Effects of the ratio of polyunsaturated and monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid concentrations

The effects of dietary monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid+MUFA/saturated fatty acid (PUFA+MUFA/SFA) ratio on plasma and liver lipid concentrations were studied. In experiment I, when rats were fed with 40% fat (energy%, PUFA/SFA ratio 1.0) and 1% (w/w) cholesterol (C) diets for 21 d, a large amount of MUFA (28.1 energy%, PUFA+MUFA/SFA=5.7) in the diet was found to increase the plasma total C, triacylglycerol (TAG), and phospholipid (PL) as compared with the low-MUFA diet (7.0 energy%, PUFA+MUFA/SFA=1.4). The plasma very low density lipoprotein (VLDL)-C, VLDL-TAG, VLDL-PL, and low density lipoprotein (LDL)-C increased significantly in the high-MUFA diet group, but high density lipoprotein (HDL)-C did not change significantly. The high-MUFA diet resulted in greater accumulation of liver C but lesser accumulation of TAG. In experiment II, when dietary SFA was fixed at a certain level (13.2 energy%; PUFA+MUFA/SFA=2.0), rats given a larger amount of MUFA (23.1 energy%; PUFA/MUFA=0.2; MUFA/SFA=1.8) showed higher plasma and liver C levels than did the low-MUFA diet (7.7 energy%; PUFA/MUFA=2.5; MUFA/SFA=0.6). When PUFA was fixed at a certain level (24.4 energy%), there was not a significant difference in the plasma C level between the high-and low-MUFA dietary groups (PUFA+MUFA/SFA=4.8 and 8.4), but the higher PUFA+MUFA/SFA diet, which was high in MUFA/SFA ratio, significantly decreased the plasma HDL-C and TAG levels. However, when MUFA content was fixed at a certain level (16.4 energy%), no significant difference was observed between the two groups with different PUFA/SFA ratios of 0.2 and 4.1, but liver C level was raised in the higher PUFA/SFA diet. It appears that the PUFA/SFA ratio alone is unsuitable to predict the change of plasma C level, because a large amount of dietary MUFA may lead to an increase of plasma and liver lipids in rats. It seems that the prerequisites for keeping low plasma and liver C are (i) low MUFA/SFA ratio, (ii) high PUFA/MUFA ratio, and (iii) PUFA+MUFA/SFA ratio not to exceed 2.     

The effects of dietary cholesterol on blood and liver polyunsaturated fatty acids and on plasma cholesterol in rats fed various types of fatty acid diet

Male rats were fed on a fat-free diet for 8 weeks and then switched to diets containing 10% hydrogenated coconut oil (HCO), safflower oil (SFO) or evening primrose oil (EPO). Half of each group was also given 1% of cholesterol in the diet. After 5 further weeks, plama, red cell and liver fatty acids were measured in the various lipid fractions. Plasma and liver cholesterol also were estimated. In almost all fractions and on all three diets, feeding cholesterol led to accumulation of the substrates of desaturation reactions and to deficits of the products of these reactions. The results were consistent with inhibition of Δ-6, Δ-5 and Δ-4 desaturation of n−6 essential fatty acids. Since the diets were deficient in n−3 fatty acids, levels were very low but were also consistent with inhibition of desaturation. In contrast, cholesterol had relatively less consistent effects on 20∶3n−9, suggesting that desaturation of n−9 fatty acids was less inhibited. Plasma cholesterol levels rose sharply in the HCO and SFO groups but not at all in the EPO group. EPO contains the product of Δ-6 desaturation, 18∶3n−6, suggesting that conversion of linoleic acid to 18∶3n−6 and possibly to further metabolites may be important for the cholesterol-lowering effect of polyunsaturates.     

How do polyunsaturated fatty acids lower plasma cholesterol levels?

For 30 years it has been known that linoleic acid can lower elevated cholesterol levels. Large increases in linoleic acid have been widely recommended as a way of reducing the risk of cardiovascular disease. Such recommendations have resulted in major dietary shifts in some countries, including the USA. Yet the precise characteristics of the linoleic acid molecule which confer on it cholesterol-lowering properties are unknown. gamma-Linolenic acid, the first essential fatty acid metabolite of linoleic acid, has been found to have cholesterol-lowering actions ca. 170 times greater than the parent molecule, suggesting that linoleic acid must be converted to gamma-linolenic acid to exert its desirable effects on cholesterol metabolism. Aging, sex, diabetes mellitus, alcohol, catecholamines and trans fatty acids and saturated fats can all modulate the delta-6-desaturase enzyme which converts linoleic acid to gamma-linolenic acid. This provides a possible unifying explanation for the actions of these known risk factors for cardiovascular disease.     

