An adaptation of the needle biopsy procedure of Beynen and Katan for human adipose tissue, which yields 2-10 mg adipose samples, is described and evaluated. Micromethods are presented for the analysis of alpha-tocopherol, cholesterol and fatty acids in each adipose specimen. The needle biopsy procedure, which uses a Vacutainer to create suction, is compared with a punch biopsy method. The needle biopsy is rapid (6 samples/hr), simple and unobjectionable to the subjects, and provides samples with reproducible ratios of cholesterol and alpha-tocopherol. Unlike the punch biopsy, the needle biopsy reliably obtains specimens with a lipid composition typical of adipocytes. The needle biopsy method is adaptable to nutritional studies of tocopherol and fatty acid metabolism in adipose, and to studies of hazardous compounds stored in adipose. The linoleic acid content of adipose from residents of the West Coast was found to be considerably higher than values reported earlier. The adipose fatty acid data indicate an increase in human adipose linoleate when compared with earlier reports and suggest a trend toward increasing linoleic acid in the American diet.

Adipose Tissue↗

PubMed1988

Vitamin E distribution in ocular tissues following long-term dietary depletion and supplementation as determined by microdissection and gas chromatography-mass spectrometry.

Vitamin E is thought to be important for protection of polyunsaturated fatty acids (PUFA) from oxidative damage. A microbiochemical procedure using microdissection and gas chromatography-mass spectrometry was developed to determine vitamin E distribution in ocular tissues in a rodent model, with the eventual goal of using it in a study of phototoxic degeneration of the retina, where PUFA oxidation is potentially the causal mechanism. Sample preparation was achieved by freeze-drying the retina followed by micro-dissection to obtain the desired structures for analysis. A deuterated alpha-tocopherol internal standard is added to the tissue sample before extraction and derivatization which are achieved in a single step. The data presented show the vitamin-E content in various structures of the retina, particularly the outer segments and retinal pigment epithelium (RPE); however, the vitamin E content of other ocular tissues is also included. Data were obtained from albino and pigmented rats receiving vitamin E-depleted, supplemented, and regular chow diets, and from rabbits and cats receiving regular chow diets formulated for each species. Within all dietary groups the highest concentration of vitamin E was located in the RPE followed by the outer segments of the photoreceptor cells. Other ocular tissues consistently contained lower amounts of vitamin E. Different tissues were depleted of vitamin E at different rates and this points out the importance of determining vitamin E levels in tissues of interest in studies on the consequences of dietary depletion.

Animals↗

PubMed1987

Detection of phospholipid peroxides in biological samples.

Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid peroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review presents an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in vivo.

Animals↗

PubMed1986

Occurrence of 4-hydroxyalkenals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry.

Malondialdehyde measurements have been the major tool for studying relationships between lipid peroxidation and tissue pathology. Recently, we presented a novel gas chromatography-mass spectrometry method for direct detection of phospholipid peroxides with picogram sensitivity based on transesterification of phospholipids or triglycerides to form pentafluorobenzyl esters. Under some circumstances the reactive primary oxidation products break down. Therefore, we developed a convenient, high sensitivity method to detect more stable secondary lipid oxidation products, the 4-hydroxyalkenals. The method accomplishes a facile extraction of 4-hydroxynonenal from tissues by forming pentafluorobenzyl oxime derivatives to displace aldehydes from Schiff base linkages. 4-hydroxynonenal was found in heart, liver, adrenal, and testis from rats and was detected to the 10-100 pg level by the current method.

Aldehydes↗

PubMed1986

Transesterification of phospholipids or triglycerides to fatty acid benzyl esters with simultaneous methylation of free fatty acids for gas-liquid chromatographic analysis.

A novel method is presented for transesterification of fatty acid esters in phospholipids and triglycerides to benzyl esters while simultaneously recovering free fatty acids as methyl esters. Transesterification is catalyzed by 0.2 M (m-trifluoromethyl phenyl)trimethyl ammonium hydroxide in methylene chloride, 10% (v/v) benzyl alcohol, and 1% (w/v) potassium tert-butoxide, and is complete in 30 min at room temperature. Methyl esters of all common fatty acids separate from the benzyl esters formed from phospholipids. This method has broad utility and is applicable to the formation of esters optimized for detection by absorbance or fluorescence (high performance liquid chromatography), electron capture (gas-liquid chromatography), or negative ion chemical ionization (gas-liquid chromatography-mass spectrometry).

Benzyl Alcohol↗

PubMed1985

Rapid analysis of the major classes of retinoids by step gradient reversed-phase high-performance liquid chromatography using retinal (O-ethyl) oxime derivatives.

A rapid step-gradient reversed-phase high-performance liquid chromatography (HPLC) method is presented for analysis of the major classes of retinoids in tissues. Retinal was converted into a new derivative, retinal (O-ethyl) oxime, since the standard derivative, retinaloxime, co-elutes with retinol on reversed-phase HPLC. The most abundant naturally occurring retinyl esters, retinyl palmitate and retinyl stearate, were eluted within 12 min to complete the separation. Retinoids were extracted in the presence of an antioxidant, butylated hydroxytoluene, and a lipid carrier, cholesterol. Recoveries of 98-100% were obtained from tissue samples by internal addition for the retinoids tested (retinol, retinal and retinyl palmitate); and the absolute recovery of endogenous retinal from rat eyecups was confirmed by spectrophotometric measurements of rhodopsin. Extraction was carried out in an air atmosphere and under subdued incandescent light rather than requiring inert atmosphere and safe-light conditions used in most methods. Cis-trans isomers were not separated under the reversed-phase HPLC conditions employed. Quantitation was carried out using retinyl acetate as internal standard and the day to day precision was better than 3.5%. A sensitivity of about 1 ng is obtained for all retinoids using absorbance monitoring at 325 nm and a C18 5 micrometers column with 12% reversed-phase loading. The tocopherols can also be separated and detected simultaneously with similar sensitivity by this method using a fluorescence detector in series [G. J. Handelman, L. J. Machlin, K. Fitch, J. J. Weiter and E. A. Dratz, J. Nutr., 115 (1985) 807].

