High Omega-3 Essential Fatty Acid Status in Nigerians and Low Status in Minnesotans Ralph T. Holman*, Susan B. Johnson**, Douglas M. Bibus* Theo C. Okeahialem and Peter O. Egwim*** *The Hormel Institute, University of Minnesota 801 16th Ave. NE Austin, MN 55913 Tel: 507 433 8804 email: rtholman@maroon.tc.umn.edu ** Present address: The Mayo Clinic Rochester, MN 55905 ***College of Medicine Univeristy of Nigeria Enugu Campus, Enugu, Nigeria To be submitted to: WWW Jour. Biol. Abstract The purpose of this study was to investigate the essential fatty acid status of a population from Africa who consume low levels of hydrogenated fat and natural sources of fat derived from fish, meats and vegetables. Plasma samples from 38 healthy adult Nigerians from Enugu, 25 rural and 13 urban, were analyzed for their essential fatty acid status. Plasma lipids from phospholipid (PL), non-esterified fatty acids(NEFA), triglyceride (TG) and cholesteryl ester (CE) were extracted, derivatized and analyzed by capillary gas chromatograph and are expressed as % composition ( SEM. The Nigerian data were compared to our standard control group which consists of 100 normal Minnesotans. Nigerian subjects had markedly higher amounts of total and individual w3 acids when compared to Minnesotans for all lipid classes tested. Plasma PL total w3 was 13.4(0.70 compared to MN controls, 5.53(0.13, p<0.001, or a 2.42 fold increase. Similar increases in total w3 were observed for TG, NEFA and CE, 2.67, 2.79 and 2.90 times control, respectively. Plasma PL 20:5w3, 22:5w3 and 22:6w3, 3.80(0.45, 1.26(0.06, 8.11(0.31, were all significantly elevated versus MN controls, 0.59(0.03, 1.13(0.03, 3.59(0.11. Increased levels of w3 acids were partially offset by significant decreases in w6 acids. Plasma PL 18:2w6 and 20:4w6, 18.61(0.62 and 8.38(0.27, were significantly decreased versus control, 23.90(0.28 and 12.81(0.19. In conclusion, Nigerians have significantly higher amounts of plasma w3 acids than do Minnesota controls, largely offset by decreased levels of w6 acids that may be related to the absence of hydrogenated fat in the Nigerian diet. Introduction The essential fatty acids are defined as linoleic (18:2w6) and alpha-linolenic (18:3w3) acids and their elongation and desaturation products. Essential fatty acids are required for membrane integrity, visual and neurological function and their deficiency is associated with neurological and immunological disease (7-11). This study was initiated with the intent to describe the fatty acid profiles in the four major classes of plasma lipids, the phospholipids (PL), cholesteryl esters (CE), triglycerides (TG) and non-esterified fatty acids (NEFA) in a population that does not consume significant levels of hydrogenated vegetable oils. This would allow evaluation of essential fatty acid (EFA) status with respect to w3 fatty acids (FA) and w6 FA, and would permit comparison to a free-living American population known to consume partially hydrogenated vegetable oils as a major dietary source of its dietary fat. The data presented here concern the essential fatty acid (EFA) status of the Ibo people of eastern Nigeria, who had little access to partially hydrogenated vegetable oils in 1989 when this study was begun, in comparison to a typical American group, for which partially hydrogenated vegetable oil is a common source of dietary fat. Methods Subjects: The subjects were healthy volunteers, aged 19 to 42 years of age. In our previous studies of EFA content of plasma lipids (1, 2) , we had observed no significant sex or age differences in the levels of individual fatty acids or of related groups of fatty acids. The present study was a comparison of adult Nigerians to adult Minnesotans. The rural subgroup comprised 25 Nigerian rural subjects,10 male and 15 female, with mean age of 33.8 yr from farming towns within 40 km from Enugu. The urban subgroup comprised 13 urban subjects, 6 male and 7 female, with a mean age of 34.3 years, who were residents of Enugu. The female subjects were non-pregnant, non-lactating, multiparous mothers, aged 30 to 40 years, who accompanied their children to the pediatric outpatient clinic at the University of Nigeria Teaching Hospital in Enugu. The males were members of their families. Only those were recruited who were found free of serious illness or health abnormalities upon medical examination by a physician. All subjects were from the Ibo ethnic group. Following informed consent, they were classified into two subgroups, rural or urban, based on the place of domicile. Information about their nutritional