The role of oleic acid is currently the subject of much discussion, particularly at the level of the control of serum lipoprotein concentrations. The effect of oleic acid has primarily been studied in reference to cardiovascular disease and the regulation of plasma LDL and their cholesterol content and ability to be oxidized (Khosla and Hayes 1993, Reaven et al. 1993 and 1994). Oleic acid may also play a role in the control of blood pressure and viscosity and in the transport of cations in the erythrocytes (Sacks et al. 1987).

Studies in different species have shown the influence of a high level of oleic acid on the fatty acid composition of tissues (Periago et al. 1990, Rao et al. 1993). Oleic acid modulates lipid metabolism (Lu and Wu 1994). Dietary oleic acid integrated into membrane phospholipids affects many enzyme activities [including desaturation of fatty acids (Giron et al. 1989), transport efficiency and receptor activities] and induces a dysfunction of pulmonary endothelial cells via oxidizing agents (Hart et al. 1993). However, oleic acid can also act by itself, affecting, for example, gap junctions in the heart and smooth muscle cells differently (Hirschi et al. 1993). It also modulates the binding to glucocorticoid receptors in the lung negative feedback (Viscardi and Max 1993). In addition, oleic acid can affect signal transduction, in particular by activating the isoenzymes of protein kinase C (Khan et al. 1993) and the phosphorylation of proteins in the hippocampus (Chen and Murakami 1994). Oleic acid also modulates the interaction between benzodiazepines and the -aminobutyric acid (a major inhibitory neurotransmitter in the mammalian central nervous system) receptor (Witt and Nielsan 1994). Oleic acid can also modulate the effect of toxins on neuroblastomas (Jourdon et al. 1989), as well as the expression of genes, for example, by stimulating the expression of the phosphoenolpyruvate carboxykinase gene in cultured adipocyte cells (Antras-Ferry et al. 1994). Oleic acid has been implicated in the structure of the endogenous digitalis factor (Young et al. 1986) and 1-oleoyl-2-acetyl glycerol in the proliferation of Schwann cells induced by myelin (Saunders and de Vries 1988).

In view of the importance of oleic acid, the aim of this work was to determine whether rats are able to synthesize all the oleic acid needed by feeding a diet totally deficient in oleic acid and measuring the amount of this acid found in various tissues, in comparison with rats fed a diet containing oleic acid.

MATERIALS AND METHODS

Table 4. Effects of dietary fatty acids on oleic acid levels in various organs and subcellular fractions from 21-d-old rats1,2,3 [View Table]

Table 5. Effects of oleic acid-deficient diet on 18:1(n-7) and 16:1(n-7) concentrations in various organs and subcellular fractions from 21-d-old rats1,2,3 [View Table]

Table 1. Fatty acid composition of the synthesized triglycerides1 [View Table]

Table 2. Composition of the diets1 [View Table]

We thus fed rats semipurified diets whose lipids (triglycerides) were synthesized chemically. These triglycerides included essential linoleic and -linolenic polyunsaturated fatty acids [added to the diets in the proportion of 6 to 1, as suggested by our previous results (Bourre et al. 1989)] and triglycerides formed with oleic acid and with stearic acid. Fatty acid composition of the various synthesized triglycerides is given in Table 1. The amount of triglycerides added to the different diets is given in Table 2. The overall composition of the semipurified diets used has already been published (levels of casein, DL-methionine, cellulose, starch, sucrose and vitamin and mineral mixtures) (Bourre et al. 1989) (Table 2).

Five groups of Wistar rats (Iffa credo, France) received different diets: Group 1 (control, R1), essential fatty acids and oleic acid (control diet); Group 2 (R2), a diet totally deficient in oleic acid; Group 3 (R3), a diet enriched with 1.6% oleic acid; and Groups 4 (R4) and 5 (R5), diets containing stearic acid (1.6 and 8.6%, respectively). All the diets contained the same amount of essential polyunsaturated fatty acids [approximately 12 g of 18:2(n-6) and 0.2 g of 18:3(n-3) per kilogram of diet]. The oleic acid concentration in the control diet was 29.09 g/kg diet. The fatty acid composition of the various diets is given in Table 3.

Table 3. Fatty acid composition of the diets1 [View Table]

Three weeks before mating, five groups of females each received one of the five diets. Half of their male pups were killed at 21 d (weaning) and half at 60 d.

Rats were killed by decapitation and exsanguination. Lipids were extracted with chloroform-methanol using the Folch method; methyl esters were obtained by the Morrisson method using methanolic boron trifluoride; fatty acids were analyzed on capillary column. All techniques have been published (Bourre et al. 1989).

For the various organs, values are the means of at least five different rats, from at least three different litters. For myelin and nerve endings, each value is the mean of at least four different preparations; each density gradient required at least four rats. Thus, each value represents at least 16 rats (from at least three different litters). Experimental protocols were approved and met the government guidelines (Ministry of Agriculture, authorization no. 03007, June 4, 1991).

Data are expressed as means ± SD, with n = 5 per group. Statistical significance of means was calculated by ANOVA using Student-Newman-Keuls test for multiple comparisons and Student's t test for comparison of two groups. The Mann-Whitney test was also used when the conditions for parametric tests were not met. Differences were considered significant at P < 0.05. The SigmaStats package (Jandel Scientific, Ekrath, Germany) was used for statistical analysis.

