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Oxidative metabolism of [1-14C] mono-trans isomers of linoleic and α-linolenic acids in the rat
Biochimica et Biophysica Acta 1390 Ž1998. 207–214
Oxidative metabolism of w1-14Cx mono-trans isomers of linoleic and a-linolenic acids in the rat Lionel Bretillon a , Jean-Michel Chardigny a , Jean-Louis Sebedio ´ ´ Jean-Pierre Noel ¨ b, Jean-Michel Vatele ` c
a,)
, Didier Poullain b,
a
b
INRA, Unite´ de Nutrition Lipidique, Dijon, France CEA-Saclay, SerÕice des Molecules Marquees, ´ ´ Gif-sur-YÕette, France c ESCIL, Laboratoire de Chimie Organique 1, Villeurbanne, France
Received 27 June 1997; revised 1 October 1997; accepted 3 October 1997
Abstract Trans polyunsaturated fatty acids are formed during processing of vegetable oils such as deodorization and frying. The oxidative metabolism of linoleic and a-linolenic acids and of their mono-trans isomers Ž9cis,12 trans-18:2, 9trans,12 cis-18:2 and 9cis,12 cis,15trans-18:3, 9trans,12 cis,15cis-18:3, respectively. was studied in fasting rats. A single dose of 18.5 MBq of each w1-14 Cx labelled fatty acid Ž260 mg. was orally given to the animals. The 14 CO 2 expired was monitored during 24 h. Radioactive countings of the CO 2-trapping agent were performed at regular intervals up to 24 h after oral administration of the radiolabelled fatty acid. Radioactive countings were also performed on several tissues Žliver, heart, brain, kidneys, sus-epidydimal fat, gastrocnemian muscle, gastrointestinal tract and carcass.. The 14 CO 2 production 24 h after oral administration of the fatty acid ranged from 55.5% to 68.7% of the radioactivity administered for the C18:2 isomers and from 69.7% to 73.5% for the C18:3 fatty acids. From 6 to 24 h, 14 CO 2 recovery was significantly higher after oral administration of 9cis,12 trans-18:2 than after giving both other octadecadienoic isomers. 14 C retention per gram of tissue in the liver and in the heart was significantly lower after feeding 9cis,12 trans-18:2 than after administration of both other C18:2 isomers. 14 C retention per gram of tissue in the muscle was significantly lower after administration of both trans C18:2 isomers compared to linoleic acid. Neither 14 CO 2 recoveries nor 14 C retentions were significantly different after administration of the three octadecatrienoic acids. The difference observed in 14 CO 2 recovery within the dienes was probably not due to a higher specificity of the enzymes involved in the b-oxidation sequence for the D12 trans double bond, as previously reported. Indeed, due to the labelling of the fatty acids on the carboxyl end, 14 C values recorded in the CO 2-trapping agent were only due to the first cycle of b-oxidation. q 1998 Elsevier Science B.V. Keywords: trans Fatty acid; b-Oxidation; Linoleic acid; a-Linolenic acid
)
Correspondence author. Fax: q33 3 8063 3223; E-mail: [email protected]
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 7 8 - 1
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L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
1. Introduction Trans polyunsaturated fatty acids are mainly produced during heat treatment of oils w1–3x. In deodorized or used frying oils, 9 cis,12 trans-18:2, 9 trans,12 cis-18:2, 9 cis,12 cis,15trans-18:3 and 9trans,12 cis,15cis-18:3 are the major isomers w1,4–6x and can also be ingested by human. The classical scheme of partitioning of dietary essential fatty acids is shared between b-oxidation on one hand and accumulation of the fatty acid itself, of its metabolites Žproduced by desaturation–elongation. and of the newly synthesized fatty acids produced from acetate units formed by b-oxidation of the essential fatty acid on the other hand w7x. Even if the trans polyunsaturated fatty acids have been found to be converted into products of desaturation andror elongation such as C18:4, C20:4 and C20:5 isomers with one trans ethylenic bond w8–15x, so far little data are available on the partitioning of these trans C18:2 and C18:3 fatty acids between b-oxidation and distribution in the