Richard O. Recknagel

[40] Spectrophotometric detection of lipid conjugated dienes

Publisher Summary Like many other substances, naturally occurring lipids exhibit simple end absorption in ultraviolet light as the wavelength is lowered toward 200 nm. The spectra of a variety of organic molecules containing conjugated dienes, however, are characterized by intense absorption, the so-called K band, which may range, with respect to peak absorption, from 215 to 250 nm, depending on nearby substituent groups. Ultraviolet spectrophometric detection of conjugated dienes has been used for many years in the food industry for the detection of autoxidized lipids. The method appears to have been applied for the first time to the problem of liver cell lipid peroxidation of toxigenic origin in 1966 and has been widely used since. For a variety of pathological processes, the question has been raised whether peroxidative decomposition of membrane lipids has occurred in vivo . A second principle of the method recognizes that for whole-animal studies involving possible lipid peroxidation, the fraction of endogenous lipids actually peroxidized may not only be low, but the process of lipid peroxidation may be confined to a particular subcellular structure.

Pathological mechanisms in carbon tetrachloride hepatotoxicity.

Liver cell injury induced by carbon tetrachloride involves initially the metabolism of carbon tetrachloride to trichloromethyl free-radical by the mixed function oxidase system of the endoplasmic reticulum. It is postulated that secondary mechanisms link carbon tetrachloride metabolism to the widespread disturbances in hepatocyte function. These secondary mechanisms could involve the generation of toxic products arising directly from carbon tetrachloride metabolism or from peroxidative degeneration of membrane lipids. The possible involvement of radical species such as trichloromethyl (.CCl3), trichloromethylperoxy (.OOCCl3), and chlorine (.Cl) free radicals, as well as phosgene and aldehydic products of lipid peroxidation, as toxic intermediates is discussed. Data do not support the view that an increase in cytosolic free calcium is important in the toxic action of carbon tetrachloride or bromotrichloromethane. In addition, carbon tetrachloride-induced inhibition of very low density lipoprotein secretion by hepatocytes is not a result of elevated levels of cytosolic free calcium.

Early onset of lipoperoxidation in rat liver after carbon tetrachloride administration

Abstract Peroxidative decomposition of rat liver microsomal lipids, as evidenced by the appearance of diene conjugation absorption, is more than half-maximal within 5 minutes after intragastric administration of carbon tetrachloride in mineral oil. In the microsome fraction the degenerative process is complete within 15 minutes. Peroxidative decomposition of mitochondrial lipids is much slower than for microsomal lipids. To our knowledge this explosive peroxidative decomposition of liver cell microsomal lipids is the most rapid pathological alteration yet noted for carbon tetrachloride liver damage under comparable conditions of dosage and route of administration of this toxic haloalkane. The significance of this finding as supporting evidence for a lipoperoxidation hypothesis for carbon tetrachloride hepatotoxicity is discussed.

Critical role of lipid peroxidation in carbon tetrachloride-induced loss of aminopyrine demethylase, cytochrome P-450 and glucose 6-phosphatase

Abstract Liver microsomal glucose 6-phosphatase. cytochrome P-450 and aminopyrine demethylase all decrease rapidly in the CCl 4 -poisoned rat. It has been suggested that such enzyme loss may be due to direct attack on enzyme catalytic sites by free radical cleavage products of CCl 4 metabolism. An alternate view has favored the suggestion that peroxidative decomposition of lipids is an intermediate link between initial homolytic cleavage of the CCl 3 -Cl bond and eventual loss of these enzymes. We have subjected these two ideas to a critical test. In an anaerobic system in vitro containing liver microsomes supplemented with an NADPH-generating system, and in the presence of EDTA, all added CCl 4 is metabolized in 90 min. About one-third appears as CHCl 3 amd most of the remainder is covalently bound to microsomal lipids and proteins. In this anaerobic system in vitro there is no evolution of malonic dialdehyde. During the period of conversion of CCl 4 to CHCl 3 , when extensive binding of 14 C from 14 CCl 4 is also taking place, there was no detectable loss of either glucose 6-phosphatase or cytochrome P-450, and aminopyrine demethylase activity decreased minimally. In the same system, under aerobic conditions and without EDTA, there is vigorous lipid peroxidation and all three of these enzyme systems decrease markedly. These experiments demonstrate conclusively that CCl 3 -Cl bond cleavage and covalent binding of products of CCl 4 metabolism do not constitute a mechanism for loss of microsomal glucose 6-phosphatase, cytochrome P-450 or aminopyrine demethylase for the particular anaerobic conditions employed in vitro . By extension they suggest, but do not prove, that covalent binding of CCl 4 carbon probably does not constitute a mechanism for CCl 4 -induced loss of these enzymes in vivo . Rather, these experiments support the view that lipid peroxidation is an obligatory link between initial CCl 3 -Cl bond cleavage and loss of these enzymes.