RESULTS AND DISCUSSION

Table 6. Effects of dietary fatty acids on oleic acid concentrations in various organs and subcellular fractions from 60-d-old rats1 [View Table]

In comparison with control rats, rats fed diets supplemented with oleic acid (R3: 1666 mg/100 g diet, Table 4) had normal levels of this fatty acid in some organs (liver, heart and sciatic nerve) but not in others (kidney, muscle, testes). Fatty acid levels in whole brain, myelin and nerve ending were not altered. This supplementation with oleic acid led to lower concentrations of 16:1(n-7), which returned to about the same levels as in the control group in all organs except liver. Dietary supplementation with stearic acid (at 1.6 or 8.6%) did not alter the level of 18:1(n-9) in the tissues compared with animals fed an oleic acid-deficient diet.

Levels of polyunsaturated fatty acids in the different diets were identical. Consequently, these fatty acids were not involved in the changes seen for saturated and monounsaturated fatty acids. For the saturated fatty acids, the level of stearic acid was the same in both the deficient diets and those supplemented with oleic acid. Thus, this acid did not play a role in the differences observed in the organs of rats fed these two diets. Moreover, the addition of stearic acid (R4 and R5, Table 4) to the diet devoid of oleic acid not only did not raise stearic acid concentrations in the tissues but also failed to increase the 18:1(n-9) concentration, while the (n-7) concentration remained elevated. Thus, -9-desaturase (which synthesizes oleic acid from stearic acid) seems to be more active on the 16:0 family than on the 18:0 family with respect to synthesis of 16:1(n-7) and 18:1(n-9), respectively [the latter fatty acid being sometimes further elongated into 18:1(n-7)]. Interestingly, in oleic acid-deficient rats, the concentration of 18:1(n-9) was always higher than the level of 16:1(n-7) + 18:1(n-7); for example, in liver it was 15.4% vs. 5.48%. Thus, -9-desaturase synthesized approximately two times more 18:1(n-9) than (n-7) monounsaturated fatty acids.

Similar results were obtained in 60-d-old oleic acid-deficient rats (Table 6). In comparison with results for 21-d-old rats, this longer period of dietary deficiency did not further lower oleic acid concentration, and in some cases slightly corrected it. The lower concentrations of 18:1(n-9) in liver, kidney, testes, muscle and sciatic nerve were 45, 38, 29, and 26%, respectively. There was one notable difference: heart 18:1(n-9), which was not significantly affected in 21-d-old rats, was significantly lower (50%) in adults. On the other hand, testes 18:1(n-9), which was significantly lower in young rats, was not affected in adults.

Dietary supplementation with oleic acid (R3: 1666 mg/100 g diet, Table 6) up to 60 d resulted in normal levels of this fatty acid in some organs (liver, testes and muscle) but not in others (kidney, heart and sciatic nerve). Levels in whole brain, myelin and nerve endings were not altered. Deficiency in oleic acid induced lower levels of 16:1(n-7) in kidney, testes, muscle, heart, myelin and sciatic nerve; 18:1(n-7) concentration was higher in all organs, except for brain, myelin and nerve endings (Table 7). Supplementation with oleic acid led to lower concentrations of 16:1(n-7), which returned to about the same levels as in the control group in liver and heart only (data not shown). Dietary supplementation with stearic acid (at two concentrations: 1.6 and 8.6%) generally did not significantly alter the level of 18:1(n-9) in the tissues in comparison with animals fed an oleic acid-deficient diet (Table 6). Thus, rats fed an oleic acid-deficient diet up to 60 d presented different alterations compared with those fed the deficient diet up to 21 d.

Table 7. Effects of oleic acid-deficient diet on 18:1(n-7) and 16:1(n-7) concentrations in various organs and subcellular fraction from 60-d-old rats [View Table]

In the brain, palmityl-CoA and stearyl-CoA desaturases have analogous activities, which decrease rapidly during the perinatal period in mice (Carreau et al. 1979). In other organs, one can speculate that the affinity of desaturase for the two fatty acids could be different.

Thus, 18:1(n-9) membrane levels are the result not only of -9-desaturase activity but also of the amount of this fatty acid in the diet. It should be noted that oleic acid deficiency resulted in little or no alteration in polyunsaturated fatty acid levels [in fact, dietary oleic acid protects the (n-3) series] (Navarro et al. 1994).

The amounts of -linolenic [18:3(n-3)] and linoleic [18:2(n-6)] acids that should be supplied in the diet to allow the development of the membrane structures of different organs, including brain, have been determined (Bourre et al. 1989 and 1993). Recent experiments have measured the amounts necessary to maintain the structures, i.e., to ensure their renewal (Bourre et al. 1990). These studies have led to advances in the industrial field: the development of a vegetable oil blend and the improvement of formula milks for infants. Oleic acid will now have to be taken into account.

Dietary oleic acid deficiency led to a decrease in this fatty acid in some organs. In this study, abnormalities observed during the gestation-lactation period persisted in adult rats that continued to receive a deficient diet. Membrane fatty acid composition did not further deteriorate; indeed, there was sometimes a slight correction. We conclude that rats (and particularly the liver) do not have sufficient synthesizing potential to guarantee the normal fatty acid composition of certain organs if oleic acid is deficient in the diet. For some organs, 1.6% 18:1 in the diet does not meet the requirements.

These results show that oleic acid should be taken into account in nutritional recommendations. Nevertheless, the relationship between cellular and subcellular physiologies and oleic acid levels in the membrane remains to be determined.

ACKNOWLEDGMENT