rat tissues. Indeed, the only data available on the catabolism of the polyenes reported the catabolism of a mixture of the mono-trans isomers of linoleic acid and of the di trans isomer in rat liver mitochondria w16x and in rats w17,18x. Results have shown that trans isomers were catabolized to a greater extent than were their cis counterparts. Thus, in order to determine if these trans fatty acids follow the same biochemical partitioning as the essential fatty acids, pure synthetic and radiolabelled 9 c is ,1 2 tr a n s -1 8 :2 , 9 tr a n s ,1 2 c is -1 8 :2 , 9cis,12 cis,15trans-18:3 and 9trans,12 cis,15cis-18:3 were orally administered to rats. 14CO 2 was monitored during 24 h and the rats were then sacrificed. Radioactive countings were performed after sacrifice in the dissected rat. 2. Materials and methods 2.1. Animals Male Wistar rats, 41–57 days old, weighing 190– 230 g, supplied by Centre d’Elevage Depre ´ ´ ŽSaint Doulchard, France. were used. The animals were housed in individual cages under controlled conditions of temperature Ž22 " 18C., and relative humidity Ž55–60%.. A 12 h light–dark cycle Žlights on 7:00
a.m. to 7:00 p.m.. was maintained. The rats were fed sterilized commercial pellet diet Ž Extralabo, Provins, France. and had free access to tap water. All the experiments were performed at the same time Žgastric tubing at 9:00 a.m.. to avoid diurnal variations of the metabolism. The day before the experiment, prior to the dark period, each rat was restricted to 10 g of food. Purified tap water was available ad libitum. 2.2. Radiolabelled fatty acids L in o le ic Ž w1 -14 C x 9 c is ,1 2 c is -1 8 :2 , 1.96 GBq mmoly 1 . and a-linolenic Žw1- 14 C x 9cis,12 cis,15cis-18:3, 1.92 GBq mmoly1 . acids were supplied by NEN Products Ž Dupont de Nemours, Les Ulis, France. . Trans isomers of linoleic acid Žw1-14Cx 9cis,12 trans-18:2, 2.00 GBq mmoly1 and w1-14Cx 9trans,12 cis-18:2, 1.99 GBq mmoly1 . and of a-linolenic acid Žw 1- 14 C x 9 cis,12 cis,15 trans-18:3, 1.94 GBq mmoly1 and w1-14Cx 9trans,12 cis,15cis18:3, 1.88 GBq mmoly1 . were obtained by total synthesis w19,20x. All the fatty acids were of 98.6% radiopurity. Each fatty acid Ž18.5 MBq, 260 mg. was dissolved in triolein Žpurity 99%, Sigma Chemicals, L’Isle d’Abeau, France. and then administered by gastric tubing Žtotal volume: 200 ml.. To determine the exact quantity which was administered, the syringe and the catheter used were weighed before and after tubing. The rats were fasted during the whole 24 h experiment, but had free access to purified tap water. 2.3. Experimental system Immediately after oral administration of the radiolabelled fatty acid, the rat was housed in an airtight plexiglass ‘‘metabolic chamber’’ Ž Fig. 1. . The current of air in the cage was devoid of carbon dioxide by passing it through soda lime Žgranulation 2–5 mm, CaŽOH. 2 Ž 75–80%., NaOH q KOH Ž5–6%. , H 2 O Ž14–20%., Prolabo, Briare, France. . The 14CO 2 expired by the rat was trapped in a bottle containing 500 ml of 2-methoxyethylamine Ž Carbamate-1 National Diagnostics, Bionis, Clamart, France. . Effluent vapours of amine, nitrogen monoxide and carbon monoxide were subsequently trapped in 300 ml of water and in 300 ml of a diluted hydrochloric acid solution. The air flux in the cage Ž 950 ml miny1 . was obtained by the use of a peristaltic pump.
L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
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Fig. 1. Schematic system used to collect the air expired from the rat. 1: soda lime tube; 2: air-tight "metabolic chamber" Žthe feeding bottle containing tap purified water has not been drawn.; 3: CO 2-trap bottle containing the 2-methoxyethylamine as CO 2-trapping agent; 4: apparatus added in order to remove aliquotes of the trapping agent without interrupting the bubbling of the air expired from the rat in the 2-methoxyethylamine; 5 and 6: bottles containing water and diluted hydrochloric acid trapping the vapors of amine, nitrogen and carbon monoxide; air flux from the soda lime tube to the peristaltic pump.
