Total Conjugated Linoleic Acid Isomers Research Articles

CLA supplementation and aerobic exercise lower blood triacylglycerol, but have no effect on peak oxygen uptake or cardiorespiratory fatigue thresholds.

This study examined the effects of 6 weeks of conjugated linoleic acid (CLA) supplementation and moderate aerobic exercise on peak oxygen uptake (VO2 peak), the gas exchange threshold (GET), the respiratory compensation point (RCP), and serum concentrations of cholesterol, triacylglycerol, and glucose in humans. Thirty-four untrained to moderately trained men (mean ± SD; age = 21.5 ± 2.8 years; mass = 77.2 ± 9.5 kg) completed this double-blind, placebo controlled study and were randomly assigned to either a CLA (Clarinol A-80; n = 18) or placebo (PLA; sunflower oil; n = 16) group. Prior to and following 6 weeks of aerobic training (50% VO2 peak for 30 min, twice per week) and supplementation (5.63 g of total CLA isomers [of which 2.67 g was c9, t11 and 2.67 g was t10, c12] or 7.35 g high oleic sunflower oil per day), each participant completed an incremental cycle ergometer test to exhaustion to determine their [Formula: see text] peak, GET, and RCP and fasted blood draws were performed to measure serum concentrations of cholesterol, triacylglycerol, and glucose. Serum triacylglycerol concentrations were lower (p < 0.05) in the CLA than the PLA group. For VO2 peak and glucose, there were group × time interactions (p < 0.05), however, post hoc statistical tests did not reveal any differences (p > 0.05) between the CLA and PLA groups. GET and RCP increased (p < 0.05) from pre- to post-training for both the CLA and PLA groups. Overall, these data suggested that CLA and aerobic exercise may have synergistic, blood triacylglycerol lowering effects, although CLA may be ineffective for enhancing aerobic exercise performance in conjunction with a 6-week aerobic exercise training program in college-age men.

Dietary linseed oil and selenate affect the concentration of fatty acids in selected tissues of sheep

The aim of the study was to determine the influence of the addition of 5% linseed oil (LO) and 2 &amp;micro;g Se as selenate (SeVI) per g of diet on body mass gain (BMG), feed conversion efficiency (FCE), and concentrations of fatty acids (FA), especially conjugated linoleic acid (CLA) isomers, in the liver, heart, m.&amp;nbsp;longissimus dorsi (MLD), m. biceps femoris (MBF), subcutaneous fat (SF), perirenal fat (PF), and intermuscular fat (IF) of sheep. Each group comprised 5 lambs with an average initial body weight of 25 &amp;plusmn; 2 kg. For 35 days the sheep were fed the control or experimental diets enriched with 5% LO, SeVI, or LO and SeVI combined. The diets with LO, regardless of the presence of SeVI, increased BMG, FCE, and the mass of MBF, MLD, and the liver. The LO treatment increased the concentration of total CLA isomers in the liver, SF, PF, and MLD in comparison with the control. Dietary LO with or without SeVI resulted in negligible changes in the concentration of total CLA isomers in MBF. The diet enriched with LO, irrespective of the presence of SeVI, increased the concentrations of cis- monounsaturated fatty acids (cis-MUFA), polyunsaturated fatty acids (PUFA), as well as unsaturated fatty acids (UFA) in the liver, heart, and especially in SF and PF (i.e. energy storage tissues) compared with the control group and SeVI-fed sheep. Dietary SeVI more effectively increased the capacity of ∆9-desaturation in all adipose tissues as the concentration of cis9,trans11CLA increased in these tissues. The addition of SeVI to the diet decreased the yield of the catabolism of fatty acids in these adipose tissues and both muscles compared with the control sheep or other experimental groups. The diets enriched with LO with or without SeVI increased the nutritional value of MBF, MLD, and adipose tissues as the ratio of unsaturated FA/saturated FA increased. Our results of a short-term study provide useful information for nutritionists carrying out further investigations to improve the nutritional quality of feed for ruminants as well as humans. &amp;nbsp;

Can the trans-18:1 and conjugated linoleic acid profiles in retail ground beef be healthier than steak?

