Pmstaghtdins kJ Longman

Leukotrienes and Essential Ltd 1992

Fatty Acids

(1992) 46, 111-121


The Influence of Dietary Essential Fatty Acids on Uterine C20 and C22 Fatty Acid Composition A. Howie’, H. A. Leaver, N. H. Wilson, P. L. Yap* and I. D. Aitken+ Department of Pharmacology, University of Edinburgh, Edinburgh EH8 9JZ, UK, *Clinical Pharmacology Unit, Department of Medicine, University of Edinburgh, UK and + Moredun Research Institute, Edinburgh, UK (Reprint requests to HAL)

The effect of dietary fatty acids on uterine fatty acid composition was studied in rats fed control diet or semi-synthetic diet supplemented with 1.5 &$ay evening primrose oii (EPO) or fish oil (FO). Diet-related changes in uterine lipid were detected within 21 days. Changes of 2- to 20-fold were detected in the uterine n-6 and n-3 essential fatty acids (EFA) and in certain saturated and monounsaturated fatty acids. The FO diet was associated with higher uterine C20 and C22 n-3, and the EPO diet, with higher uterine n-6 fatty acid. High uterine Cl&2 n-6 was detected in neutral lipid (NL) of rats fed high concentrations of this fatty acid, but there was little evidence of selective incorporation or retention of Cl&2 n-6 by uterine NL. The incorporation of EFA into uterine phosphoiipids (PL) was greater than NL EFA incorporation, and uterine PL n-3/n-6 ratios showed greater diet dependence. Tiiu&iet fatty acid ratios in NL and PL also indicated preferential incorporation/synthesis of C16:l n-9, and C16:0, and there was greater incorporation of C12:O and C14:O into uteri of rats fed EPO and FO. Replacement of 5040% of arachidonate with n-3 EFA in uterine PL may inhibit n-6 EFA metabolism necessary for uterine function at parturition.



bition of uterine PGE2 synthesis was also detected in fish oil-fed rats. Desaturation and chain elongation of dietary EFA play an important role in determining the fatty acid composition of mammalian tissue. The n-6 EFA and n-3 EFA are not interconvertible and form two distinct families of EFA. In the rat the main site of elongation and desaturation is the liver. The n-3 and n-6 EFA compete for liver desaturase and elongase enzymes, the n-3 fatty acids having a greater affinity for desaturase (11-13). The D6 desaturase is the rate determining enzyme of the EFA elongase-desaturase pathway, and gammalinoleic acid in evening primrose oil (EPO) does not require the D6 desaturase enzyme for arachidonate production. The dietary C20 and C22 fatty acids of the n-3 series in fish oil also bypass D6 and D5 desaturase activities. D5 desaturase activity is important for arachidonic acid production from dietary precursors in both the rat and the human. The accumulation of 20:3 n-6 suggests that low D5 desaturase activity is present in the testis (14), thymus (15) and adrenal (16). Analysis of the specificity of incorporation and metabolism of the n-6 EFA in the uterus indicates

Arachidonic acid metabolism in the uterus is characterised by the selective esterification of arachidonic acid by uterine phospholipids (l-3) and the quantity of prostaglandins released at parturition (4, 5). Both arachidonic acid esterification and prostaglandin (PG) release are influenced by gonadotrophins (1,4-7). An inhibitory effect of n-3 fatty acids on uterine activity was identified in rats fed alpha-linolenic acid as the major essential fatty acid (EFA) (8, 9). These rats showed major defects in initiating and sustaining labour, and fetal mortality was high, but if caesarian section was carried out early in labour, live animals were delivered. We observed similar effects on the initiation of parturition when fish oil (FO) was given as the major dietary EFA (5, 10). In these experiments, inhi-

’ Present address: Efamol Research Institute, PO Box 818, Kentville, Nova Scotia, Canada BN4 4H8.