2.4. In ÕiÕo recoÕery of
14
CO 2 expired by the rat
Without interrupting the bubbling of the expired air in the trapping agent, 1 ml was removed every 30 min during the first 6 h, hourly during the six following hours, each 3 h up to 21 h and then hourly up to 24 h. 9 ml of the scintillation cocktail Ž Monoflow 4 National Diagnostics. were immediately added and the solution was counted using a Tri-Carb 2000CA liquid scintillation analyzer Ž Packard, Groningen, Netherlands.. 2.5. RecoÕery of radioactiÕity in the tissues After 24 h, the rat was slightly anesthetized under a diethyl ether flux and blood was withdrawn by venipuncture into a syringe containing 2 ml of an anticoagulant agent ŽCitric acid 0.8%, sodium citrate 2.2%, D-glucose 2.45%, wrwrw.. Then liver, kidneys, heart, brain, gastrocnemian muscles and sus-epidydimal fat were removed, blotted and weighed before homogenization. The gastrointestinal tract was divided into two parts: from the stomach to the small
intestine Župper portion. on one hand and from the colon to the rectum including faeces Žlower portion. on the other hand. Each gastrointestinal tract portion was weighed separately and homogenized, the remainder of the animal Žcarcass. was weighed before homogenization. In order to determine the radioactivity in all tissues of the rat, three aliquots of 30–80 mg of each tissue, five aliquots of 50–100 mg of carcass and gastrointestinal tract portions were digested with 1 ml of Solusol ŽNational Diagnostics. from 4 to 5 h at 508C. When the digestion was achieved, 10 ml of the scintillation cocktail ŽMonoflow 4. were added. Three aliquots of 200 ml of blood were digested with 1 ml of a mixture of isopropanolrSolusol Ž1:1, vrv. for 15–30 min at 508C. 50 ml of hydrogen peroxide Ž 25%. were added under gentle stirring. Radioactive counting was performed after addition of 10 ml of the scintillation cocktail ŽMonoflow 4.. The urine from the 24 h experimental period was collected by rinsing the cage with water. The mixture of urine with water was divided in 1 ml fractions. 10 ml of scintillation
L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
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cocktail ŽUltima-Gold, Packard, Groningen, Netherlands. were added to each fraction. All the fractions were counted as described above.
statistical analysis. A P value - 0.05 was considered significant.
2.6. RecoÕery of total radioactiÕity
3. Results
2.6.1. Total 14CO 2 recoÕery The volume of the trapping agent present at the time of sampling was determined by carrying out the 24 h experiment three times without any rat in the cage. The values were used to calculate the 14CO 2 production. The values of 14CO 2 recovery were expressed per 100 g of rat in order to take into account the metabolic activity of each animal.
3.1. Body weight and weights of tissues of the dissected rats
2.6.2. RecoÕery of radioactiÕity in rat tissues The values of radioactivity in all parts of the rats were expressed as percentage of the administered radioactivity per gram of tissue.
The weights of the rats before oral administration of the fatty acids and of tissues of the rats after were not significantly different within the experimental groups Ž data not shown, P ) 0.05.. The weight losses during the 24 h experiment were not significantly different between the different groups of animals Ždata not shown, P s 0.32 and 0.41 for the octadecadienoic acids and for the octadecatrienoic acids, respectively.. 3.2.
14
C recoÕeries
2.7. Statistical analysis Values were reported as means "standard deviation Ž SD. . One-way analysis of variance Ž ANOVA. followed by paired Student’s t-test was employed for
3.2.1. 14C distribution in rat tissues The recovery of radioactivity per gram of tissue 24 h after oral administration of the w1-14Cx fatty acids is presented in Table 1: 94–99% of the administered
Table 1 Recovery of radioactivity per gram of tissue 24 h after oral administration of the w1-14Cx radiolabelled fatty acids to the fasting rat 18:2cc .a
18:2ct .b
18:2tc
18:3ccc
18:3cct
18:3tcc
CO 2 Žfinal value at 24 h Liver Brain Heart Kidneys Sus-epidydimal fat Muscle Upper GIT portion Lower GIT portion Carcass Urine Blood
27.04 Ž2.052. 0.23 Ž0.030. 0.04 Ž0.003. 0.17 Ž0.050. 0.17 Ž0.016. 0.20 Ž0.068. 0.11 Ž0.035. b 0.31 Ž0.101. 1.27 Ž1.402. 0.12 Ž0.011. 0.17 Ž0.071. 0.03 Ž0.002.
33.95 Ž0.873 0.12 Ž0.005. c 0.03 Ž0.002. 0.08 Ž0.008. b 0.14 Ž0.038. 0.30 Ž0.111. 0.05 Ž0.007. 0.14 Ž0.080. 0.99 Ž0.513. 0.09 Ž0.004. 0.23 Ž0.112. 0.03 Ž0.002.
