published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Studies were conducted to determine the effects of cyclopropenoid fatty acids (CPFA) on the microsomal membrane of livers of rainbow trout (Salmo gairdneri). Slab and tube gel electrophoresis of microsomes from trout fed a CPFA diet (CPFA-microsomes) for varying time periods showed a decrease in the number of protein bands resolved in the high molecular weight region. This disappearance of high molecular weight proteins was not due to increased proteolysis in the CPFA-microsomes. Antibodies against whole microsomal protein from livers of trout fed 300 ppm CPFA were produced in rabbits. Microsomal proteins were first separated by polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose sheets (NC) and analyzed by the peroxidase-antiperoxidase (PAP) immunochemical staining procedure. Immunoabsorption of antisera directed against CPFA-microsomes by control-microsomes did not reveal any new proteins induced by the CPFA diets. However, the intensity of PAP staining was much greater in CPFA microsomes after immunoabsorption. Hydrolysis of phospholipids in the microsomal membrane by phospholipase A₂ failed to reveal any differences between control and CPFA fed trout. Proteolysis of microsomal membrane proteins had similar effects on NADPH cytochrome reductase and cytochrome P-450 activity on fish fed the different diets. PAGE analysis of these digests did show some differences in digested proteins between the control and CPFA group. These results may reflect a possible change in orientation of microsomal membrane proteins brought about by CPFA in the diet. Additional evidence for altered orientation of proteins was found with PAGE analysis of trypsin-digested microsomes. Moreover incubation of trypsin-digested microsomes with antisera and stained with PAP showed that proteolytic attack was different between control and CPFA microsomes. A final study with incubation of transferred proteins from control and CPFA-microsomes with antisera directed against purified cytochrome P-450 (P-450) and cytochrome P-448 (P-448) showed that CPFA had an effect on the concentration of P-448 but not P-450. / Graduation date: 1983

In this five-week feeding study male New Zealand rabbits were fed diets containing cyclopropene fatty acids (CPFA), diets containing cholesterol, and diets with both CPFA and cholesterol added to test the effect of CPFA on cholesterol metabolism and atherosclerosis induction. When CPFA-fed animals were compared with control animals, they tended to have higher plasma cholesterol levels, higher liver cholesterol levels and a high incidence of aortic atherosclerosis. Control animals had no atherosclerosis. A similar pattern was seen when animals fed cholesterol were compared with those fed both cholesterol and CPFA. Mean triglyceride levels were higher in CPFA-fed rabbits than in controls. A wide range of sensitivity to the CPFA was observed. Some animals had plasma cholesterol levels eight to ten times normal after three weeks of feeding; others showed only slight changes after five weeks. Histological examination of liver cells from CPFA-fed animals showed evidence of alteration in cellular morphology and, in some cases, extensive damage. In some liver cells of CPFA-fed rabbits, subcellular organelles appear to be aligned in fiber-like structures. The most extreme signs of toxicity were noted in livers of rabbits fed CPFA and cholesterol. After five weeks on experimental diets, adipose tissue of the rabbits contained approximately 2% CPFA. Ratios of 16:0 to 16:1 and 18:0 to 18:1 fatty acids increased in liver lipid and erythrocyte ghost lipid after CPFA feeding. Over the five-week study there were no differences in rate of weight gain or in feed conversion ratios in CPFA-fed animals. / Graduation date: 1974

Cyclopropenoid fatty acids (CPFA) are natural components of cottonseed oil, a major food oil in the United States. The ability of CPFA to cause abnormal biochemical and physiological effects when fed to laboratory and farm animals has prompted an investigation of their effects on mice. Between 0.05 and 0.55% CPFA were fed as glycerines of Sterculia foetida oil (SFO, containing 55% CPFA) to mice to determine their effect on certain aspects of growth, lipid metabolism and mitochondrial function. One-half percent SFO fed to weanling mice caused a small temporary decrease in growth rate when compared to controls. Many mice fed 1% SFO stopped growing and died by the end of a 9-week feeding trial. Mice fed less than 1% SFO, or 1% SFO for shorter periods of time, showed increased liver-to-body-weight ratios, accumulation of CPFA in adipose tissue and increased ratios of 16:0/16:1, 18:0/18:1 and total saturated to unsaturated fatty acids in liver and depot fat. Erythrocytes from CPFA-fed mice hemolyzed more slowly than erythrocytes from control mice in isotonicnonelectrolytes, implying an effect of CPFA on membrane lipid composition. One-half percent SFO fed for 9 to 31 days inhibited almost completely the incorporation of [¹⁴c] from labeled palmitate or acetate into liver monounsaturated fatty acids. At the same time, 0.5% SFO retarded the incorporation of label from acetate into ¹⁴C₂ and total liver lipid, but stimulated twofold the incorporation into liver sterols. The oxidation of labeled palmitate was also reduced. CPFA caused lipid accumulation in livers. P:0 ratios of liver mitochondria from mice fed 1% SFO for 6 to 15 days were 1.06- 1.45 while control P:0 ratios were 2.30-2.85. The decrease was due to decreased phosphorylation, but increased respiration. The relationship between the observed results and membrane fatty acid composition was discussed. / Graduation date: 1975