Date received 8 August 1991 Date accepted 13 November 1991 111




and Essential Fattv Acids

that there is selective turnover of n-6 fatty acids in various lipid pools of the uterus (2, 3, 5, 8-10, 17). There is also evidence of selective release of n-6 fatty acids during parturition (2, 10, 17-19). These studies of fatty acid uptake and release indicate that the enzymes of uterine phospholipid metabolism regulate the supply of n-6 fatty acids at parturition. We have proposed that the n-3 effect on parturition may be explained in terms of the competitive inhibition of n-6 specific cyclooxygenase metabolism (5). However, evidence for the highest level of uterine n-6 specificity appears to lie at the level of uptake and esterification, rather than at the level of release and cyclooxygenase specificity (2, 5, 20). This selectivity may involve acyl and acetyl transferases and the transesterification and remodelling of phospholipids. In this study, the effects of dietary n-3 and n-6 EFAs on uterine fatty acid composition were investigated. The effects of diets containing two naturally acid preparations containing occurring fatty predominantly n-6 fatty acids (evening primrose oil, EPO), or n-3 fatty acids (fish oil, FO) on uterine fatty acid composition were compared with a control diet. The dietary EFA composition of the semisynthetic diets with EFA supplementation was either predominantly (92%) n-6 in the EPO diet or predominantly (81%) n-3 in the FO diet. The source of n-6 fatty acids used in this study, EPO, was characterised by its content of gamma-linolenic acid, C18:3n-6, (8.7%) which bypasses the rate limiting delta-5 desaturase enzyme. The kinetics of diet induced effects on uterine EFA were studied by comparing short-term feeding (21 days) with longterm feeding (220 days). The fatty acid content and composition of uterine lipid of rats in the three diet groups were compared. The preferential incorporation of specific fatty acids was investigated by comparison of diet and tissue fatty acid composition. The lipid pools in which changes in EFA metabolism occurred were investigated by analysing phospholipid and neutral lipid fatty acid composition after 21 days of dietary EFA manipulation. The influence of dietary EFA on developmental changes in the fatty acids of rat uterus are described in an accompanying paper (21).



Materials Evening primrose oil (EPO, Efamol) and Hi-EPA (FOz) were donated by Efamol Ltd, Guildford, Surrey, and MaxEPA (FOi), by Seven Seas Health Care Ltd, Marfleet, Hull. Analytical grade reagents were purchased either from Rathburn Chemicals Ltd, Walkerburn, Scotland or from BDH Ltd,

Poole, England. Fatty acid methyl ester standards were from Sigma, Poole, England. Silicic acid (lOO200 mesh) was purchased from Unisil, Clarkson Chemical Company, Williamsport, USA. Animals and diet Female Sprague-Dawley rats of three age groups were used: adult (240 f 19 days, n = 39), young (46 + 0 days, n = 15) and newly weaned (23 + 0.65 days, n = 10). Rats from the adult and young groups were randomly divided into three groups, and weaned onto three different diets consisting of either a control pelleted diet or a semi-synthetic diet low in EFA supplemented with EPO or FO. Rats of the newly weaned group were born from mothers of the three different diet groups. Rats of Diet Group 1 (control) were fed a normal pelleted diet (CRM diet, BSS Ness, Edinburgh). Groups 2 and 3 were fed a semi-synthetic diet depleted of EFAs, and supplemented with 1.5 ml/kg rat body weight per day of EPO or FO. The EPO and FO supplement was stored under nitrogen at 4°C to minimise oxidation and administered orally using 10 cm Portex infant feeding tubes (Kent, UK). The semi-synthetic diet fed to rats in Groups 2 and 3 consisted of fat 16.5 en %, of which 13.5 en% was saturated fat (hydrogenated coconut oil, Pilsbury’s, Birmingham, UK) and 3.3 en% was essential fatty acid supplement (EPO or FO); protein 15.7 en% (fat-free casein, BDH, Poole, UK); carbohydrate 68.7 en% (D-glucose) with non-digestible fibre (cellulose 11.02 g/kcal, kaolin 5.5 g/kcal); DLmethionine (2.6 g/kcal), BDH; Vitamin premix (2.76 s/k ca 1) an d mineral premix (12.9 g/kcal) were from Special Diet Services, Cambridge, UK. Adult rats received MaxEPA as a fish oil supplement (FOi) and young rats were given Hi-EPA (F02). The fatty acid composition of the diets containing FOi and F02 was similar (Table l), however FOz had a slightly higher n-3 content (Table 10). The essential fatty acid composition of the EPO diet consisted of 99.5% n-6 and 0.5% of n-3 fatty acid; the pelleted diet, 84% n-6 and 16% n-3; and, in the FO diet, 25% of EFA were n-6 and 75% were n-3 fatty acids. In addition to the uterine fatty acids identified, several minor fatty acids were detected which did not vary significantly between diet groups, including an unidentified peak with shorter retention time than C12:O (2.7-2.9% of total fatty acid). There was no evidence of EFA deficiency in rats fed the EFA supplemented semisynthetic diet as detected by weight loss, fur condition or the 20:3 n-9 content of uterine lipid (Tables 2-4). The 20: 3 n-9/20:4 n-6 ratio in the FO and EPO fed rats was not greater than 0.2 in any of the rats (a ratio of greater than 0.4 indicates fatty acid deficiency (17).