A new relationship between total/high density lipoprotein cholesterol and polyunsaturated fatty acids

Dietary and plasma fatty acids have been linked to total cholesterol but not to the ratio of total/high-density lipoprotein cholesterol (TC/HDLC). To evaluate the relationship between dietary and plasma levels of polyunsaturated fatty acids (PUFA) and TC/HDLC, we analyzed cross-sectional and longitudinal data using 519 plasma samples (50% men, 50% women) from subjects participating in the Framingham Heart Study and results from a study feeding diets rich in either n-6 linoleic acid or n-3 α-linolenic acid with or without fish oil supplements (n-3 derivatives). Values of TC/HDLC are inversely related to the percent of plasma PUFA when both variables are measured at the same time in different subjects,R=0.82,P<0.000001. PUFA in phospholipids increase in response to increased dietary intake of different PUFA, either n-3 or n-6 or fish oils. There was a highly significant inverse relationship between TC/HDLC and the percent of PUFA in phospholipids,R=0.97,P<0.001. The relationship was similar regardless of the source and type of dietary fatty acids. A similar relationship existed when only the baseline points were considered. When plasma PUFA % increases, either in response to a diet high in PUFA or across different subjects, TC/HDLC ratios decline. Evaluation of plasma fatty acid profiles and increased balanced dietary intake of PUFA to bring fatty acid profiles of subjects with low PUFA plasma levels closer to the profile of a healthy reference group is an effective approach to reduce high TC/HDLC. Reductions of more than 50% in TC/HDLC appear feasible with dietary modification alone. Further research into fatty acid metabolic activity may determine the biochemical basis of common dysplipidemias.     

The cardiac antiarrhythmic effects of polyunsaturated fatty acid

Each year in the United States alone some 250,000 persons die within one hour of an acute myocardial infarction. These deaths are largely due to ischemia-induced ventricular arrhythmias, primarily ventricular fibrillation (VF). Thus a safe, simple means of preventing such arrhythmias has considerable public health benefit potential. We have demonstrated that the intravenous infusion of n-3 polyunsaturated fatty acids (PUFA) from fish oils will prevent ischemia-induced VF in prepared, nonanesthetized, exercising dogs, confirming earlier feeding studies in rats. We show that this protective effect is due to an action of the free acidic form of the PUFA to alter the electrophysiology of individual cardiac myocyte so that the cells are electrically more stable. The electrophysiologic effects, in turn, result from direct and specific effects of the PUFA to block the fast voltage-dependent sodium channels. The binding of the free fatty acids is directly to the protein of the sodium channels and results in prolongation of the inactivated state of these channels. Other ion channels are also affected by the PUFA. Two clinical trials with n-3 PUFA are mentioned which inadvertently support the antiarrhythmic potential of PUFA ingestion.     

Diets rich in lean beef increase arachidonic acid and long-chain ω3 polyunsaturated fatty acid levels in plasma phospholipids

