Systematic isolation and identification of membrane lipid oxidation products.

This report discusses the analytical procedure by which it is possible to isolate and identify the oxidation products of cellular and subcellular membrane lipids. The key point of this procedure is the method used for the transmethylation of the lipid material isolated from the tissues. In effect, both the conversion of the glycerides into methyl esters and the reduction of the hydroperoxyl groups into the corresponding hydroxyl groups is performed in one step, without breaking any oxirane rings that may be present. The methyl esters containing functional groups introduced by oxidative processes are separated from the non-modified ones by preparative TLC and are identified by GLC and GC-MS.

Lipid peroxidation in cancer cells: chemical and physical studies.

Our studies on the biochemical composition and the structural organization of smooth and rough endoplasmic reticulum isolated from Morris hepatomas 9618A and 3924A confirm the results obtained employing the total microsomal fraction. We have definitely established the following facts: (1) Tumor subcellular organelles exhibit the very low degree of peroxidizability that has been shown to be related to the growth rate of the tumor. (2) Associated with such a low susceptibility to peroxidation are (a) changed lipid composition of cellular membranes, whose content in polyunsaturated fatty acid is markedly decreased, and (b) changed static and dynamic properties of the membrane. Previously it was also found that cellular oxy-radical scavenging enzymes are markedly reduced. From these data, it is possible to infer that tumor membranes are altered structurally and functionally in part as the result of an oxy-radical-induced damage that occurs in vivo under conditions of oxygen toxicity. This seems to be supported by recent findings that the spontaneous increase in growth rate of the originally very slow-growing Morris hepatoma 9618A results also in the loss of cytochrome P-450 (an important intramembraneous propagator of lipid peroxidation) as well as of C20:4 and C22:6. Studies performed by GLC and GC-MS on the fatty acid residues of phospholipids of rat liver microsomes show the presence of C20:3-OH and C18:1-OH, but no hydroxyl derivatives of low molecular weight aldehydes. The hydroxyl derivatives of arachidonic acid and linoleic acid are present in much smaller amounts in the microsomes isolated from H9618A and H3924A.

Synthesis of highly pure dolichyl and cholesteryl esters.

Highly pure dolichyl and/or cholesteryl esters can be synthesised at room temperature by reacting a defined pure dolichol and/or pure cholesterol with an acyl chloride in the presence of pyridine. During the reaction some acyl anhydride originates as a secondary product. It can be hydrolysed by methanolic KOH and removed by a rapid percolation of the reaction mixture through a small alumina column. A final purification of the raw product obtained by silicic-acid/celite column chromatography makes it possible to obtain highly pure esters with a satisfactory yield.

Phospholipid composition of photosynthetic membranes of Rhodopseudomonas capsulata.

The phospholipids and the fatty acids present in membranes of cells of Rhodopseudomonas capsulata, grown photosynthetically in anaerobiosis, were analyzed by thin layer chromatography and gas chromatography-mass spectrometry. The three phospholipids detected, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol, contained about 80% of a single monounsaturated C18 fatty acid, cis-vaccenic acid. These membranes offer therefore a naturally occurring model system endowed with an extremely simplified phospholipid complement. The data indicate moreover that the biosynthetic pathway of unsaturated fatty acids present in these facultative aerobic bacteria proceeds only via the 3-hydroxydecanoyl acyl carrier protein dehydratase (E.C. 4.2.1.60).

Hydrocarbons in hens injected with inactivated oil adjuvant vaccine.

The radioactivity of different organs and tissues of laying hens injected with inactivated oil adjuvant vaccine containing [n-1-14C] octadecane was measured. It was shown that the hydrocarbons injected with the vaccination diffuse in relatively short periods of time to all the tissues, especially to those of the organs with greater blood supply, and that the hydrocarbons are largely eliminated by means of the eggs.

Investigation of the hydrocarbons found in the tissues of chickens injected with inactivated oil adjuvant vaccine.

Dead virus vaccines emulsified with mineral oils and given by intramuscular or subcutaneous routes are widely used in prophylaxis of several conditions in poultry. In order to establish in what amount and how long mineral oils remain in the vicinity of the injection site, an investigation was made on broilers vaccinated against Newcastle disease with this type of vaccine. At different time intervals between the 10th and 282th day after vaccination, soft tissues were removed from over the bone surface of the tibia of inoculated legs and of control legs, and were subsequently examined. The results showed that the hydrocarbons introduced by vaccination remain in considerable amounts in the tissues examined, even 282 days after vaccination.

Simultaneous reversed-phase high-performance liquid chromatographic separation of non-polar isoprenoid lipids and their determination.

A procedure for the rapid identification and determination of non-polar isoprenoid lipids from animal tissues was developed. The complete determination can be carried out by reversed-phase HPLC of just two samples. The first, extracted from unaltered tissues and suitably processed by column chromatography, provides information about free cholesterol, cholesteryl esters, coenzymes Q, free dolichols and dolichyl esters. The second, obtained from saponified tissues, can be used to detect both total cholesterol and total dolichols. Specific calibration graphs were constructed for the determination of the different constituents.

Anomalous behaviour of lycopene by HPLC on cyano-amino polar phase.

By reversed phase HPLC the highly pure lycopene produces only one peak, that corresponds to the all-trans polyunsaturated system. Commercial standards, however, show generally two peaks that correspond to the molecule in all-trans configuration (about 90%) and probably to its 13-cis geometrical isomer (about 10%), respectively, independent of the solvent used to inject the sample. An anomalous HPLC behaviour was observed by analysing a commercial sample of lycopene by normal-phase HPLC. In this case the chromatogram showed different profiles depending on the solvent used to prepare the solution to be injected. A relationship between such unusual behaviour and the polarity of the solvent used to dissolve the sample was tentatively found. It is worth emphasizing that in the same HPLC conditions the behaviour of beta-carotene does not show the above described phenomenon.

Proposal of an analytical method for the study of the oxidation products of membrane lipids.

A method of lipid sample preparation for GLC and GC-MS analysis is presented which would seem particularly suitable for studying the chemical composition of the oxidation products of membrane lipids. The method requires small amounts of lipid, is quite rapid and avoids the formation of oxidation artifacts during the different analytical steps. Due to the small quantities of lipid material used it is possible that the method is not rigorously quantitative. Transmethylation of lipids is carried out with methanolic NaBH4 in the presence of NaOH. The reaction is complete in 20 min both on neutral and on polar lipids. In the course of the transmethylation the hydroperoxidic groups are reduced to the corresponding hydroxy groups and can be located through the GC-MS spectra of the corresponding TMS derivatives. The epoxidic rings that may be present are not hydrolyzed. They are located by opening the ring with BF3/MeOH and by GC-MS analysis of the corresponding methoxy-hydroxy derivatives.