The Influence of Dietary Essential Fatty Acids on Uterine C20 and C22 Fatty Acid Composition

Fatty acid extraction and methylation After the rats were killed, uteri were placed immediately in chloroform : methanol (2 : 1). Lipids were extracted four times from the ground tissue using chloroform: methanol 2: 1, under nitrogen. In samples collected from the weaned and adult chloroform : methanol contained the groups, OS-5 mg C17:O internal standard. Uterine lipid from young rats was separated into phospholipid and neutral lipid fractions by silicic acid column chromatography, using columns dehydrated prior to use with 20 ml diethylether, 20 ml 1: 1 diethylether : acetone then 60 ml diethylether. Neutral lipids were eluted using 30 ml of ether, and phospholipids were eluted using 50 ml of methanol. The Cl7:O internal standard was added to neutral lipid (20 pg) and to phospholipid (200 pg). The fatty acids of total lipid, neutral lipid and phospholipid samples were released by alkaline hydrolysis in methanol and methylated using Boron Trifluoride (10). The fatty acid methyl esters were extracted four times using petroleum ether (60-80”). Gas chromatography Samples in hexane were injected into a Pye 204 gas chromatograph with flame ionisation detector fitted with a 15 m DB wax bonded carbowax capillary column (J. and W. Scientific, Ranch0 Cordova, California, USA), using Helium as a carrier gas (5 ml/min) and injector and detector temperatures of 175°C and 250°C respectively. Oven temperature, 180°C for 1 min, increased to 230°C at CC/min. Fatty acids in samples were identified by comparison with retention times of standard fatty acid methyl esters. The quantitation of fatty acids was carried out using a Supergrator 1A Integrator (Kentronix UK Ltd, Compton, Berkshire, England), using C17:O as the internal standard.

predominant polyunsaturated fatty acid in uteri of all diet groups. Small quantities of short-chain fatty acids comprising ~1% of total fatty acids, were detected in the uterus of adult rats, one of which, C16:l n-9, was influenced by diet, being higher in FO fed rats. Feeding rats the EPO and FO diets resulted in changes in uterine saturated fatty acids, monounsaturated fatty acids, and n-3 and n-6 polyunsaturated fatty acids (Table 2). Significant differences between diet groups were detected in the uterine distribution of the saturated Cl2 and Cl4 fatty acids, which were present in higher concentrations in the EPO and FO groups, and in C16:0, which was higher in the control diet group. Uterine lipid from the FO group contained more C16:l n-9 than uterine lipid of rats fed the control diet. The uterine content of saturated fatty acids was dependent on the stage of uterine development (see 21). In uterine n-6 EFA, two major diet-related changes were detected: the EPO diet was associated with 1.5- to 9-fold increases in the proportion of long-chain n-6 EFA C20:2, 20:3, 20:4, 22:4 and 22:5, compared with controls; and the FO diet was associated with lower proportions of the long-chain n-6 EFA C20:4 and C22:4. The greatest dietrelated changes in uterine n-3 fatty acids were detected in the FO diet group, which contained approximately three times the concentrations of long-chain n-3 EFA C20: 5, C22 : 5, C22: 6 detected in control and EPO groups.

Table 1 Percentage fatty acid composition of rat diets Fatty acid


Adult EPO

Young EPO

Adult FO,

Young FO*

0.01 0.4 17.0 0.7 2.1 20.6 45.6

51.7 17.8 8.2 0.3 6.8 3.0 7.2 0.84 0.03

49.2 17.0 8.2 0.29 6.6 3.3 10.2 1.22 0.03

50.4 18.7 9.5 1.5 6.8 3.9 0.98

51.4 19.1 9.0 1.4 6.9 3.3 0.8

The effect of dietary fatty acids on uterine fatty acid composition in adult rats

12:o 14:o 16:0 16:l 18:0 18:l 18:2 18:3 18:3 20:o 2O:l 20:4 20:5 2215 22~6

0:5 0.05 < <

The influence of dietary essential fatty acids on uterine C20 and C22 fatty acid composition.

The effect of dietary fatty acids on uterine fatty acid composition was studied in rats fed control diet or semi-synthetic diet supplemented with 1.5 ...
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