Diets rich in meat are claimed to contribute to the high tissue arachidonic acid (20∶4ω6) content in people in Westernized societies, but there are very few direct data to substantiate this assertion. Because meat contains a variety of long-chain polyunsaturated fatty acids (PUFA) that are susceptible to oxidation, we initially examined the effect of cooking on the long-chain PUFA content of beef, and then determined the effect of ingestion of lean beef on the concentration of long-chain PUFA in plasma phospholipids (PL). First, we examined the effect of grilling (5–15 min) and frying (10 min) different cuts of fat-trimmed lean beef on the long-chain PUFA content. Second, we investigated the effect of including 500 g lean beef daily (raw weight) for 4 wk on the fatty acid content and composition of plasma PL in 33 healthy volunteers. This study was part of a larger trial investigating the effect of lean beef on plasma cholesterol levels. In the first two weeks, the subjects ate a very low-fat diet (10% energy) followed by an increase in the dietary fat by 10% each week for the next 2 wk. The added fat consisted of beef fat, or olive oil (as the oil or a margarine) or safflower oil (as the oil or a margarine). This quantity of beef provided 60, 230, 125, 140 and 20 mg/d, respectively, of eicosatrienoic acid (20∶3ω6), 20∶4ω6, eicosapentaenoic acid (20∶5ω3), docosapentaenoic acid (22∶5ω3) and docosahexaenoic acid (22∶6ω3). Grilling for 10–15 min, but not frying, of the fat-trimmed lean beef resulted in 20–30% losses of the 20 and 22 carbon PUFA. The consumption of the lean beef during the first two-week period, when there was a very low level of dietary fat, was associated with significant increases in the proportion and concentration of 20∶3ω6, 20∶4ω6, 20∶5ω3 and 22∶5ω3 in the plasma PL and a significant decrease in the proportion and content of 18∶2ω6. The addition of beef fat or olive oil to the diets containing lean beef did not alter the plasma PL fatty acid profile compared with the very low-fat diet, whereas the addition of safflower oil maintained the significant increases in 20∶4ω6 and 22∶5ω3 but led to decreases in 18∶3ω3 and 20∶5ω3 compared with the very lowfat diet. The results showed that diets rich in lean beef increased the 20∶3ω6, 20∶4ω6 and the long-chain ω3 PUFA levels in the plasma PL. A high level of linoleic acid in diets rich in lean beef prevented the rise in the plasma level of 20∶3ω6 and 20∶5ω3, two fatty acids known to antagonize the effects of 20∶4ω6 on platelet aggregation.     

Linoleic acid-induced fatty acid changes in platelet and aorta of the rat: Effect of age and cholesterol

The influence of age and cholesterol on polyunsaturated fatty acids (PUFa) levels was studied in young and old male Sprague-Dawley rats. Animals were fed a fat-free diet supplemented with 10% (by wt) safflower oil with or without 1% cholesterol for 8 wk. As a result of cholesterol feeding, proportions of linoleic acid (18∶2n−6) and dihomo-γ-linolenic acid (30∶3n−6) were increased and and that of arachidonic acid (20∶4n−6) was decreased in the liver and platelet phospholipids in 64-wk-old rats, suggesting inhibitory effects of cholesterol on 20∶4n−6 synthesis from 18∶2n−6. The prominent age-dependent effect on the levels of PUFA was a retention of C−22 n−3 PUFA, accompanied by decreased C−22 n−6 PUFA and increased 20∶3n−6 in the liver and platelet phospholipids. Ratio of 20∶3n−6/20∶4n−6 increased in 64-wk-old rats regardless of dietary cholesterol, suggesting depressed Δ5-desaturase with age. In aorta phospholipids, 20∶3n−6 content and 20∶3n−6/20∶4n−6 ratio increased with cholesterol supplementation, but not with age. These results suggest that changes of PUFA composition of platelet phospholipids with age are closely linked with changes in liver phospholipids. The 20∶4n−6 content in both platelet and aorta phospholipids is kept constant, despite other n−6 and n−3 PUFA being affected by age.     

Brain development and assessing the supply of polyunsaturated fatty acid

Membrane lipids are necessary for structure and function of the developing nervous system. Rapid synthesis of brain tissue occurs during the last trimester of development of the human brain and the early postnatal weeks. This synthesis of brain structure involves the formation of complex lipids, many of which contain significant quantities of chain-elongated desaturated homologs of essential fatty acids. The present report discusses the implications of change in nutritional status on processes of brain development and metabolic events that involve lipids.     

Trans and polyunsaturated fatty acid content of some bakery fats

Twenty-seven commercial bakery products marketed in Belgium were analyzed by gas-liquid chromatography for their trans (TFA) and polyunsaturated fatty acid (PUFA) content. The mean PUFA level for the bakery fats in our study was 8.85% (S.D. = 9.90%) and the TFA content ranged from 0.0–18.81% (mean = 5.99%; S.D. = 5.02%). The unfavourable fatty acid profile of most of the analyzed bakery fats can be explained by the frequent use of animal fat and partially hydrogenated oils. The average daily intake of TFA from bakery products by the Belgian population was calculated at 0.43g/person/day. As it is expected that their consumption will increase in the future, lower TFA levels in bakery fats are necessary to avoid that these products become a more important dietary source of TFA.     