A seasonal survey of western Canadian retail ground beef was undertaken with emphasis on trans fatty acid ( trans-FA) and conjugated linoleic acid (CLA) isomer profiles. Thirty lean ground beef samples were collected in the winter and summer from major grocery stores in Calgary (Alberta, Canada). The total fat content was affected by the collection period. Samples collected in summer had significantly higher fat content (14.4%) than samples collected during winter (12.4%; P < 0.01), but very few differences in fatty acid profile were observed on a percentage basis. The average n-6/n-3 ratio (3.17) was healthier than previously reported strip-loin steaks (backfat (5.81) and longissiumus lumborum (6.01)). The average trans-FA content in ground beef was 0.526 g/100 g serving size and 11 t-18:1 was the major trans isomer representing 33% of total trans-FA, and 10 t-18:1 was the second most abundant isomer. The 11 t-/10 t-ratio in ground beef was higher (average of 2.04) compared to values previously reported for strip-loin steaks (average of 0.65). The CLA content of ground beef was 0.63% and rumenic acid (RA, 9 c,11 t-18:2) represented 72% of total CLA isomers, which compared on average to 0.52% of total CLA, and 60% RA of total CLA isomers in the strip-loin. Overall, this study suggests that the n-6/n-3 ratio, trans-18:1 and CLA profiles were healthier in ground beef than in strip-loin steaks collected one day in winter and one day in summer at the same retail outlets. Ground beef nonetheless still contains an inherently high level of total and saturated fat, and this has to be taken into consideration when evaluating its overall nutritional profile.

Conjugated linoleic acids content in M. longissimus dorsi of Hanwoo steers fed a concentrate supplemented with soybean oil, sodium bicarbonate-based monensin, fish oil

We hypothesized that increasing ruminal pH would lead to enrichment of adipose tissue with conjugated linoleic acid (CLA). Twenty-four Korean native (Hanwoo) steers were used to investigate the additive effects of monensin (30ppm, SO-BM) and/or fish oil (0.7%, SO-BMF) in the diets along with soybean oil (7%) and sodium bicarbonate (0.5%, SO-B) on cis-9, trans-11 and trans-10, cis-12 CLAs in adipose tissue. The steers were assigned to randomly four groups of six animals each based on body weight. The control group (CON) was fed a commercial concentrate for the late fattening stage. Supplementation of oil and sodium bicarbonate reduced feed intake and daily gain, and fish oil further decreased feed intake (P<0.001) and daily gain (P<0.087) compared to steers fed other diets. Total CLA and CLA isomers in M.longissimus dorsi were not affected when steers were fed SO-B and SO-BM diets compared with those of steers fed CON and SO-BMF diets. However, total poly unsaturated fatty acids were higher (P=0.03) in steers fed SO than in CON steers.

Conjugated Linoleic Acid (CLA) content of meat from three muscles of Massese suckling lambs slaughtered at different weights

Eighteen Massese male lambs, fed mainly maternal milk were slaughtered at 11, 14 and 17 kg. Samples of Longissimus Dorsi (LD), Triceps Brachii (TB) and Semimembranosus (Sm) muscles were collected. Milk from the lamb’s dams was sampled weekly. Fatty acid composition of milk and meat was determined. TB was the fattest muscle, Sm the leanest one and LD showed an intermediate value of total lipids, while the weight at slaughter did not influence total intramuscular fat content in any muscle. Although slaughter weight slightly affected overall fatty acid composition of muscles, rumenic acid and total CLA content in TB and Sm, but not in LD, significantly increased with slaughter weight. As regard milk fatty acid composition, the contents of total CLA, RA and others minor CLA isomers decreased during the first four weeks after lambing and then increased at the last control (five weeks). The animals slaughtered at a live weight of 14 and 17 kg showed a greater SCD enzyme activity (estimated by product/substrate ratio) and a higher rumen activity (estimated by means of branched chain and odd chain fatty acid content in meat) than animals slaughtered at 11 kg. Cis-7, trans-9 CLA content significantly increased with the slaughter age in TB and SM, while trans-7, trans-9 CLA, only increased in TB, and cis-8, cis-10 CLA, only increased in SM. Further studies are needed in order to verify weather the different behaviour of RA in LD muscle may be due to differences in muscle metabolism or fatty acid utilisation.

Pilot‐Scale Production of Conjugated Linoleic Acid‐Rich Soy Oil by Photoirradiation

