investigated in vivo in unstarved, starved and diabetic ketotic rats. Hexanoic, octanoic and decanoic acids were converted to C6-, C6-C8- and C6-C10-dicarboxylic acids, respectively, in fed and 72-h-starved rats. Lauric acid was converted to C6-C8-dicarboxylic acids in starved rats but not in unstarved rats. Decanoic and lauric acids were converted to relatively high amounts of C6-C8-dicarboxylic acids compared with myristic acid in myristic acid in ketotic diabetic rats, while radioactivity from [1-14C]-and [16-(14)] palmitic acid was not incorporated into C6-C8-dicarboxylic acids in diabetic ketotic rats. C6-C12-monocarboxylic acids in hydrolysed rat adipose tissue wee determined by gas-liquid chromatography-mass spectrometry (selected ion monitoring). Decanoic and lauric acids were found in amounts of 7.6-9.1 and 85.9-137.5 micrometers/100 mg tissue, respectively, whereas the amounts of hexanoic and octanoic acids were negligible. It is concluded that the biological origin of the C6-C8-dicarboxylic aciduria seen in ketotic rats are C10-C14-monocarboxylic acids, which are initially omega-oxidised solely or partly as free acids and subsequently beta-oxidised to adipic and suberic acids. The in vitro omega-oxidation of C6-C16-monocarboxylic acids to corresponding dicarboxylic acids in the 100,000 Xg supernatant fraction of rat liver homogenate was measured by selected ion monitoring. 0.09, 0.14, 16.1, 5.8, 7.0 and -6.9% of, respectively, hexanoic, octanoic, decanoic, lauric, myristic and palmitic acid were omega-oxidised to dicarboxylic acids of corresponding chain lengths after 90 min of incubation, when correction for the production of dicarboxylic acids in control assays was made. An in vitro production of C12-C16-dicarboxylic acids was detected in all assays ()including control assays), probably formed from"endogenous' monocarboxylic acids preexistent in the homogenate. Ths "endogenous' production of dicarboxylic acids was inhibited by C10-C16-monocarboxylic acids, where palmitic acid had the strongest effect. In fact, palmitic acid inhibited its own omega-oxidation when added in concentrations above 0.6 mM. Starvation of rats for 72 h did not alter the "endogenous' in vitro production of hexadecanedioic acid.     

Role of Lipase from Community-Associated Methicillin-Resistant Staphylococcus aureus Strain USA300 in Hydrolyzing Triglycerides into Growth-Inhibitory Free Fatty Acids

Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid.     

Effect of dietary oils on lipid peroxidation and on antioxidant parameters of rat plasma and lipoprotein fractions.

In order to investigate the influence of fatty acid pattern and antioxidants other than vitamin E on lipid peroxidation and antioxidant levels of plasma very low density and low density lipoproteins (VLDL + LDL), the effects of three diets (equalized for vitamin E) containing soybean oil, olive oil, or an oleate-rich mixture of triglycerides (triolein) were studied in rats. A significantly lower concentration of thiobarbituric acid-reactive substances (TBA-RS) in plasma and lipoproteins was found after the olive oil diet (soybean oil, 3.7 +/- 0.4 nmol/ml; triolein, 2.1 +/- 0.5 nmol/ml; olive oil, 1.5 +/- 0.3 nmol/ml, in plasma) (soybean oil, 0.99 +/- 0.16 nmol/ml; triolein, 0.96 +/- 0.13 nmol/ml; olive oil, 0.38 +/- 0.12 nmol/ml, in the VLDL + LDL fraction). Furthermore, the results from in vitro copper-induced lipid peroxidation, expressed in terms of conjugated dienes, lipid hydroperoxides, and TBA-RS content, showed that VLDL + LDL particles from olive olive oil-fed rats were remarkably resistant to oxidative modification. The results suggest that the fatty acid unsaturation of dietary oils is not the only determining factor of the antioxidant capacity of lipoproteins in this animal model. The maximal protection observed after the olive oil diet may be explained by the presence of other unidentified antioxidants in addition to vitamin E, derived from oil intake. Therefore, the optimal balance between the content of unsaturated fatty acids and natural antioxidants in dietary oils appears to be of major importance.     

Identification and quantitation of urinary dicarboxylic acids as their dicyclohexyl esters in disease states by gas chromatography mass spectrometry

Clinical studies were conducted by gas chromatography mass spectrometry selected ion monitoring of urinary dicarboxylic acids as dicyclohexyl esters. The dicyclohexyl esters of the dicarboxylic acids give characteristic electron impact mass spectra suitable for selected ion monitoring. The mass spectra exhibit a prominent acid + 1H ion and an (acid + 1H)-H2O ion for use as quantitating and confirming ions. The cyclohexyl esters are stable for days at room temperature and have excellent chromatographic properties. Dicarboxylic acid quantitation is performed within one hour using only 50 microliter of unpurified urine. A rapid method specifically for methylmalonic acid quantitation is described which has assisted physicians in the diagnosis of pernicious anemia and methylmalonic aciduria. This procedure is applicable for screening urinary organic acids for detection of inborn errors of metabolism. The detection of a child with elevated medium length dicarboxylic acids in the terminal urine specimen is reported. This condition, previously described as an inborn error, is attributed to a terminal event. Finally, an increase in urinary succinic acid paralleling putrescine levels is described during a response to cancer chemotherapy.     