Long-chain polyunsaturated fatty acids in plasma lipids of obese children

Fatty acid composition of plasma phospholipids (PL), triglycerides (TG), and sterol esters (STE) was determined by high-resolution capillary gas-liquid chromatography in 22 obese children (age: 13.7±1.4 y, body weight relative to normal weight for height: 170±24%, mean ±SD) and compared with data obtained in 25 age-matched healthy controls. There were no differences in the levels of linoleic acid (LA, C_18∶2n-6) in any of the plasma fractions from the obese children and the controls. Obese children exhibited significantly higher values of arachidonic acid (AA, C_20∶4n-6) than controls both in PL (12.6 [2.4] vs. 8.3 [1.4], % wt/wt, [median (interquartile range)],P<0.001) and STE (7.3 [1.8] vs. 6.0 [1.1],P<0.05). Similarly, obese children showed higher values than controls for dihomo-γ-linolenic acid (DHGLA, C_20∶3n-6) in PL (4.0 [0.5] vs. 3.0 [0.6],P<0.001), TG (0.4 [0.1] vs. 0.2 [0.1],P<0.001), and STE (0.9 [0.1] vs. 0.7 [0.1],P<0.01), and for γ-linolenic acid (C_18∶3n-6) in STE (1.1 [0.2] vs. 0.8 [0.2],P<0.001). The AA/LA ratios were higher in obese children than in controls in PL (0.68 [0.16] vs. 0.42 [0.09],P<0.0005) and STE (0.16 [0.04] vs. 0.12 [0.02],P<0.05), whereas the AA/DHGLA ratios were lower in TG of obese children than in controls (3.40 [0.64] vs. 5.10 [1.75],P<0.005). Plasma glucose concentrations were inversely related to AA in TG (r=0.53,P<0.05), and plasma TG concentrations were inversely related to AA in PL and STE (r=−0.49,P<0.05 andr=−0.48,P<0.05) and to the AA/DHGLA ratios in PL (r=−0.57,P<0.01),TG (r=−0.56,P<0.01) and STE (r=−0.56,P<0.01). We conclude that the significantly higher values of n-6 long-chain polyunsaturated fatty acids (LCP) in plasma lipids of obese children than in age-matched controls may be caused by an enhanced activity of Δ6-desaturation, and we speculate that elevated fasting immunoreactive insulin seen in obese children (19.4±8.0 μU/mL) may stimulate synthesis of n-6 LCP fatty acids.     

Application of new methods and analytical approaches to research on polyunsaturated fatty acid homeostasis

New methods and analytical approaches are important to challenge and/or validate established beliefs in any field including the metabolism of polyunsaturated fatty acids (PUFA; polyunsaturates). Four methods that have recently been applied toward obtaining a better understanding of the homeostasis of PUFA include the following: whole-body fatty acid balance analysis, magnetic resonance imaging (MRI), ¹³C nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Whole-boby balance studies permit the measurement of both the percentage of oxidation of linoleate and α-linolenate and their conversion to long-chain PUFA. This method has shown that β-oxidation to CO₂ is normally the predominant metabolic fate of linoleate and α-linolenate. Furthermore, models of experimental undernutrition in both humans and animals show that β-oxidation of linoleate and α-linolenate markedly exceeds their intake, despite theoretically sufficient intake of linoleate or α-linolenate. Preliminary results suggest that by using MRI to measure body fat content, indirect whole-body linoleate balance can be done in living humans, ¹³C NMR spectroscopy provided unexpected evidence that linoleate and α-linolenate were metabolized into lipids synthesized de novo, an observation later quantified by tracer mass balance done using GC-C-IRMS. This latter method showed that within 48 h of dosing with ¹³C-α-linolenate, >80% underwent β-oxidation to CO₂ by suckling rats, whereas 8–9% was converted to newly synthesized lipids and <1% to docosahexaenoate. Further application of these recently developed methods in different models should clarify the emerging importance of β-oxidation and carbon recycling in PUFA homeostasis in mammals including humans.     