Dietary cyclopropenoid fatty acids (CPFA) have long been known to cause pronounced physiological disorders in both farm and laboratory animals. Past work has shown CPFA to be a powerful promoter of carcinogenesis in trout, rats and mice. Sterculic and malvalic acids (CPFA's) are found in seed lipids of plants from the order Malvales. Two members of this order are cotton and kapok, both of which are used extensively as cooking oils for human consumption. Present chemical and instrumental methods of analysis for CPFA are effective only at CPFA levels above 0.1%, and accurate only at levels above 1%. A more sensitive method of analysis was developed exploiting recent technological advances in glass capillary gas chromatography (GCGC). By the use of cold on-column injection, and positioning of the column at the base of the detector flame, this method eliminates two problem areas found in other GC methods. The other common component involved in CPFA decomposition, during GC analysis, is the column. Vitrious silica columns with an inert stationary phase, SE-30, were shown to separate the highly reactive CPFA without decomposition. Proof of stability during analysis was obtained by changing the variables of relative time on column and column temperature. This method not only allows individual determination of sterculic and malvalic acid concentrations, it is rapid, accurate (to the 70 parts per million range), and is superior to other instrumental and chemical methods. The CPFA concentration for okra, hollyhock, cheese weed, seashore mallow, kapok, and white cap cottonseed oil are as follows: 0.3% to 0.92%, 0.33%, 2.6%, 2.6%, 12.8%, and 75ppm, respectively. The two different values for okra were found because seeds from two different growing seasons were analyzed. No CPFA could be detected in Diet Imperial Margarine, raw cocoa beans, cocoa butter or Lucca's winterized cottonseed oil. / Graduation date: 1987

The cyclopropene fatty acids, sterculic and malvalic, occur naturally in a major food lipid, cottonseed oil, and have been implicated in several physiological disorders. Rainbow trout in this study were fed semipurified diets containing 200 or 300 ppm methyl sterculate, and the effects on hepatic lipid and glycogen levels, lipid incorporation of radioactive phosphate and fatty acids, and mitochondrial energy production were investigated. Chronic ingestion of methyl sterculate caused numerous histological changes and some necrosis in the liver. Glycogen accumulated in irregular pockets, but the level in whole liver was not increased over controls. Hepatic lipid accumulation was noted after several weeks, reaching a level six times that of controls after six months. Liver weight of the sterculate-fed fish decreased in proportion to body weight during the first three weeks on the sterculate diet, but after three months became significantly greater than controls. Ingestion of 200 ppm methyl sterculate for two weeks by trout caused reduced incorporation of both phosphate and fatty acids into phospholipids. The major reductions were found in minor fractions. Label incorporation into cardiolipin, important for mitochondrial function, was reduced ten-fold by the dietary sterculate. Incorporation into a minor unknown phospholipid which constituted less than 1% of total phospholipids was reduced as much as 135-fold in the mitochondrial fraction. Incorporation of 1-¹⁴C-oleate into diglycerides was also inhibited. The major changes caused by methyl sterculate appear to occur in mitochondria. Neither the level of cytochrome P⁴⁵⁰ nor the activity of NADPH neotetrazolium reductase, both of which occur in the endoplasmic reticulum, was altered. P/0 ratios and the ability to oxidize oleic acid to CO₂ were curtailed after only two weeks on a diet containing methyl sterculate. / Graduation date: 1974

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