background and dietary habits and preferences were solicited and recorded. Sample preparation: Fasting venous blood (8-10 ml) was drawn from the left arm and collected directly in a heparinized tube. Plasma samples were prepared by low-speed centrifugation (1000 rpm 5 min.) within one hour of collection. Plasma (4-5 ml) was withdrawn by syringe, extracted twice with 5 ml portions of chloroform-methanol (2:1, v/v). The combined chloroform extract was injected directly into a labeled vial which was wrapped individually in cotton batting for shipment. All stoppers were tested for leak. The tubes were shipped in a padded cardboard box, designed for safe international transportation. The box was sealed, clearly marked, tightly tied and kept in a deep freezer (-10(C) until dispatch. A colleague traveling to New York hand-carried the package, and mailed it to the Hormel Institute from his port of entry. During storage and transit, the extracted lipids dissolved in chloroform-methanol were protected by dilution from the autooxidative chain reaction which occurs in neat lipids. Lipid Analysis : Total lipid extracts were washed with water, the aqueous layer was removed, and the chloroform layer was blown to dryness under nitrogen (2). The lipids were then redissolved in a minimal volume of chloroform and applied to a silicic acid thin-layer chromatography plate. The lipids were developed in a mixture of petroleum ether (b.p. 30-60°C), diethyl ether and acetic acid (80:20:1, vol, vol, vol) to separate the phospholipids (PL), cholesteryl esters (CE), triglycerides (TG) and non-esterified fatty acids (NEFA). Each lipid fraction was scraped from the plate and esterified with 12% BF3 in methanol, converting it to the fatty acid methyl esters (FAME), and then extracted with petroleum ether (b.p. 30-60°C) for capillary gas chromatographic analysis (15,16). A model 428 Packard gas chromatograph equipped with a 50 m x 0.25 mm bonded 007 FFAP-based silica capillary column (Quadrex, New Haven, CT) was used to separate the FAME. Its temperature was programmed from 170° to 220° C/min with the final hold of 25 min, separating the methyl esters of fatty acids ranging from 12:0 to 24:1w7. The detector and injector temperatures were 250°C. Helium was used as carrier gas at a flow rate of 1.4 mL/min and a split ratio of 1:65. FAME were identified by comparing with authentic FAME standards (NuChek Prep, Elysian, MN) and the peak areas were integrated as wt% with a dedicated microprocessor. Individual peaks were distinguished and measured, and peaks as small as 0.05% of the FAME were measured. The analyses of the plasma lipids of the Nigerian subjects were made in 1989. The control group of 100 omnivorous healthy adults, volunteers among the students and staff of the University of Minnesota, were the control group in a study investigating the effects of vegetarianism upon EFA status, also conducted in 1989 ( 3 ). Analysis of Data: Data are expressed as percentage of total fatty acids (% composition), because the percentage of fatty acid within a lipid class better expresses the concentration of substrate available to an interfacial enzyme at a two-dimensional surface, than does the concentration of the substrate within the aqueous space. The ratio of experimental to control values, or normalcy ratio (NR), equivalent to a Z score, indicates relative concentration change in disease. In the figures, NR is plotted on a logarithmic scale so that increases and decreases of the same proportion are indicated by the same length. The vertical axis is the control or normal value. Open bars indicate a change which does not reach significance (ns); bars with wide striations indicate p < 0.05, bars with close striations p < 0.01, and black bars: p < 0.001. Changes greater than ten-fold are shown as 10-fold changes. Statistical probabilities were calculated using Student's t-test. Results Comparison of Rural and Urban Nigerian Populations. The plasma PL of the group of 25 rural subjects and of the group of 13 urban Nigerians were first compared with each other to reveal differences in fatty acid pattern due to place of residence. The profile of 25 rural Nigerians compared to 13 urban Nigerians is shown in Figure 1. Of all the detected fatty acids, only two minor fatty acids, 14:0 and 22:0, were found to differ significantly between the two groups. Because no significant difference was found for any of the w3 or w6 fatty acids, we considered the two groups as equal, so the entire composite Nigerian population of 38 subjects was grouped together for comparison to our group