Conjugated linoleic acid (CLA) is found naturally in dairy and beef products at levels of 0.2% to 2% of the total fat. A more concentrated source of dietary CLA, low in saturated fat, would be highly desirable to obtain optimum CLA levels of about 3 g/d. We recently reported photoisomerization of soy oil with iodine catalysis to be a simple way of producing CLA in laboratory without high-energy input or expensive enzymes and microorganisms. However, a long irradiation time of 144 h has been a limitation for this technique to be of practical value. The objectives of this study were to build a pilot plant unit to rapidly produce high-CLA soy oil by photoirradiation and optimize the processing parameters to obtain high-CLA soy oil. Degassed oil with dissolved-iodine catalyst was irradiated by UV lamps in an illuminated laminar flow unit (ILFU). The ILFU consists of 2 borosilicate glass plates in a silicone lined stainless steel frame. The static mode of operation yielded 5.7% of total CLA isomers and performed twice as well than the continuous mode with 2.5% of total CLA. Irradiating oil in a static mode with reflective surfaces increased the CLA yields 3-fold to 16.4%. About 22% of total CLA isomers can be rapidly produced from soy oil linoleic acid with 0.35% iodine catalyst in a 0.5-cm-thick oil layer maintained at 48 degrees C for 12 h. The peroxide value and GC-MS analysis did not identify any volatile compounds characteristic of lipid oxidation. This study is a definitive step toward the commercialization of large-scale production of CLA-rich soy oil.

Effects of Dietary Conjugated Linoleic Acid on the Fatty Acid Profile and Cholesterol Content of Egg Yolks from Different Breeds of Layers

Brown Dwarf hens and White Leghorn hens were fed corn- and soybean meal-based diets containing 0, 2.5, or 5.0% conjugated linoleic acid (CLA) for 56 d to explore the effects of dietary CLA on the fatty acid profile and cholesterol content of egg yolks from laying hens of different breeds. Four hens were placed in 1 cage, and 3 cages were grouped as 1 replicate, resulting in 6 replicates per treatment. After feeding the experimental diets for 11 d, eggs were collected to determine the fatty acid composition of the egg yolks. From d 54 to 56, eggs were collected to measure the cholesterol content of yolks, and on d 56, a hen was selected randomly from each replicate and bled to determine the cholesterol content in plasma. There was a significant effect of layer breed on layer performance and egg composition. Concentrations of stearic, arachidonic, and docosahexaenoic acids were higher in the yolks of Brown Dwarf hens than in those of White Leghorn hens. Enrichment of cis-11, trans-13 was higher in the yolks of White Leghorns, but cis-10, cis-12 was higher in those of Brown Dwarf hens. In contrast, feed intake and egg weight, as well as yolk weight and its ratio to egg, were decreased by the 5% dietary CLA treatment. Egg production and feed efficiency were not affected by dietary CLA. Concentrations of total CLA and CLA isomers in the yolk lipids increased (P < 0.01) with increasing dietary CLA. Furthermore, yolk cholesterol was increased with increasing dietary CLA (P < 0.01), but this was significantly decreased in Brown Dwarf hens (P < 0.01) by feeding 2.5% CLA. There was no apparent correlation between yolk cholesterol content and serum cholesterol content. In conclusion, Brown Dwarf layers had the breed-specific characteristics of enrichment of CLA isomers and fatty acids in yolk lipids in response to dietary CLA.

Utilization of Oil By-Product from the Purification Process of Conjugated Linoleic Acid as Feeding Supplements for the Accumulation of Conjugated Linoleic Acid in the Egg Yolk

This study was performed to examine an efficient way to use oil by-product which is obtained during the purification process of conjugated linoleic acid (CLA) from safflower oil. The CLA by-product (CBP) was fed to the laying hens to accumulate CLA in the egg yolk. Egg yolk samples from 3 different dietary groups were analyzed: control; CBP, 2%; and CLA-80 (which contained 80% free form of CLA), 2%. Fatty acids from the yolk were analyzed by gas chromatography, and the parameters of egg quality were determined. During the feeding trial, there was little alteration in the egg quality and egg production of the tested groups. The CLA contents in the test group with CBP, which contain CLA as glyceride form, significantly increased in the first week of feeding and had the highest level among the tested groups throughout the feeding trials, whereas the CLA group showed an increase in CLA contents from the third week. Moreover, the contents of total CLA isomers in CLA-80 and CBP oils were decreased by 12.92 and 0.51% after heat treatment in 48 h, and the contents of linoleic acid (LA) isomer in LA-80 (which contained 80% free form of LA) and LA by-product (LBP) oils were decreased by 19.63 and 5.78%, respectively. It was confirmed that a major form of fats in CBP was mainly esterified forms, whereas the fats in CLA-80 and LA-80 were composed of free fatty acids. There was no significant difference in the egg quality and production among the tested groups. It is meaningful that the oil by-product could be utilized as a source for functional foods of animal origin without influencing egg quality and production.