29.59 Ž2.864. 0.18 Ž0.014. 0.06 Ž0.012. 0.13 Ž0.024. 0.14 Ž0.016. 0.51 Ž0.407. 0.06 Ž0.009. 0.27 Ž0.030. 0.93 Ž0.813. 0.11 Ž0.012. 0.12 Ž0.076. 0.03 Ž0.001.
35.09 Ž2.052. 0.17 Ž0.076. 0.04 Ž0.024. 0.11 Ž0.079. 0.15 Ž0.044. 0.12 Ž0.061. 0.03 Ž0.006. 0.28 Ž0.050. 2.02 Ž1.146. 0.07 Ž0.010. 0.25 Ž0.044. 0.02 Ž0.003.
34.30 Ž1.886. 0.11 Ž0.020. 0.05 Ž0.006. 0.06 Ž0.008. 0.17 Ž0.015. 0.13 Ž0.020. 0.05 Ž0.014. 0.18 Ž0.054. 0.85 Ž0.343. 0.08 Ž0.007. 0.15 Ž0.049. 0.03 Ž0.002.
32.75 Ž3.588. 0.11 Ž0.007. 0.05 Ž0.006. 0.07 Ž0.009. 0.19 Ž0.031. 0.11 Ž0.028. 0.03 Ž0.006. 0.36 Ž0.049. 0.19 Ž0.057. 0.07 Ž0.002. 0.19 Ž0.055. 0.02 Ž0.009.
Total activity recovery
94.9 Ž2.66.
93.8 Ž3.10.
96.6 Ž1.72.
99.4 Ž8.26.
95.1 Ž3.1.
96.3 Ž3.11.
Results are expressed as means of the percentage of the radioactivity administered per gram of tissue Ž"SD. from three animals Ž9cis,12 cis-18:2: 18:2cc; 9cis,12 trans-18:2: 18:2ct; 9trans,12 cis-18:2: 18:2tc; 9cis,12 cis,15cis-18:3: 18:3ccc; 9cis,12 cis,15trans-18:3: 18:3cct; 9trans,12 cis,15cis-18:3: 18:3tcc.. GIT: gastrointestinal tract. a Obtained by dividing the value of 14CO 2 production by the weight of the rat at t s 0. b Significantly different from the corresponding values 24 h after administration of both other octadecadienoic acids at P - 0.05. c Significantly different from the corresponding values 24 h after administration of both other octadecadienoic acids at P - 0.01.
L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
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Fig. 2. 14CO 2 recovery per 100 g of rat after oral administration to the fasting rat of: ŽA. w1-14Cx radiolabelled linoleic acid and of its D9trans and D12 trans isomers; ŽB. w1-14Cx radiolabelled a-linolenic acid and of its D9trans and D15trans isomers. Data have been obtained from three male Wistar rats for each fatty acid. Results are expressed as means of the radioactivity administered recovered as 14 CO 2 per 100 g of rat "SD. ) and ) ) significantly different from the corresponding values after administration of both other octadecadienoic acids at P - 0.05 and 0.01, respectively.
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dose was recovered. The three octadecatrienoic acids were similarly oxidized Ž P ) 0.05.. On the other hand, 9cis,12 trans-18:2 was significantly more catabolized into 14CO 2 than were linoleic acid and its D9trans isomer Ž P s 0.019.. The 14C activities in the liver 24 h after administration of 9trans,12 cis-18:2 and of linoleic acid were similar but it was significantly higher than the 14C retention after administration of 9cis,12 trans-18:2 Ž P s 0.0025.. The radioactivity per gram of heart was significantly lower after administration of 9cis,12 trans-18:2 when compared to what was observed after ingestion of 9trans,12 cis18:2 and to linoleic acid Ž P s 0.04. . In the gastrocnemian muscles, the radioactivity recovered was significantly higher 24 h after oral administration of linoleic acid than after feeding its D9trans or D12 trans isomers Ž P s 0.01.. No difference of radioactivity per gram of tissue was found in any compartment of the fasting rat after administration of the three octadecatrienoic acids Ž P ) 0.05.. 3.2.2.
14
CO 2 recoÕery
3.2.2.1. Metabolic oxidation of linoleic acid and of its mono-trans isomers. Fig. 2ŽA. presents the 14CO 2 recovery after administration of linoleic acid and of its D9 and D12 trans isomers. The three curves presented a similar pattern with asymptotic profiles.