of 100 Minnesota control subjects. The Fatty Acid Profile of Plasma Phospholipids (PL), for all 38 Nigerian subjects in comparison to our 100 Minnesota normal controls (3), is shown graphically in Figure 2 and in tabular form in Table 1. The levels of all w6 fatty acids in PL were found to be less than in our Minnesota controls, all of which were significant, except for 18:3w6. In contrast to the w6 EFA, the levels of all the individual w3 EFA were significantly elevated above the Minnesota control group. The mean total of w3 FA in the phospholipid FA of the Nigerians was 2.43 times as high as in our Minnesota control, p < 0.001. In contrast, the mean total w6 EFA in the PL FA of Nigerians was 0.72 times that of Minnesotans, p < 0.001. Although these major differences in the proportions of the two families of EFA occurred, they did not cause major changes in total saturated fatty acids or monounsaturated acids. Total saturated fatty acids were higher in the Nigerians than in the Minnesotans by the small but significant factor of 1.16, p < 0.001. Total monoenoic acids were lower in Nigerians, at 98% of the Minnesotans' content, p < 0.05. The Fatty Acid Profile of Plasma Cholesteryl Esters (CE), for all the Nigerian subjects compared to Minnesota controls is shown graphically in Figure 3 and given numerically in Table 2. The graphic profile of the CE indicates that 18:2w6, 20:3w6 and 20:4w6 were significantly lower in Nigerians than in Minnesotans, whereas all w3 FA were significantly elevated. The total of w6 FA in the CE of Nigerians was 79% of the level in Minnesotans, whereas the total of w3 FA was 2.9 times the level found in Minnesotans. Accompanying shifts in the saturated and monounsaturated acids also occurred in this comparison. Increased 16:0, but decreased 18:0, accompanied the low w6 level and the high w3 level in the Nigerians. The Fatty Acid Profile of Plasma Triglycerides (TG), for all the Nigerian subjects compared to the Minnesota controls is shown graphically in Figure 4 and given numerically in Table 3. The graphic profile of the TG indicates that the individual w6 acids and the total w6 acids were lower in the Nigerians than in the Minnesotans. The total of w6 FA in the TG of Nigerians was 73% of the level found in Minnesotans, whereas the total of w3 FA was 2.67 times the level found in Minnesotans. Accompanying shifts in the saturated and monounsaturated fatty acids also occurred in this comparison. In plasma TG, 16:0, 18:0, 20:0 and 22:0 were significantly lower in Nigerians than in Minnesotans. Among the monoenoic acids, a major acid 16:1 was significantly lower, whereas a minor acid 22:0, was significantly higher in the Nigerians. The Fatty Acid Profile of Plasma Non-esterified Acids (NEFA), for all the Nigerian subjects compared to the Minnesota controls, is shown graphically in Figure 5 and shown numerically in Table 4. The fatty acid profile of the NEFA indicates a significantly lower 18:2w6 and significantly higher 20:2w6 and 22:4w6 in Nigerians than in Minnesotans. However, among the w3 EFA, 18:3w3 was not significantly different in the two populations , but its metabolic products were all dramatically and significantly higher in the Nigerians. The total of w6 FA was lower in the Nigerians, 86% as much as in Minnesotans, but the total of w3 EFA was 2.79 times higher in the Nigerians than in the Minnesotans. The data from all four lipid classes show that the w3 EFA status of Nigerians is more than twice as high as is the status of our Minnesota controls. For PL the ratio is 2.43, for CE 2.9, for TG 2.67, and for NEFA 2.79. Increased levels of w3 FA were predominantly offset by decreased levels of w6 FA, and they were accompanied by increased levels of 16:0, p < 0.001. Discussion Food selection and habits in rural and urban Nigeria: The level of intake and the selection of local food staples depends upon the seasonal availability of foodstuffs. The caloric intake is high, but may fail to meet nutritional requirements, due to cost constraints, especially in the rural communities. The major carbohydrate-rich staples are the starchy tubers such as yams, cocoa-yams and cassava, the cereals rice and maize, and minor foods such as plantains and bananas. The major protein staples include legumes such as beans and pulses, seeds, nuts, cereal proteins and leaf proteins, some of which are rich in 18:3w3. Animal protein sources such as milk and eggs are virtually nil for rural communities, and are very limited for the urban population. Meats and fish, good sources of polyunsaturates, are in limited supply. Crayfish