{Omega}-oxidation of {alpha}-chlorinated fatty acids: identification of {alpha}-chlorinated dicarboxylic acids

Myeloperoxidase-derived HOCl targets tissue- and lipoprotein-associated plasmalogens to generate α-chlorinated fatty aldehydes, including 2-chlorohexadecanal. Under physiological conditions, 2-chlorohexadecanal is oxidized to 2-chlorohexadecanoic acid (2-ClHA). This study demonstrates the catabolism of 2-ClHA by ω-oxidation and subsequent β-oxidation from the ω-end. Mass spectrometric analyses revealed that 2-ClHA is ω-oxidized in the presence of liver microsomes with initial ω-hydroxylation of 2-ClHA. Subsequent oxidation steps were examined in a human hepatocellular cell line (HepG2). Three different α-chlorinated dicarboxylic acids, 2-chlorohexadecane-(1,16)-dioic acid, 2-chlorotetradecane-(1,14)-dioic acid, and 2-chloroadipic acid (2-ClAdA), were identified. Levels of 2-chlorohexadecane-(1,16)-dioic acid, 2-chlorotetradecane-(1,14)-dioic acid, and 2-ClAdA produced by HepG2 cells were dependent on the concentration of 2-ClHA and the incubation time. Synthetic stable isotope-labeled 2-ClHA was used to demonstrate a precursor-product relationship between 2-ClHA and the α-chlorinated dicarboxylic acids. We also report the identification of endogenous 2-ClAdA in human and rat urine and elevations in stable isotope-labeled urinary 2-ClAdA in rats subjected to intraperitoneal administration of stable isotope-labeled 2-ClHA. Furthermore, urinary 2-ClAdA and plasma 2-ClHA levels are increased in LPS-treated rats. Taken together, these data show that 2-ClHA is ω-oxidized to generate α-chlorinated dicarboxylic acids, which include α-chloroadipic acid that is excreted in the urine.     

C6-C10-dicarboxylic acids in liver and kidney tissue in normal, diabetic ketotic and clofibrate-treated rats

A combined gas chromatographic-mass spectrometric method (selected ion monitoring) to determine C6-C10-dicarboxylic acids in liver and kidney tissue is reported. Alterations in tissue concentrations of the dicarboxylic acids were reflected in urinary excretions, i.e., diabetic rats with 'ketotic dicarboxylic aciduria' had corresponding elevated concentrations of short-chain dicarboxylic acids in liver and kidney tissue. Stimulation of the enzymes of fatty acid oxidation by clofibrate was, as a sole event, not sufficient to cause elevated tissue concentrations of dicarboxylic acids, nor did it result in dicarboxylic aciduria, probably because of a relative lack in substrate (fatty acids) compared to the diabetic ketotic state, where lipolysis is increased. These results strongly indicate that 'ketotic dicarboxylic aciduria' parallels the activity of the lipid metabolism at cellular level, and that it is not just a matter of renal handling.     

The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleoyl phosphatidylcholine causes hemolysis of human red blood cells

Dioleoyl phosphatidylcholine (PC) liposomes were ozonized and the ozonized liposomes were tested for their lytic potency on human red blood cells (RBC). Ozonation of PC liposomes generated approximately 1 mole equivalent of hydrogen peroxide (H2O2) and 2 mole equivalents of aldehydes, based on the moles of ozone consumed. The time necessary for 50% hemolysis induced by ozonized liposomes (a convenient measure of hemolytic activity) was found to depend on the extent of ozonation of the PC liposomes, indicating the formation and accumulation of hemolytic agents during ozonation. Hemolysis was also observed when RBC were incubated with nonanal, the expected product of the ozonation of oleic acid, the principle unsaturated fatty acid in the liposomes. Hydrogen peroxide, another product of PC ozonation, did not induce hemolysis; however, a combination of H2O2 and nonanal was significantly more hemolytic than nonanal alone. A ratio of 1:2 H2O2/nonanal (the ratio observed in the ozonized liposomes) provided hemolytic activity comparable to that observed with ozonized dioleoyl PC. Among different antioxidants tested, ascorbate, catalase, and glutathione peroxidase partially inhibited hemolysis induced by ozonized liposomes and by H2O2/nonanal mixtures, but they were not protective against the nonanal-induced hemolysis. Identification of H2O2 and aldehydes as cytotoxic chemical species generated from the ozonation of unsaturated fatty acids may have an important bearing on the in vivo toxicity of ozone on the lung as well as on extrapulmonary tissues.     

Degradation of trilinolein by laccase enzymes

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.     

Lipid composition of liver peroxisomes isolated from untreated and clofibrate-treated mice and rats

Peroxisomes were isolated from liver tissue of control and clofibrate-treated adult male NMRI mice and Sprague-Dawley rats. Phospholipids, cholesterol, triglycerides and free fatty acids were measured in the peroxisomes. The fatty acid profiles of the phosphatidylethanolamine, the phosphatidylcholine, the triglyceride and the free fatty acid fractions were also analyzed. Phosphatidylethanolamine was the dominating phospholipid in peroxisomes from untreated animals. The fatty acid profiles of phosphatidylethanolamine, free fatty acids and triglycerides were similar for untreated mice and rats but differences between the species were observed in the pattern derived from phosphatidylcholine. Phosphatidylcholine was the most abundant phospholipid after clofibrate treatment. Clofibrate treatment caused an increase in the concentrations of phospholipids and unsaturated long-chain fatty acids and a decrease in the concentrations of triglycerides, free fatty acids, cholesterol and shorter saturated fatty acids.