They reached a plateau 12 h after the oral administration. The values of 14CO 2 recovery from 6 to 24 h were significantly higher after administration of 9cis,12 trans-18:2 compared to the other octadecadienoic acids Ž P - 0.05. . 3.2.2.2. Metabolic oxidation of a-linolenic acid and of its D 9trans and D15trans isomers. The catabolic profiles of the octadecatrienoic acids exhibited an asymptotic pattern Ž Fig. 2Ž B.. with a plateau 12 h after the oral administration, as observed for the octadecadienoic acids. However, no significant differences were found at any time between the three curves Ž P ) 0.05..
4. Discussion Most part of the radioactivity administered has been recovered as 14CO 2 Žfrom 55.5% to 73.5% of the radioactivity administered, data not shown. . Even if these data have to be related to the fasting status of the rat, the differences observed between the fatty acids regarding the values of 14CO 2 production reflect some differences about the ability of the animal to oxidize the different isomers studied. a-linolenic acid was more oxidized than linoleic acid Ž P s 0.0086, data 24 h after oral administration not shown. . This result was consistent with previous data reported
Fig. 3. 14CO 2 recovery per 100 g of rat per 3 h during 24 h after oral administration to the fasting rat of w1-14Cx radiolabelled linoleic acid and of its D9trans and D12 trans isomers. Data have been obtained from three male Wistar rats for each fatty acid. Results are expressed as means of the percentage of the radioactivity administered recovered as 14CO 2 per 100 g of rat "SD. ) significantly different from the corresponding values after administration of 9trans,12 cis-18:2 and linoleic acid at P - 0.05.
L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
with 21-day-old rats w21x. Along the 24 h period, CO 2 was monitored more often in our study Ž hourly during the first 12 h, at 15, 18, 21, 22, 23 and 24 h. than what was previously done w17,21x. The main part of the fatty acids was oxidized in the first 12 h Ž Fig. 2A and B.. Moreover, our results indicate that the catabolism of linoleic acid occurred mainly during the first 3 h ŽFig. 3., as observed for a-linolenic acid and 9cis,12 cis,15trans-18:3 Ž data not shown. . 9cis,12 trans-18:2 and 9trans,12 cis-18:2 exhibited a maximum 14CO 2 recovery between 3 and 6 h after oral administration Fig. 3. . A similar trend was observed for 9trans,12 cis,15cis-18:3 Ždata not shown.. This delay in maximum 14CO 2 recovery suggested some differences in bioavailability of these trans fatty acids. A lower specificity of the intestinal fatty acid binding protein towards 9cis,12 trans-18:2, 9trans,12 cis-18:2 and 9trans,12 cis,15cis-18:3, compared to linoleic and a-linolenic acids, may be explained by the above observation. Moreover, the catabolism of 9cis,12 trans-18:2 and of 9trans,12 cis18:2 seemed to be slightly different: the 14CO 2 production 6 h after administration of 9cis,12 trans-18:2 was higher than after ingestion of 9trans,12 cis-18:2 ŽFig. 2ŽA... During the b-oxidation of polyunsaturated fatty acids Že.g. linoleic acid. a trans intermediate is formed Žstep catalyzed by the D3-D2 trans-enoylCoA isomerase. . This trans compound may occur during the b-oxidation of 9cis,12 trans-18:2, without action of the enzyme. Thus, it was previously suggested that the catabolism of 9cis,12 trans-18:2 was higher than that of linoleic acid as this step does not occur w16x. However, this hypothesis cannot explain our results. Indeed, as the radioactivity recovered as 14 CO 2 was only due to the first cycle of b-oxidation, the D12 trans double bond was not able to influence the 14CO 2 recovery. Another hypothesis might be the specificity of the enzymes involved in the transport through the outer and the inner mitochondrial membranes and in the activation of the fatty acids ŽAcylCoA synthase, Carnitine Palmitoyltransferase I and II, 3-ketoacyl-CoA thiolase, etc.. . 14 C retention in the liver, in the heart and in the gastrocnemian muscle was lower after administration of 9cis,12 trans-18:2 than after ingestion of linoleic acid ŽTable 1.. These organs are the major sites of b-oxidation and are rich in mitochondria. A higher 14
213
activity of the enzymes involved in fatty acid acylation or activation towards 9cis,12 trans-18:2 than towards linoleic acid may explain the differences observed in 14C retention and in 14CO 2 recovery. Consequently, further studies in rat mitochondria would be useful to ascertain the specificity of the Carnitine Palmitoyltransferase I towards each trans fatty acid as this enzyme is known to regulate the uptake of the fatty acids. In order to fully elucidate the partitioning of the trans fatty acid between oxidation, conversion in higher metabolites and lipid neosynthesis, additional studies using perfused rat liver will be carried out.