and dried fish are important but cost constraints limit intake. The major dietary source of fat in the area is fresh palm oil, which is most affordable, is produced by rural families and is unhydrogenated. Unprocessed groundnut (peanut) oil is popular but expensive. Imported oils, many of which are hydrogenated, are infrequently consumed due to cost, especially for rural families. Minor seasonal fat and oil sources include the oil seeds such as groundnuts, melon, pumpkin, maize, bush mango seed and coconut. The latter items are more available to the rural population than to the urban population. Breadfruit and pears are also available to the rural population in greater amount than to the urban population. Fats and oils from unusual protein sources of animal origin include the snail Vicapara quadrata , the crayfish Palamonetes varians, grasshoppers, and locusts. Edible leaf- or stem-larvae are available rurally, whereas only snails and crayfish are available in urban markets. From these dietary notes it was apparent that the rural and urban Nigerian populations are similar with respect to major foodstuffs available, their food preferences and habits. Their major sources of calories, lipoproteins and lipids are relatively similar. Actual intake of calories may not be significantly different in the two subgroups. Urban subjects were more likely to be exposed to the processed vegetable oils, especially the imported varieties which would contain isomerized unsaturated fatty acids, which may explain the slightly lower levels of the w3 EFA measured in the plasma PL of the urban subjects as compared to rural subjects. Rural subjects were more likely to ingest a variety of unusual seasonal sources of animal and plant protein sources such as snails, mushrooms, edible larvae, locusts and grasshoppers. Information on their constituent fatty acids are not available, but it would seem likely that they include w3 EFA, derived from fresh leaves, generally known contain linolenic acid. It would appear that 18:3w3 may be the predominant w3 FA present in the diet of both groups. It is apparent from the above dietary notes and from the comparison of the rural and urban groups presented in Figure 1, that the rural and urban subgroups are not significantly different with respect to their available foodstuffs and that they are not significantly different with respect to the profiles of their plasma phospholipids. Therefore for purposes of comparison with Americans from Minnesota, we combined the urban and rural groups into one group of Nigerians. EFA Status of Other Ethnic Control Groups Studied in This Laboratory: Our first attempt at assessing EFA status was a survey of the four major lipid classes from 200 patients hospitalized for reasons not related to metabolic diseases (1). We found no significant correlation versus either age or sex for any individual fatty acid or group of fatty acid, although graphic correlations showed slight trends. In that study, conducted in 1978, we found that the total w6 acids in plasma PL were 37.47 ( 5.51 % of the total FA, and that total w3 acids were 4.28 ( 3.49 %. The variation in the individual values was considerable, probably due to the fact that the subjects were patients, and that disease affects the EFA pattern, as we now know. The total w3 EFA (Sw3) of this group of hospitalized patients without metabolic diseases was lower, 77% of the value for our 100 Minnesota healthy controls . In a study of Sj(gren-Larsson Syndrome, a genetic neuropathy occurring in northern Sweden, we had occasion to measure the EFA status of a normal adult local population as a standard of comparison ( 3 ). Those healthy controls from the same area, Ume(, were found to have 12.43 ( 1.70 % total w3 EFA and 35.8 ( 1.06 % total w6 FA. An unpublished study of the Keralites of the Malabar Coast of India, made in cooperation with Parinandi, Raj and Ramasarma, revealed that Keralites' plasma PL contained 10.4 ( 0.63 % total w3 EFA. An unpublished study in cooperation with Malmros and Fex found that a normal population from Malm( in southern Sweden had 8.68 ( 0.73 % total w3 EFA in the plasma PL, 1.88 times that of Minnesotans. Normal Australians ( 4 ) have been found to have 7.35 ( 0.33 % total w3 EFA in their plasma PL, 1.33 times that of Minnesotans. We have found, in an unpublished cooperation with Bussarow and Berberian, that normal Bulgarians have 5.26 ( 0.42 % total w3 EFA in their plasma PL, 95% as much as Minnesotans. In an unpublished study with Kretchmer and his colleagues, we have observed Australian aborigines to have 