Contents of conjugated linoleic acid isomers in ruminant-derived foods and estimation of their contribution to daily intake in Portugal

The present study provides a detailed overview of the contents of conjugated linoleic acid (CLA) isomers in the most consumed Portuguese CLA-rich foods (milk, butter, yoghurt, cheese, beef and lamb meat), by using silver ion-HPLC. In addition, the contribution of these ruminant-derived foods to the daily intake of CLA isomers was estimated based on Portuguese consumption habits. The total CLA concentration in milk and dairy products ranged from 4.00 mg/g fat in yoghurt to 7.22 mg/g fat in butter, and, regarding meats, from 4.45 mg/g fat in intensively produced beef to 11.29 mg/g fat in lamb meat. The predominant CLA isomers identified in these products were cis-9, trans-11 (59.89-79.21 %) and trans-7, cis-9 (8.04-20.20 %). The average estimated total CLA intake for the Portuguese population was 73.70 mg/d. Milk and cheese are probably the two products with the highest contribution to the final CLA intake, as a result of their high fat content and consumption values. The results also suggested that cis-9, trans-11 and trans-7, cis-9 are the isomers most represented, with, respectively, 76.10 and 12.56 % of the total CLA intake. Being the first detailed report on the contents of total and individual CLA isomers in Portuguese commercial ruminant-derived foods, we further discuss the implication of the results for diet characteristics and human health.

한국 유아에서 분리한 Bifidobacteria에 의한 발효유의 Conjugated Linoleic Acid (CLA) 함량 증가

More than 200 Bifidobacterium sp. originated from human intestine were investigated for their ability to produce conjugated linoleic acid (CLA). Of the Bifidobacteria tested, 1 of culture type strain and 12 isolated strains from Korean infants showed CLA producing ability. cis-9, trans-11 octadecadienoic acid presented more than 90% of the total CLA isomers produced by the Bifidobacteria. CLA content in fermented milk by Bifidobacterium sp. KHU 141 increased by 39.6 mg/l00 g, which showed the potential use for producing fermented milk containing high content of CLA. In fermented milk, little changes showed in lauric acid, myristric acid, palmitic acid, oleic acid, and linolenic acid contents, whereas the content of linoleic acid (LA) decreased and the content of CLA increased. Bifidobacterium sp. KHU 141 converted 86.0% and 84.8% of LA consumed to CLA for 24 hr and 48 hr fermentation, respectively. Prolonging incubation from 24 to 48 hours did not appear to enhance CLA formation and CLA producing ability was stable whether bottle, test tube, or fermenter was used for making fermented milk by Bifidobacterium sp. KHU 141.

Effects of conjugated linoleic acid levels and feeding intervals on performance, carcass traits and fatty acid composition of finishing barrows

Two experiments were conducted to investigate the effects of dietary conjugated linoleic acid (CLA) on performance, carcass traits, fatty acid composition and subcutaneous adipose tissue cellularity in finishing barrows. In Experiment 1, 54 crossbred barrows were allotted to one of three treatments, with six pens per treatment and three barrows in each pen. The pigs were fed a diet containing 0, 2, or 4% CLA oil for 6 weeks. Daily gain (P < 0.01) and feed efficiency (P < 0.01) improved with dietary CLA. Loin muscle area (P = 0.01) and intramuscular fat (P = 0.01) increased while 10th rib fat (P = 0.03) and last rib fat (P = 0.02) thickness decreased with increasing dietary CLA. Total CLA isomers increased (P < 0.01) with increasing dietary CLA. Myristic, palmitic and stearic acid levels were increased while oleic, linoleic, linolenic and arachidonic acid decreased in loin muscle and subcutaneous adipose tissue. In Experiment 2, barrows (n = 54) were allotted to one of two treatments with nine pens per treatment and three pigs in each pen. Pigs were fed a diet supplemented with 4% CLA for 3 or 6 weeks before slaughter. Over the entire experimental period, daily gain and feed efficiency were higher (P < 0.01) when CLA was fed for a longer period. Loin muscle area (P < 0.01) and intramuscular fat (P < 0.01) increased while backfat thickness at the 10th (P = 0.03) and last rib (P = 0.04) decreased when CLA was fed for 6 vs. 3 weeks. The number of cells in subcutaneous adipose tissue was not affected while adipocyte volume decreased (P = 0.01) with longer feeding time on dietary CLA. The increased CLA content of pork from CLA fed pigs provides the pork industry with an opportunity to provide value-added, healthful meat products for human consumption with respect to CLA intake and potential improvements in human health.

Effects of supplemental rumen-protected conjugated linoleic acid or corn oil on fatty acid composition of adipose tissues in beef cattle.