Animals↗

PubMed1985

Gas chromatography-mass spectrometry method for determination of phospholipid peroxides; I. Transesterification to form methyl esters.

The purpose of this study is to develop methods for determining the chemical species of lipid peroxides that occur in various types of tissue pathology. Experiments are aimed at determining the phospholipid peroxides associated with retinal degeneration as the initial test case. Phospholipid hydroperoxides are synthesized by photosensitized oxidation, chemically characterized and used to develop an effective and simplified method to identify and measure phospholipid hydroperoxides by gas chromatography-mass spectrometry (GC-MS). A sensitive reverse phase high performance liquid chromatography (HPLC) method is also presented to separate peroxidized phospholipids from phospholipids. For GC-MS, phospholipid peroxides are reduced with sodium borohydride and transesterified to form fatty acid methyl esters using a mild quaternary ammonium hydroxide catalyst. The hydroxyl groups produced by reducing the hydroperoxides are formed into trimethylsilyl ethers and GC-MS is employed (with electron ionization and negative ion chemical ionization) to identify oxidized fatty acids at the 10 ng level. Photooxidation of (palmitoyl)(linoleoyl) phosphatidylcholine yielded equal amounts of the conjugated (9 and 13 isomers) and the nonconjugated (10 and 12 isomers) linoleoyl hydroperoxides. Photooxidation of rat retina total lipids yielded oxidation products of oleolyl (18:1) esters as well as the conjugated and nonconjugated oxidation products of arachidonoyl (20:4) and docosahexaenoyl (22:6) esters virtually all of which arise from phospholipids. The nonconjugated products are of interest as indicators of photosensitized light damage in retina and other tissues. It is notable that all the possible singly oxidized products are found with the exception of the 4, 5 and 7 hydroperoxides of 22:6 and the 5 hydroperoxide of 20:4. It appears that the approach of singlet oxygen is strongly inhibited in the sterically hindered region near the phospholipid head groups.

Animals↗

PubMed1985

Gas chromatography-mass spectrometry method for determination of phospholipid peroxides; II. Transesterification to form pentafluorobenzyl esters and detection with picogram sensitivity.

The purpose of this study is to develop a higher sensitivity method to detect phospholipid peroxides in tissues to more effectively investigate the role of lipid peroxidation in pathology. We recently presented a gas chromatography-mass spectrometry (GC-MS) method for identification and measurement of phospholipid peroxides at the 10 ng level (van Kuijk, et al. J. Free Rad. Biol. Med., 1:215-225, 1985) based on transesterification to form fatty acid methyl ester derivatives. While this method is quite useful under some circumstances, more sensitivity was desirable to analyse trace amounts of lipid peroxidation products in small samples of various tissues. We present a method to prepare pentafluorobenzyl esters of fatty acids and hydroxy fatty acids directly from phospholipids by transesterification at room temperature or from triglycerides at 60 degrees C. Oxidized lipids are extracted into dichloromethane and peroxide functions are reduced with sodium borohydride. Transesterification of phospholipids is carried out in dichloromethane with 20% (v/v) pentafluorobenzyl alcohol, 1% (w/v) potassium tert-butoxide and 0.2 N (m-trifluoromethyl phenyl) trimethyl ammonium hydroxide at room temperature for 30 min. Pentafluorobenzyl esters are purified on open silica columns, O-trimethyl silyl derivatives of the alcohol functions are formed, and GC-MS is carried out with negative ion chemical ionization detection. The single or double oxidation products of oleate, linoleate, arachidonate, and docosahexaenoate were detected with 10 pg sensitivity in oxidized rat retina phospholipids or synthetic phospholipids, by negative ion chemical ionization with specific ion monitoring of carboxylate anions.

Borohydrides↗

PubMed1985

Consecutive action of phospholipase A2 and glutathione peroxidase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes.

The purpose of this study was to investigate the ability of selenium-dependent glutathione peroxidase to reduce phospholipid hydroperoxides in membrane bilayers and to develop a method to measure the peroxide content of phospholipids. Phospholipid hydroperoxides were synthesized by photooxidation of 1-palmitoyl 2-linoleoyl phosphatidylcholine and characterized by gas chromatography-mass spectrometry. Phospholipid hydroperoxides in phosphatidylcholine bilayers showed no detectable reactivity with Se-dependent glutathione peroxidase (the reaction is at least 65,000 times slower than with an available hydroperoxide). However, after the phospholipid hydroperoxides were preincubated with phospholipase A2, the free fatty acid hydroperoxides became available as a substrate for Se-dependent glutathione peroxidase. The enzyme assay can be used for convenient determination of peroxide values in phospholipids at the 1 nmole level and free fatty acid hydroperoxides can be distinguished from phospholipid hydroperoxides by omitting phospholipase A2. The accuracy of the enzymatic method was confirmed using an improved colorimetric chemical assay to measure peroxide values of phospholipid hydroperoxides to the same sensitivity. The chemical assay was not linear in the presence of high levels of lipid, but at low levels of lipid the peroxide values of phospholipid hydroperoxides measured by both methods agreed to within 1%. Since high levels of lipid inhibited the chemical assay, the enzyme assay is more accurate for determination of peroxides in membranes and tissues. The possible role of phospholipase deficiencies as a causal factor in degenerative diseases thought to be due to lipid peroxidation, such as Neuronal Ceroid Lipofuscinosis (Battens disease), is discussed.

Colorimetry↗