4.69 ( 0.12 % total w3 in their plasma PL, 85 % of the level in Minnesotans. In a study of malnourished Argentine children ( 5 ), we had occasion to measure the fatty acid profile in normal Argentine children, and found them to have 4.79 ( 0.25 % w3 EFA in their plasma PL. In our study of normal American newborn infants ( 6 ) we found 3.57 ( 0.19 % of total w3 EFA in their plasma PL. These data are arranged in Table 5 for easy comparison. This laboratory has also been involved in a study of a case of w3 deficiency (7). A girl 6 years of age who developed neurologic impairments after a gunshot wound to the abdomen and multiple surgeries for repair, was maintained by prolonged total parenteral nutrition (TPN) with a preparation very high in linoleic acid, but extremely low in linolenic acid. After several weeks, neurological impairments appeared, and an analysis of plasma lipids was requested. The child was found to have plasma PL content of 1.82% Sw3 FA , or 34 % of the "normal" level measured in our Minnesota control population. Administration of TPN based on soybean oil which contains adequate linolenic acid, (18:3w3) restored the child's system to normal, proving the efficacy and essentiality of w3 FA in humans. The above data from our laboratory indicates a range of total w3 EFA , from 13.4 ( 0.70 (Nigerians) down to 3.57 ( 0.19 (American infants) in presumably normal humans, a range of 9.83 %. The Minnesota control group value we customarily use as standard, lies at 5.53 % of total FA of plasma PL, or 1.96 above the lowest "normal" value. Our Minnesota controls lie at the 20th percentile of the range we have encountered in this laboratory. We question whether the 20th percentile of the current range is truly "normal" or adequate. We also wonder whether we should continue to use it as a standard for comparison of other populational groups. We know of no other element or required substance, where the criterion is set at 20% of its known range of concentration in the human body. Recently our laboratory has been also studying the EFA status of patients suffering from a variety of diseases. The progress of that research has been summarized and reviewed periodically (8-11). The w3 status of patients suffering from a variety of diseases associated with impairment of the immune system and/or with neuropathy has been found to be significantly below "normal", in comparison to the level found in our Minnesota "normal" population. That is to say, neuropathies and impairment of the immune response are associated with w3 levels which are significantly lower than our Minnesota controls, significantly lower than the 20th percentile of the observed range of normal groups we have studied, significantly lower than 5.35% of total fatty acids of plasma PL. In Figure 6 the content of Sw3 has been plotted against the content of Sw6 fatty acids for each of the above ostensibly "normal" control groups. From this figure it is clear that there is a negative correlation between Sw6 and Sw3 fatty acids in plasma PL from "normal" human populations. As Sw6 increases in the plasma of the population, the Sw3 decreases! Conversely, as the dietary supply and, consequently, the plasma Pl content of w3 EFA increases, the Sw6 of plasma is suppressed. This is exactly what was demonstrated three decades ago in this laboratory, using pure linoleic and linolenic acids at several levels in synthetic diets of rats. With dietary linoleic acid, 18:2w6, held constant, increasing the dietary level of 18:3w3 suppressed the levels of w6 metabolic products ( 12). With dietary linolenic acid, 18:3w3, held constant, increasing the level of dietary 18:2w6 suppressed the levels of w3 metabolites (13). These studies led to the concept of competition between w6 and w3 fatty acids at each level of the metabolic cascade (14). The fatty acid pool available for synthesis of structural lipids contains all the fatty acids which occur in the lipids of the diet, and all the components of the fatty acid pool compete for the same enzymes as do the w3 and w6 EFA (15). The structures of the fatty acids and their relative concentrations govern their competition. Diets rich in saturated fat (16), or monounsaturated fat (17) promote the onset of EFA deficiency symptoms in rats, indicated by an increase of a trienoic acid, now known as Mead's acid, 20:3w9 (18). This acid, synthesized endogenously via stearic and oleic acids, is a major polyunsaturated fatty acid of tissue lipids in the dietary absence of the essential linoleic and linolenic acids. The ratio of 20:3w9 / 20:4w6 has been used as