Acknowledgements This study was supported by a grant from the European Community Žcontract FAIR CT95-0594.. Lionel Bretillon was funded by an INRA and LESIEUR fellowship.
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Oxidative metabolism of w1-14Cx mono-trans isomers of linoleic and a-linolenic acids in the rat Lionel Bretillon a , Jean-Michel Chardigny a , Jean-Louis Sebedio ´ ´ Jean-Pierre Noel ¨ b, Jean-Michel Vatele ` c
a,)
, Didier Poullain b,
a
b
INRA, Unite´ de Nutrition Lipidique, Dijon, France CEA-Saclay, SerÕice des Molecules Marquees, ´ ´ Gif-sur-YÕette, France c ESCIL, Laboratoire de Chimie Organique 1, Villeurbanne, France
Received 27 June 1997; revised 1 October 1997; accepted 3 October 1997
Abstract Trans polyunsaturated fatty acids are formed during processing of vegetable oils such as deodorization and frying. The oxidative metabolism of linoleic and a-linolenic acids and of their mono-trans isomers Ž9cis,12 trans-18:2, 9trans,12 cis-18:2 and 9cis,12 cis,15trans-18:3, 9trans,12 cis,15cis-18:3, respectively. was studied in fasting rats. A single dose of 18.5 MBq of each w1-14 Cx labelled fatty acid Ž260 mg. was orally given to the animals. The 14 CO 2 expired was monitored during 24 h. Radioactive countings of the CO 2-trapping agent were performed at regular intervals up to 24 h after oral administration of the radiolabelled fatty acid. Radioactive countings were also performed on several tissues Žliver, heart, brain, kidneys, sus-epidydimal fat, gastrocnemian muscle, gastrointestinal tract and carcass.. The 14 CO 2 production 24 h after oral administration of the fatty acid ranged from 55.5% to 68.7% of the radioactivity administered for the C18:2 isomers and from 69.7% to 73.5% for the C18:3 fatty acids. From 6 to 24 h, 14 CO 2 recovery was significantly higher after oral administration of 9cis,12 trans-18:2 than after giving both other octadecadienoic isomers. 14 C retention per gram of tissue in the liver and in the heart was significantly lower after feeding 9cis,12 trans-18:2 than after administration of both other C18:2 isomers. 14 C retention per gram of tissue in the muscle was significantly lower after administration of both trans C18:2 isomers compared to linoleic acid. Neither 14 CO 2 recoveries nor 14 C retentions were significantly different after administration of the three octadecatrienoic acids. The difference observed in 14 CO 2 recovery within the dienes was probably not due to a higher specificity of the enzymes involved in the b-oxidation sequence for the D12 trans double bond, as previously reported. Indeed, due to the labelling of the fatty acids on the carboxyl end, 14 C values recorded in the CO 2-trapping agent were only due to the first cycle of b-oxidation. q 1998 Elsevier Science B.V. Keywords: trans Fatty acid; b-Oxidation; Linoleic acid; a-Linolenic acid
)
Correspondence author. Fax: q33 3 8063 3223; E-mail: [email protected]
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 7 8 - 1
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L. Bretillon et al.r Biochimica et Biophysica Acta 1390 (1998) 207–214
1. Introduction Trans polyunsaturated fatty acids are mainly produced during heat treatment of oils w1–3x. In deodorized or used frying oils, 9 cis,12 trans-18:2, 9 trans,12 cis-18:2, 9 cis,12 cis,15trans-18:3 and 9trans,12 cis,15cis-18:3 are the major isomers w1,4–6x and can also be ingested by human. The classical scheme of partitioning of dietary essential fatty acids is shared between b-oxidation on one hand and accumulation of the fatty acid itself, of its metabolites Žproduced by desaturation–elongation. and of the newly synthesized fatty acids produced from acetate units formed by b-oxidation of the essential fatty acid on the other hand w7x. Even if the trans polyunsaturated fatty acids have been found to be converted into products of desaturation andror elongation such as C18:4, C20:4 and C20:5 isomers with one trans ethylenic bond w8–15x, so far little data are available on the partitioning of these trans C18:2 and C18:3 fatty