Response of plasma lipids to dietary cholesterol and wine polyphenols in rats fed polyunsaturated fat diets

This experiment was designed to evaluate the effects of dietary red wine phenolic compounds (WP) and cholesterol on lipid oxidation and transport in rats. For 5 wk, weanling rats were fed polyunsaturated fat diets (n−6/n−3=6.4) supplemented or not supplemented with either 3 g/kg diet of cholesterol, 5 g/kg diet of WP, or both. The concentrations of triacylglycerols (TAG, P<0.01) and cholesterol (P<0.0002) were reduced in fasting plasma of rats fed cholesterol despite the cholesterol enrichment of very low density lipoprotein + low density lipoprotein (VLDL+LDL). The response was due to the much lower plasma concentration of high density lipoprotein (HDL) (−35%, P<0.0001). In contrast, TAG and cholesteryl ester (CE) accumulated in liver (+120 and +450%, respectively, P<0.0001). However, the cholesterol content of liver microsomes was not affected. Dietary cholesterol altered the distribution of fatty acids mainly by reducing the ratio of arachidonic acid to linoleic acid (P<0.0001) in plasma VLDL+LDL (−35%) and HDL (−42%) and in liver TAG (−42%), CE (−78%), and phospholipids (−28%). Dietary WP had little or no effect on these variables. On the other hand, dietary cholesterol lowered the α-tocopherol concentration in VLDL+LDL (−40%, P<0.003) and in microsomes (−60%, P<0.0001). In contrast, dietary WP increased the concentration in microsomes (+21%, P<0.0001), but had no effect on the concentration in VLDL+LDL. Cholesterol feeding decreased (P<0.006) whereas WP feeding increased (P<0.0001) the resistance of VLDL+LDL to copper-induced oxidation. The production of conjugated dienes after 25 h of oxidation ranged between 650 (WP without cholesterol) and 2,560 (cholesterol without WP) μmol/g VLDL+LDL protein. These findings show that dietary WP were absorbed at sufficient levels to contribute to the protection of polyunsaturated fatty acids in plasma and membranes. They could also reduce the consumption of α-tocopherol and endogenous antioxidants. The responses suggest that, in humans, these substances may be beneficial by reducing the deleterious effects of a dietary overload of cholesterol.     

The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis

Dietary polyunsaturated fatty acid (PUFA) supplementation has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in a recent randomized, controlled trial. These compounds are known to modulate the inflammatory cascade and to influence intestinal health in a variety of ways. Although the pathophysiology of NEC is not well understood, recent evidence suggests that platelet-activating factor (PAF) is a key endogenous mediator of intestinal necrosis in animals. Using a neonatal rat model of NEC that includes the key risk factors of asphyxia and formula feeding, we investigated the role of dietary PUFA supplementation on the incidence and pathophysiology of NEC. Our findings suggest that PUFA reduce the incidence of NEC by modulating PAF metabolism and endotoxin translocation.     

Effects of dietary n−3 fatty acid-enriched chicken eggs on plasma and tissue cholesterol and fatty acid composition of rats

The purpose of this study was to examine the effects of feeding n−3 polyunsaturated fatty acid (PUFA)-enriched chicken eggs on plasma and liver cholesterol levels and fatty acid composition in rats. Eggs were collected from laying hens fed diets containing 10% flax seed (Hn−3), 12% sunflower seed (Hn−6), or wheat and soybean meal control (CON). Yolk powders were prepared and fed at the 15% level to weanling female Sprague-Dawley rats for 28 days. Consumption of n−3 PUFA-enriched yolks significantly reduced both plasma and liver total cholesterol. Liver total lipids and phospholipids of rats fed Hn−3 diet were enriched with linolenic, eicosapentaenoic, and docosahexaenoic acids with a concomitant reduction of arachidonic acid in liver phospholipids. The plasma cholesterol of rats fed yolk powders enriched with n−6 PUFA (mainly linoleic acid) was reduced to the same extent as in those fed the n−3 enriched, but the liver cholesterol was significantly increased, indicating differential effects of dietary n−3 and n−6 PUFA. The results demonstrated that the cholesterolemic and tissue lipid modulating properties of chicken eggs could be modified in a favorable way by altering the fatty acid composition of yolk lipids through manipulation of laying hen diets.     