a measure of EFA deficiency and was used to set the minimum dietary requirement of linoleic acid at about 1 % of calories (19, 20). The conversion of oleic acid to 20:3w9 is inhibited by even low levels of linoleic acid in the diet, but the conversion of linoleic acid to arachidonic acid can be inhibited only by high ratios of oleic to linoleic acid in the diet (21). That is, oleic acid is a relatively weak competitor to linoleic acid. When oils containing PUFA are subjected to metal-catalyzed hydrogenation, the catalysts cause movement of the unsaturation up and down the carbon chain. If the hydrogenation is stopped before it is complete, it leaves a series of positional monoenoic acid isomers with the double bond located randomly within the fatty acid chain. Two-thirds of each positional isomer is trans, and one third is cis. Most of these isomers have not been found in nature, and these monoenoic acids are also competitive with natural fatty acids for enzyme sites (22) and are precursors of unusual polyunsaturated fatty acids (23). The American food supply is increasingly turning toward partially hydrogenated fats and toward oils containing 18:2w6 as sole or dominant polyunsaturated acid, for reasons of economy and convenience, ignoring the essentiality of the w3 PUFA. This tendency is driven by the merchandising system which attempts to provide products which are heat stable, incapable of autooxidation, and have an extended shelf-life. This has been accomplished at the cost of losing the w3 EFA necessary for protection against disease, notably diseases which adversely affect the nervous and immune systems (10). The Nigerian population clearly had a richer w3 supply than did our Minnesota controls 7 years ago. The current states of Nigerians and of Minnesotans have not been measured. Conclusions Analysis of the fatty acid composition of the plasma lipids of a group of healthy adult Nigerians and a comparable groups of healthy Minnesotans has revealed that the Nigerians have more than twice as much essential w3 EFA in their plasma lipids as do Minnesotans. Minnesotans lie at the 10th percentile of the range encountered in presumably healthy humans. American newborn infants are the lowest group of healthy humans in our list. We question whether these are adequate levels for optimum health and development. Literature Cited 1. Holman, R. T. , Smythe, L. and Johnson, S. Effect of Sex and Age on Fatty Acid Composition of Human Serum Lipids. Am. J. Clin. Nutr. 32: 2390-2399 (1979) 2. Phinney, S.D., Odin, R.S., Johnson, S. B., and Holman, R.T. Reduced Arachidonate in Serum Phospholipids and Cholesteryl Esters Associated With Vegetarian Diets in Humans. Am. J. Clin. Nutr. 51: 385-392, (1990) 3. Hernell, O., Holmgren, G., Jagell, S.F., Johnson, S.B., and Holman, R.T. Suspected Faulty Essential Fatty Acid Metabolism in Sj(gren-Larsson Syndrome. Pediatr. Res. 6: 45-59, (1982) 4. Sinclair, A. J., Johnson, L., O'Dea, K., and Holman, R.T. Diets Rich in Lean Beef Increase Arachidonic Acid and Long-Chain w3 Polyunsaturated Fatty Acid Levels in Plasma Phospholipids. Lipids 29, 337-343 (1994) 5. Holman, R.T., Johnson, S.B., Mercuri, O., Itarte, H. J., Rodrigo, M. L., and De Tomas, M.E. Am. J. Clin. Nutr. 34: 1534-1539 (1981) 6. Lloyd-Still, J.D., Johnson, S.B., and Holman, R.T. Essential Fatty Acid Status and Fluidity of Plasma Phospholipids in Cystic Fibrosis Infants. Am. J. Clin. Nutr. 54: 1029-1035 (1991) 7. Holman, R. T., Johnson, S.B. and Hatch, T.F. A Case of Human Linolenic Acid Deficiency Involving Neurological Abnormalities. Am. J. Clin. Nutr. 35, 617-623 (1982) 8. Holman, R. T. A Long Scaly Tale - The Study of Essential Fatty Acid Deficiency at the University of Minnesota. in "Essential Fatty Acids and Eicosanoids" Andrew Sinclair and Robert Gibson, Eds. American Oil Chemists Society, Champaign, IL, pp 3-17, (1993) 9. Holman, R. T. Omega 3 Deficiencies in Humans. In Proc. 55th Flax Institute of the United States, J. F. Carter, Ed., North Dakota State Univ., Fargo, N.D. pp 4-11 (1994) 10. Holman, R. T. Omega 3 and Omega 6 Essential Fatty Acid Status in Human Health and Disease. in "Fatty Acids: Biochemistry and Behavior", Yehuda, S. and Mostofsky, D.I., eds. Humana Press, Totowa, N.J. (1996) 11. Holman, R. T. Deficiencies of Omega 3 Fatty Acids in Humans Are Not Rare. In Proc. 56 Flax Institute of the United States, J. F. Carter, Ed. North Dakota State Univ., Fargo, N.D. pp 1-8 (1996) 12. Rahm, Joseph J. and Holman, R. T. The effect of Linoleic Acid upon the metabolism of linolenic acid. J. Nutr. 84: 15-19 (1964) 13. Mohrhauer, H. and Holman, R. T. Effect of Linolenic acid upon the metabolism of linoleic acid. J. Nutr. 81, 67-74 (1963) 14. Holman, R.T. and Mohrhauer, H. A Hypothesis Involving Competetive Inhibitions in the Metabolism of Polyunsaturated Acids. Acta Chem. Scand. 