acids between b-oxidation and distribution in the rat tissues. Indeed, the only data available on the catabolism of the polyenes reported the catabolism of a mixture of the mono-trans isomers of linoleic acid and of the di trans isomer in rat liver mitochondria w16x and in rats w17,18x. Results have shown that trans isomers were catabolized to a greater extent than were their cis counterparts. Thus, in order to determine if these trans fatty acids follow the same biochemical partitioning as the essential fatty acids, pure synthetic and radiolabelled 9 c is ,1 2 tr a n s -1 8 :2 , 9 tr a n s ,1 2 c is -1 8 :2 , 9cis,12 cis,15trans-18:3 and 9trans,12 cis,15cis-18:3 were orally administered to rats. 14CO 2 was monitored during 24 h and the rats were then sacrificed. Radioactive countings were performed after sacrifice in the dissected rat. 2. Materials and methods 2.1. Animals Male Wistar rats, 41–57 days old, weighing 190– 230 g, supplied by Centre d’Elevage Depre ´ ´ ŽSaint Doulchard, France. were used. The animals were housed in individual cages under controlled conditions of temperature Ž22 " 18C., and relative humidity Ž55–60%.. A 12 h light–dark cycle Žlights on 7:00
a.m. to 7:00 p.m.. was maintained. The rats were fed sterilized commercial pellet diet Ž Extralabo, Provins, France. and had free access to tap water. All the experiments were performed at the same time Žgastric tubing at 9:00 a.m.. to avoid diurnal variations of the metabolism. The day before the experiment, prior to the dark period, each rat was restricted to 10 g of food. Purified tap water was available ad libitum. 2.2. Radiolabelled fatty acids L in o le ic Ž w1 -14 C x 9 c is ,1 2 c is -1 8 :2 , 1.96 GBq mmoly 1 . and a-linolenic Žw1- 14 C x 9cis,12 cis,15cis-18:3, 1.92 GBq mmoly1 . acids were supplied by NEN Products Ž Dupont de Nemours, Les Ulis, France. . Trans isomers of linoleic acid Žw1-14Cx 9cis,12 trans-18:2, 2.00 GBq mmoly1 and w1-14Cx 9trans,12 cis-18:2, 1.99 GBq mmoly1 . and of a-linolenic acid Žw 1- 14 C x 9 cis,12 cis,15 trans-18:3, 1.94 GBq mmoly1 and w1-14Cx 9trans,12 cis,15cis18:3, 1.88 GBq mmoly1 . were obtained by total synthesis w19,20x. All the fatty acids were of 98.6% radiopurity. Each fatty acid Ž18.5 MBq, 260 mg. was dissolved in triolein Žpurity 99%, Sigma Chemicals, L’Isle d’Abeau, France. and then administered by gastric tubing Žtotal volume: 200 ml.. To determine the exact quantity which was administered, the syringe and the catheter used were weighed before and after tubing. The rats were fasted during the whole 24 h experiment, but had free access to purified tap water. 2.3. Experimental system Immediately after oral administration of the radiolabelled fatty acid, the rat was housed in an airtight plexiglass ‘‘metabolic chamber’’ Ž Fig. 1. . The current of air in the cage was devoid of carbon dioxide by passing it through soda lime Žgranulation 2–5 mm, CaŽOH. 2 Ž 75–80%., NaOH q KOH Ž5–6%. , H 2 O Ž14–20%., Prolabo, Briare, France. . The 14CO 2 expired by the rat was trapped in a bottle containing 500 ml of 2-methoxyethylamine Ž Carbamate-1 National Diagnostics, Bionis, Clamart, France. . Effluent vapours of amine, nitrogen monoxide and carbon monoxide were subsequently trapped in 300 ml of water and in 300 ml of a diluted hydrochloric acid solution. The air flux in the cage Ž 950 ml miny1 . was obtained by the use of a peristaltic pump.
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Fig. 1. Schematic system used to collect the air expired from the rat. 1: soda lime tube; 2: air-tight "metabolic chamber" Žthe feeding bottle containing tap purified water has not been drawn.; 3: CO 2-trap bottle containing the 2-methoxyethylamine as CO 2-trapping agent; 4: apparatus added in order to remove aliquotes of the trapping agent without interrupting the bubbling of the air expired from the rat in the 2-methoxyethylamine; 5 and 6: bottles containing water and diluted hydrochloric acid trapping the vapors of amine, nitrogen and carbon monoxide; air flux from the soda lime tube to the peristaltic pump.