Destruction of liver microsomal calcium pump activity by carbon tetrachloride and bromotrichloromethane

Abstract Disturbed cellular calcium homeostatis has been observed during carbon tetrachloride (CCl4) poisoning, with large alterations in calcium content occurring 8 hr after administration. Moore el at. [10] have shown that the hepatic smooth endoplasmic reticulum can sequester calcium and that this ability is decreased severely within 30 min after CCl4 administration to rats. It was suggested that disturbed endoplasmic reticulum calcium pump activity may have a critical role in the expression of CCl4 hepatotoxicity. We examined the effect of bromotrichloromethane (BrCCl3) and CCl4 metabolism on the calcium pump of Fe2+-free rat liver microsomes. It was determined that severe deficits in calcium uptake can be correlated with minimal lipid peroxidation induced by these agents. At a given level of lipid peroxidation, calcium uptake was affected more severely than were the activities of the microsomal enzymes glucose-6-phosphatase and aminopyrine demethylase. Calcium uptake was increased 7-fold by the presence of 5 mM ATP in incubations prior to assay of calcium sequestration. Lipid peroxidation induced by BrCCl3-NADPH was accompanied by leakage of calcium from calcium-loaded microsomes. These results strengthen the possibility that disturbances in intracellular calcium homeostasis may be a key event in liver injury induced by BrCCl3 and CCl4.

Early incorporation of carbon-labeled carbon tetrachloride into rat liver particulate lipids and proteins☆

Abstract After intragastric administration to rats of 14 C-labeled carbon tetrachloride in mineral oil, the lipids of the liver microsome fraction become labeled very rapidly. The process of microsomal lipid labeling is complete within 5 minutes. There is a slight decline in the degree of labeling of microsomal lipids from 5 to 15 minutes, after which the level of radioactivity remains constant up to 90 minutes when measurements were terminated. Labeling of mitochondrial lipids was one-fourth to one-seventh that of microsomal lipids. Also, labeling of microsomal proteins was much less than labeling of microsomal lipids. The rapid incorporation into microsomal lipids of 14 C from intragastrically administered 14 CCl 4 , and the rapid progress of microsomal lipid peroxidation known to accompany the labeling of the microsomal lipids, fit the expectations of the lipoperoxidation hypothesis. To our knowledge, these two events occur with an explosive rapidity far beyond any other known consequences of the reaction of carbon tetrachloride with the liver. The experimental results offer strong presumptive evidence that cleavage of the carbon-chlorine bond and consequent microsomal lipid peroxidation are the keys to carbon tetrachloride hepatotoxicity.

No chemical evidence of hepatic lipid peroxidation in acute ethanol toxicity.

Abstract The appearance of abnormal diene conjugation absorption in rat liver microsomal lipids peroxidized in vitro , and following acute carbon tetrachloride poisoning in vivo , as reported earlier from this laboratory, was confirmed. If peroxidative decomposition of liver lipids is also involved in acute ethanol hepatitis, then the characteristic diene conjugation absorption should be present in liver lipids after ethanol administration. With this method, we were unable to detect any evidence of lipid peroxidation in any subcellular fraction from 1 to 24 hours after ethanol feeding. Peroxidation of lipids also leads to the appearance of lipid peroxides. By use of a semimicro iodimetric procedure, we could readily detect small amounts of lipid peroxides in peroxidized oils and in rat liver microsomes peroxidized in vitro . However, we could not detect any peroxides in rat liver lipids from 1 to 18 hours after ethanol feeding. These negative findings greatly complicate the current situation with regard to the molecular pathobiology of acute ethanol hepatitis.

Carbon tetrachloride hepatotoxicity:status quo and future prospects

A new line of work has opened up in the study of CCl4 hepatotoxicity, centred on the possibility that the action of this hepatotoxin may involve a disturbance in hepatocellular calcium homeostasis.

Effect of carbon tetrachloride on release of free fatty acids by rat adipose tissue.

Conclusions 1. Administration of epinephrine to rats increased release of free fatty acid from adipose tissue in vitro. 2. Administration of carbon tetrachloride had no effect on release of free fatty acids from adipose tissue in vitro. 3. These results are not consistent with the hypothesis that carbon tetrachloride administration elicits a supporting adrenal discharge.

New data on the question of lipoperoxidation in carbon tetrachloride poisoning.

Abstract Administration of antioxidants to rats prior to carbon tetrachloride protects against lethality of the hepatotoxin. The protective effect implicates destructive peroxidations as a vector of the toxic action. However, when evidence for increased lipoperoxidation was sought through use of the thiobarbituric acid reaction, none could be found throughout the first 25 hours after intragastric carbon tetrachloride administration. However, it was shown that rat liver whole homogenates, prepared in saline-phosphate buffer at pH 7.4 and containing EDTA, when incubated at 38° could induce disappearance of TBA-positive material generated by a prior incubation. Authentic malonic dialdehyde, added as substrate, was also metabolized. The enzyme system is localized in the mitochondrial fraction and was inactive in the absence of ATP, Mg ++ , and inorganic phosphate. The significance of the failure to find TBA-positive material in livers of carbon tetrachloride-poisoned rats is thus greatly lessened, since evidently the TBA-positive material can be further metabolized. It was also shown that the system in the whole homogenate, which generates TBA-positive material in mild acid media containing no EDTA, is inhibited by small amounts of carbon tetrachloride added in vitro . However, the loss of this capacity in whole homogenates prepared from livers of carbon tetrachloride-poisoned rats is probably a secondary phenomenon not due to a direct effect of the hepatotoxin.