17, S84-S90 (1963) 15. Holman, R.T. Nutritional and metabolic interrelationships between fatty acids. Fed. Proc. 23, 1062-1067 (1964) 16. Peifer, J.J. and Holman, R. T. Effect of saturated fat upon essential fatty acid metabolism of the rat. J. Nutrition 68, 155-167 (1959) 17. Dhopeshwarkar, G. A. and Mead, J.F. Role of Oleic Acid in the Metabolism of Essential Fatty Acids. J. Am. Oil Chem. Soc. 38, 297-301 (1961) 18. Fulco, A. and Mead, J. F. Metabolism of Essential Fatty Acids VIII. Origin of 5,8,11- Eicosatrienoic Acid in the Fat-Deficient Rat. J. Biol. Chem. 234, 1411- 1416 (1959) 19. Holman, R.T., The Ratio of Trienoic:Tetraenoic Acids in Tissue Lipids as a Measure of Essential Fatty Acid Requirement. J. Nutrition 70, 405-410 (1960) 20. Mohrhauer, H., Rahm, J.J., Seufert, J. and Holman, R.T. Metabolism of Linoleic Acid in relation to Dietary Monoenoic Fatty Acids in the Rat. J. Nutrition. 91, 521-527 (1967) 21. Hill, E. G., Johnson, S.B. and Holman, R.T. Intensification of Essential Fatty Acid Deficiency by dietary Trans Fatty Acids. J. Nutrition 109, 1759-1765 (1979) 22. Hill, E. G., Johnson, S. B., Lawson, L. D., Mahfouz, M.M. and Holman, R.T. Perturbation of the Metabolism of Essential Fatty Acids by Partially Hydrogenated Vegetable Oil. Proc. Natl. Acad. Sci. USA 79, 953-957 (1982) 23. Holman, R. T., Pusch, F., Svingen, B., and Dutton, H.J. Unusual Isomeric Polyunsaturated Fatty Acids in Liver Phospholipids in Rats Fed Hydrogenated Oil. Proc. Natl. Acad. Sci. USA 88, 4830-4834 (1991) TABLE 1 Plasma Phospholipid Fatty Acid Profile of Normal Adult Nigerians Compared with Normal Adult Minnesota Controls. ________________________________________________________ Fatty Nigerians n = 38 MN Controls n = 100 acid Mean SEM Mean SEM p = _________________________________________________________ 18:2w6 18.61 ( 0.62 23.90 ( 0.28 < 0.001 18:3w6 0.08 ( .01 0.12 ( 0.01 20:2w6 0.35 ( 0.02 0.47 ( 0.02 < 0.05 20:3w6 2.45 ( 0.13 3.41 ( 0.08 < 0.001 20:4w6 8.38 ( 0.27 12.81 ( 0.19 < 0.001 22:4w6 0.24 ( 0.01 0.76 ( 0.03 < 0.001 22:5w6 0.20 ( 0.01 0.60 ( 0.02 < 0.001 Sw6 30.32 ( 0.78 42.07 ( 0.23 < 0.001 18:3w3 0.25 ( 0.02 0.21 ( 0.01 20:5w3 3.80 ( 0.45 0.59 ( 0.03 < 0.001 22:5w3 1.26 ( 0.06 1.13 ( 0.03 < 0.05 22:6w3 8.11 ( 0.31 3.59 ( 0.11 < 0.001 Sw3 13.42 ( 0.70 5.53 ( 0.13 < 0.001 20:3w9 0.18 ( 0.01 0.13 ( 0.01 < 0.01 S PUFA 39.11 ( 1.63 47.73 ( 0.26 < 0.001 14:0 0.20 ( 0.02 0.20 ( 0.01 16:0 26.34 ( 0.62 21.05 ( 0.20 < 0.001 18:0 11.96 ( 0.21 12.53 ( 0.16 20:0 0.34 ( 0.02 0.33 ( 0.02 22:0 0.73 ( 0.05 1.11 ( 0.04 < 0.001 24:0 0.01 ( 0.01 0.88 ( 0.07 < 0.001 ( Sat. 40.60 ( 0.78 36.29 ( 0.22 < 0.001 16:1w7 0.48 ( 0.04 0.70 ( 0.02 < 0.001 18:1w9 9.72 ( 0.22 8.87 ( 0.16 < 0.01 20:1w9 0.09 ( 0.00 0.22 ( 0.02 < 0.001 22:1 0.08 ( 0.02 0.01 ( 0.00 <0.001 24:1 1.25 ( 0.06 1.20 ( 0.04 ( Mono. 13.22 ( 0.23 13.83 ( 0.15 < 0.05 ( Branched 0.96 ( 0.08 1.05 ( 0.05 ( Oddchain. 1.01 ( 0.32 0.19 ( 0.01 < 0.001 TABLE 2 Plasma Cholesteryl Ester Fatty acid Profile of Normal Adult Nigerians Compared with Normal Adult Minnesota Controls. ________________________________________________________ Fatty Nigerians, n = 37 MN Controls, n = 99 acid Mean SEM Mean SEM p = _________________________________________________________ 18:2w6 45.37 ( 1.20 56.59 ( 0.42 < 0.001 18:3w6 0.88 ( 0.07 0.90 ( 0.04 20:2w6 0.11 ( 0.01 0.05 ( 0.02 20:3w6 0.60 ( 0.03 0.87 ( 0.04 < 0.001 20:4w6 5.45 ( 0.19 7.84 ( 0.18 < 0.001 22:4w6 0.01 ( 0.00 0.01 ( 0.00 22:5w6 0.03 ( 0.01 0.02 ( 0.00 Sw6 52.46 ( 1.18 66.27( 0.37 < 0.001 18:3w3 0.44 ( 0.02 0.43 ( 0.02 20:5w3 2.50 ( 0.24 0.51 ( 0.03 < 0.001 22:5w3 0.10 ( 0.02 0.02 ( 0.00 < 0.001 22:6w3 1.11 ( 0.05 0.47 ( 0.03 < 0.001 Sw3 4.14 ( 0.27 1.43 ( 0.05 < 0.001 20:3w9 0.00 ( 0.00 0.00 ( 0.00 S PUFA 56.60 ( 1.14 67.70 ( 0.38 < 0.001 14:0 0.57 ( 0.04 0.66 ( 0.03 16:0 14.09 ( 0.21 9.76 ( 0.14 < 0.001 18:0 0.73 ( 0.03 0.96 ( 0.03 < 0.001 20:0 0.03 ( 0.01 0.02 ( 0.01 22:0 0.03 ( 0.01 0.00 ( 0.00 < 0.001 24:0 0.00 ( 0.00 0.00 ( 0.00 ( Sat. 14.69( 0.70 11.67 ( 0.16 < 0.001 16:1w7 2.96 ( 0.25 2.64 ( 0.08 18:1w9 23.15 ( 0.43 15.62 ( 0.18 < 0.001 20:1w9 0.00 ( 0.00 0.00 ( 0.02 22:1 0.00(0.00 0.00 ( 0.00 24:1 0.00 ( 0.00 0.00 ( 0.00 ( Mono. 26.54 ( 0.60 19.44 ( 0.25 < 0.001 ( Branched 0.43 ( 0.05 0.74 ( 0.05 <.0.001 ( Oddchain. 