Specificity of polyunsaturated fatty acid release from rat brain synaptosomes

Release of specific polyunsaturated fatty acids from cell membranes may have a significant implication in biological function, considering the involvement of various fatty acids in cell signal transduction. In the present study, release of polyunsaturated fatty acids from rat brain synaptosomes by endogeneous synaptosomal lipase activity was examined in comparison to that by cobra venom phospholipase A₂ (Naja naja naja). Cobra venom phospholipase A₂ (Naja naja naja) preferentially hydrolyzed docosahexaenoic acid (22∶6n−3) from both synaptosomes and lipid muxtures containing similar classes of lipids commonly found in the brain. Arachidonic acid (20∶4n−6) and oleic acid (18∶1n−9) were also hydrolyzed; however, monoene species was hydrolyzed slower than were polyenoic species in synaptosomes. Phosphatidylethanolamine was the most preferred phospholipid class for release of 22∶6n−3 fatty acid from both lipid mixtures and synaptosomes. In contrast to hydrolysis by cobra venom phospholipase A₂, endogenous synaptosomal lipase activity preferentially hydrolyzed 20∶4−6 from rat brain synaptosomes, despite the high abundance of 22∶6n−3 in synaptosomal membranes. Preferential release of 20∶4n−6 was observed over a wide range of pH values and calcium concentrations. Synaptosomal 22∶6 species appeared to be resistant to hydrolysis even after stimulation with various agents such as phorbolmyristate, suggesting that physiological importance of 22∶6−3 in neuronal membranes may not be as the release fatty acid.     

Response of free and esterified plasma cholesterol levels in the mongolian gerbil to the fatty acid composition of dietary lipid

The present study was undertaken to investigate the potential suitability of the Mongolian gerbil as a useful animal model to study the effects of dietary fats on plasma cholesterol levels. Semipurified diets containing either 20% lard, 20% safflower oil, or 19.5% beef tallow +0.5% safflower oil were equalized to contain 0.01% cholesterol and 0.05% plant sterol and were fed for a four week experimental period. The proportions of total calories contributed by fat, protein and carbohydrate (starch/sucrose ratio of 2∶1) were 40, 14 and 46%, respectively, so as to approach the distribution of calories within the average North American diet. Free, esterified, and total plasma cholesterol levels of male gerbils were determined weekly by gas liquid chromatography after drawing blood via a serial sampling technique. After 1, 2, 3, and 4 weeks of feeding the experimental diets, total cholesterol levels were lowest in the safflower oil fed animals; the corresponding values were 19–64% greater in gerbils fed lard and 68–91% greater in those consuming the beef tallow diet. Cholesterol in the free form generally responded more dramatically to the type of dietary lipid than did cholesterol in the ester form. Irrespective of the type of dietary lipid or the length of the feeding trial, 18–23% of the total plasma cholesterol was in the free form and 77–82% was present as the ester. In view of the similarity to the human of the relative proportions of free versus esterified cholesterol, the type of cholesteryl esters, and their response to dietary manipulation, the gerbil appears to be a useful animal model for studying the regulatory effect of dietary lipid on plasma cholesterol levels. Presented in part at the A.O.C.S. Annual Meeting, San Francisco, CA, May 1979.     

Models and methods for studying behavior in polyunsaturated fatty acid research

This report examines a range of models and procedures applicable to polyunsaturated fatty acid (PUFA) research and considers their relative merits. Considerations pertaining to cost, efficiency, and scientific rigor are of particular interest. parallel activities in other areas of behavioral neuroscience, such as behavioral pharmacology and toxicology, that have profitably exploited various behavior designs for the study of human and animal cognition are noted. Special attention is given to the utility of operant conditioning models and schedules of reinforcement, which are currently underrepresented in PUFA research. Investigations of analogs of complex human behavior as well as implications for generalizing laboratory results to clinical phenomena are addressed.     

Homo-gamma-linolenic acid: A major polyunsaturated fatty acid of swine adrenal cholesteryl esters

Homo-gamma-linolenic acid is a major polyunsaturated constituent of the cholesteryl esters isolated from swine adrenals. The identity of this acid was confirmed by hydrogenation, reductive ozonolysis and a combination of thin layer and gas liquid chromatographic techniques.