Thirty-six Angus x Hereford heifers (365 +/- 60 kg) were used to determine the effects of supplemental dietary lipid sources on fatty acid composition of i.m., perianal (p.a.), and s.c. lipid depots. Lipid was supplied to diets as either corn oil or a rumen-protected conjugated linoleic acid (CLA) salt for two specific treatment periods of either the final 32 or 60 d on feed. Following an initial 56-d feeding period, heifers were fed one of three dietary treatments (DM basis): 1) basal diet containing 88% concentrate and 12% grass hay (CON), 2) basal diet plus 4% corn oil (OIL), or 3) basal diet plus 2% rumen-protected CLA salt (RPCLA) containing 31% CLA. The trans-10, cis-12 CLA concentration was greatest (P < 0.05) for heifers fed RPCLA and OIL diets and least (P < 0.05) for CON, regardless of time on dietary treatment. Heifers fed supplemental RPCLA had greater (P < 0.05) total CLA content than either CON- or OIL-fed heifers. Adipose tissue concentration of trans-11 vaccenic acid (TVA) was less (P < 0.05) for CON than OIL or RPCLA, which did not differ (P > 0.05). Percentages of C18:1 trans-10 were least (P < 0.05) in i.m. lipid compared with p.a. and s.c., which did not differ (P > 0.05). Following 60 d of lipid supplementation, heifers fed OIL and RPCLA had lower (P < 0.05) concentrations of oleic acid and total monounsaturated fatty acids (MUFA) compared with CON. The ratio of cis-9, trans-11 CLA:TVA was higher (P < 0.05) for heifers fed 60 vs. 32 d, but did not differ (P > 0.05) between adipose depots. Feeding OIL increased (P < 0.05) adipose concentration of C18:2 fatty acid, whereas feeding RPCLA increased (P < 0.05) total CLA isomers by 22%. Intramuscular lipid contained the lowest (P < 0.05) percentage of cis-9, trans-11 CLA, total CLA, C18:1 cis-9, C18:1 trans-10, and TVA. Total CLA and cis-9, trans-11 CLA isomers were increased (P < 0.05) in p.a. and s.c. adipose depots, whereas i.m. adipose tissue contained increased (P < 0.05) amounts of total PUFA. Results from this study indicate that short-term lipid supplementation to feedlot cattle can increase adipose tissue CLA concentrations, but only marginally (8.3 to 17.5%). Moreover, observed decreases in oleic acid and total MUFA concentrations of adipose tissues from heifers fed rumen-protected CLA or corn oil suggest that lipid supplementation may decrease delta9 desaturase activity in adipose tissues, which in turn would lower the conversion of TVA to cis-9, trans-11 CLA isomer.

Preferential incorporation oftrans,trans-conjugated linoleic acid isomers into the liver of suckling rats

The present study was designed to compare the conjugated linoleic acid (CLA) isomeric distribution pattern in the liver of suckling rats in relation to those in the milk and maternal diet. Silver-ion HPLC was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the milk was very similar to that in the maternal dietary fat. However, the CLA isomeric distribution patterns in the liver phospholipids (PL) and triacylglycerols were different from those in the diet and milk. In the liver PL, totalcis/transisomers accounted for 63·6–63·9 % of total CLA, which was in contrast to the values of 88·1–89·1 % in the milk and diet. In the liver PL, totaltrans/transisomers were 20·6–20·8 % of the total CLA isomers whereas they were only 2·6–3·7 % in the milk and diet. It is concluded thattrans/trans-CLA were preferentially incorporated into the liver whereas for the incorporation ofcis/trans-CLA there was partial discrimination.

Preferential incorporation of trans, trans-conjugated linoleic acid isomers into the liver of suckling rats.

The present study was designed to compare the conjugated linoleic acid (CLA) isomeric distribution pattern in the liver of suckling rats in relation to those in the milk and maternal diet. Silver-ion HPLC was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the milk was very similar to that in the maternal dietary fat. However, the CLA isomeric distribution patterns in the liver phospholipids (PL) and triacylglycerols were different from those in the diet and milk. In the liver PL, total cisltrans isomers accounted for 63.6-63.9% of total CLA, which was in contrast to the values of 88.1-89.1% in the milk and diet. In the liver PL, total transltrans isomers were 20.6-20.8% of the total CLA isomers whereas they were only 2.6-3.7% in the milk and diet. It is concluded that trans/trans-CLA were preferentially incorporated into the liver whereas for the incorporation of cis/trans-CLA there was partial discrimination.

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography.

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag+-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag+- HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans, 10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans, 10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.