0.22 ( 0.02 0.20 ( 0.01 TABLE 3 Plasma Triglyceride Fatty acid Profile of Normal Adult Nigerians Compared with Normal Adult Minnesota Controls. ________________________________________________________ Fatty Nigerians, n = 37 MN Controls, n = 101 acid Mean SEM Mean SEM p = _________________________________________________________ 18:2w6 14.97 ( 0.79 21.02 ( 0.44 < 0.001 18:3w6 0.36 ( 0.03 0.51 ( 0.02 < 0.01 20:2w6 0.25 ( 0.02 0.25 ( 0.02 20:3w6 0.36 ( 0.03 0.43 ( 0.02 20:4w6 1.33 ( 0.07 1.64 ( 0.06 < 0.05 22:4w6 0.18 ( 0.02 0.22 ( 0.02 22:5w6 0.16 ( 0.02 0.19 ( 0.02 Sw6 17.61 ( 0.80 24.27( 0.46 < 0.001 18:3w3 0.81 ( 0.15 0.93 ( 0.04 20:5w3 0.90 ( 0.14 0.15 ( 0.01 < 0.001 22:5w3 0.61 ( 0.05 0.34 ( 0.02 < 0.001 22:6w3 2.49 ( 0.32 0.37 ( 0.03 < 0.001 Sw3 4.81 ( 0.07 1.80 ( 0.06 20:3w9 0.14 ( 0.01 0.14 ( 0.01 S PUFA 22.56 ( 0.96 26.24 ( 0.49 < 0.001 14:0 1.57 ( 0.13 1.48 ( 0.08 16:0 26.54 ( 0.69 21.75 ( 0.34 < 0.001 18:0 4.14 ( 0.17 3.61 ( 0.11 < 0.05 20:0 0.15 ( 0.02 0.03 ( 0.01 < 0.001 22:0 0.17 ( 0.08 0.04 ( 0.01 < 0.05 24:0 0.00 ( 0.00 0.00 ( 0.00 ( Sat. 23.86 ( 1.08 27.33 ( 0.40 < 0.001 16:1w7 3.05( 0.22 4.14 ( 0.11 < 0.001 18:1w9 37.07 ( 0.85 37.83 ( 0.37 20:1w9 0.02 ( 0.01 0.04 ( 0.01 22:1 0.11 ( 0.03 0.00 ( 0.00 < 0.001 24:1 0.01 ( 0.00 0.00 ( 0.00 ( Mono. 41.92 ( 0.65 44.38 ( 0.36 < 0.01 ( Branched 0.45 ( 0.04 1.06 ( 0.06 < 0.001 ( Oddchain 1.09 ( 0.63 0.32 ( 0.01 < 0.05 TABLE 4 Plasma Non-esterified Fatty Acids Profile of Normal Adult Nigerians Compared with Normal Adult Minnesota Controls. ________________________________________________________ Fatty Nigerians, n = 37 MN Controls n = 98 acid Mean SEM Mean SEM p = _________________________________________________________ 18:2w6 15.13 ( 0.74 18.39 (0.28 < 0.001 18:3w6 0.19 ( 0.07 0.15 ( 0.03 20:2w6 0.36 ( 0.04 0.15 ( 0.02 < 0.001 20:3w6 0.38 ( 0.04 0.52 ( 0.05 20:4w6 2.52 ( 0.25 2.42 ( 0.13 22:4w6 0.13 ( 0.02 0.06 ( 0.01 < 0.05 22:5w6 0.04 ( 0.01 0.05 ( 0.01 Sw6 18.74 ( 0.81 21.74( 0.35 < 0.001 18:3w3 0.94 ( 0.14 1.08 ( 0.05 20:5w3 0.91 ( 0.12 0.05 ( 0.01 < 0.001 22:5w3 0.32 ( 0.03 0.10 ( 0.02 < 0.001 22:6w3 2.16 ( 0.19 0.32 ( 0.06 < 0.001 Sw3 4.33 ( 0.34 1.55 ( 0.09 <0.001 20:3w9 0.07 ( 0.01 0.01 ( 0.00 <0.001 S PUFA 23.15 ( 0.97 23.31 ( 0.39 14:0 0.99 ( 0.09 1.14 ( 0.06 16:0 24.38 ( 0.73 19.37 ( 0.27 < 0.001 18:0 11.31 (0.56 12.10 ( 0.31 20:0 0.41 ( 0.06 0.02 ( 0.01 < 0.001 22:0 0.30 ( 0.04 0.06 ( 0.02 < 0.001 24:0 0.01 ( 0.00 0.00 ( 0.00 ( Sat. 38.12 ( 1.11 32.99 ( 0.46 < 0.001 16:1w7 2.29 ( 0.21 2.86 ( 0.10 < 0.01 18:1w9 31.51 ( 1.09 37.34 ( 0.52 < 0.001 20:1w9 0.05 ( 0.01 0.01 ( 0.00 < 0.001 22:1 0.02( 0.01 0.01 ( 0.01 24:1 0.13 ( 0.03 0.01 ( 0.00 < 0.001 ( Mono. 34.35( 1.15 42.27 ( 0.55 < 0.001 ( Branched 0.75 ( 0.11 0.83 ( 0.07 ( Oddchain. 0.64 ( 0.06 0.25 ( 0.02 < 0.001 TABLE 5 Content of Total w3 EFA and Total w6 EFA in Fatty Acids of Plasma Phospholipids of "Normal" Groups Studied Thus Far. All are Adults Unless Stated Otherwise. Groups are Arranged in Order of Decreasing Sw3. . Group n Sw3 SEM Sw6 SEM w6/w3 ratio Nigerians 38 13.4 ( 0.70 30.3 ( 0.78 2.26 Swedes, northern 17 13.1 ( 1.36 35.8 ( 1.06 2.73 Keralites, India 39 10.4 ( 0.63 29.2 ( 0.72 2.81 Swedes, southern 19 8.68 ( 0.73 37.4 ( 0.98 4.31 Australians 33 7.35 ( 0.33 39.9 ( 0.53 5.43 Minnesota males 38 5.65 ( 0.23 42.6 ( 0.39 7.54 Minnesota omnivores (m+f) 100 5.53 ( 0.13 42.1 ( 0.23 7.61 Minnesota vegetarians 23 5.48 ( 0.31 41.0 ( 0.42 7.48 Minnesota females 62 5.45 ( 0.16 41.7 ( 0.28 7.65 Bulgarians 30 5.26 ( 0.42 39.0 ( 0.46 7.41 Australian Aborigines 93 4.69 ( 0.12 37.7 ( 0.36 8.04 Argentine infants 48 4.78 ( 0.25 34.9 ( 0.98 7.3 American infants 38 3.57 ( 0.19 38.1 ( 0.68 10.67 Legends for Figures: Figure 1. Fatty Acid Profile of Plasma Phospholipids of 25 Rural Nigerians, Compared with 13 13 Urban Nigerian Controls. Figure 2. Fatty Acid Profile of Plasma Phospholipids of 38 Nigerian Adults, compared with 100 Minnesota adults. Figure 3. Fatty Acid Profile of Plasma Cholesteryl Esters of 37 Nigerians, Compared with 99 Minnesota Controls. Figure 4. Fatty Acid Profile of Plasma Triglycerides of 37 Nigerians, Compared with 101 Minnesota Controls. Figure 5. Fatty Acid Profile of Plasma Non-esterified Fatty Fatty Acods pf 37 Nigerians, Compared with 98 Minnesota Controls. Figure 6. Plot of Mean Values for Total w3 Fatty Acids versus Mean Values for Total w6 Fatty acids in the 13 Ethnic "Normal" Control Groups Listed in Table 5. Correspondance to: Ralph T. Holman The Hormel Institute 801 16th Ave NE Austin, MN 55912 507-433-8804 rtholman@maroon.tc.umn.edu bibus002@maroon.tc.umn.edu Key words :essential fatty acids, polyunsaturated, hydrogenated, ethnic, African, American, omega-3, omega-6 1