2.4. In ÕiÕo recoÕery of
14
CO 2 expired by the rat
Without interrupting the bubbling of the expired air in the trapping agent, 1 ml was removed every 30 min during the first 6 h, hourly during the six following hours, each 3 h up to 21 h and then hourly up to 24 h. 9 ml of the scintillation cocktail Ž Monoflow 4 National Diagnostics. were immediately added and the solution was counted using a Tri-Carb 2000CA liquid scintillation analyzer Ž Packard, Groningen, Netherlands.. 2.5. RecoÕery of radioactiÕity in the tissues After 24 h, the rat was slightly anesthetized under a diethyl ether flux and blood was withdrawn by venipuncture into a syringe containing 2 ml of an anticoagulant agent ŽCitric acid 0.8%, sodium citrate 2.2%, D-glucose 2.45%, wrwrw.. Then liver, kidneys, heart, brain, gastrocnemian muscles and sus-epidydimal fat were removed, blotted and weighed before homogenization. The gastrointestinal tract was divided into two parts: from the stomach to the small
intestine Župper portion. on one hand and from the colon to the rectum including faeces Žlower portion. on the other hand. Each gastrointestinal tract portion was weighed separately and homogenized, the remainder of the animal Žcarcass. was weighed before homogenization. In order to determine the radioactivity in all tissues of the rat, three aliquots of 30–80 mg of each tissue, five aliquots of 50–100 mg of carcass and gastrointestinal tract portions were digested with 1 ml of Solusol ŽNational Diagnostics. from 4 to 5 h at 508C. When the digestion was achieved, 10 ml of the scintillation cocktail ŽMonoflow 4. were added. Three aliquots of 200 ml of blood were digested with 1 ml of a mixture of isopropanolrSolusol Ž1:1, vrv. for 15–30 min at 508C. 50 ml of hydrogen peroxide Ž 25%. were added under gentle stirring. Radioactive counting was performed after addition of 10 ml of the scintillation cocktail ŽMonoflow 4.. The urine from the 24 h experimental period was collected by rinsing the cage with water. The mixture of urine with water was divided in 1 ml fractions. 10 ml of scintillation
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cocktail ŽUltima-Gold, Packard, Groningen, Netherlands. were added to each fraction. All the fractions were counted as described above.
statistical analysis. A P value - 0.05 was considered significant.
2.6. RecoÕery of total radioactiÕity
3. Results
2.6.1. Total 14CO 2 recoÕery The volume of the trapping agent present at the time of sampling was determined by carrying out the 24 h experiment three times without any rat in the cage. The values were used to calculate the 14CO 2 production. The values of 14CO 2 recovery were expressed per 100 g of rat in order to take into account the metabolic activity of each animal.
3.1. Body weight and weights of tissues of the dissected rats
2.6.2. RecoÕery of radioactiÕity in rat tissues The values of radioactivity in all parts of the rats were expressed as percentage of the administered radioactivity per gram of tissue.
The weights of the rats before oral administration of the fatty acids and of tissues of the rats after were not significantly different within the experimental groups Ž data not shown, P ) 0.05.. The weight losses during the 24 h experiment were not significantly different between the different groups of animals Ždata not shown, P s 0.32 and 0.41 for the octadecadienoic acids and for the octadecatrienoic acids, respectively.. 3.2.
14
C recoÕeries
2.7. Statistical analysis Values were reported as means "standard deviation Ž SD. . One-way analysis of variance Ž ANOVA. followed by paired Student’s t-test was employed for
3.2.1. 14C distribution in rat tissues The recovery of radioactivity per gram of tissue 24 h after oral administration of the w1-14Cx fatty acids is presented in Table 1: 94–99% of the administered
Table 1 Recovery of radioactivity per gram of tissue 24 h after oral administration of the w1-14Cx radiolabelled fatty acids to the fasting rat 18:2cc .a
18:2ct .b
18:2tc
18:3ccc
18:3cct
18:3tcc
CO 2 Žfinal value at 24 h Liver Brain Heart Kidneys Sus-epidydimal fat Muscle Upper GIT portion Lower GIT portion Carcass Urine Blood
27.04 Ž2.052. 0.23 Ž0.030. 0.04 Ž0.003. 0.17 Ž0.050. 0.17 Ž0.016. 0.20 Ž0.068. 0.11 Ž0.035. b 0.31 Ž0.101. 1.27 Ž1.402. 0.12 Ž0.011. 0.17 Ž0.071. 0.03 Ž0.002.
33.95 Ž0.873 0.12 Ž0.005. c 0.03 Ž0.002. 0.08 Ž0.008. b 0.14 Ž0.038. 0.30 Ž0.111. 0.05 Ž0.007. 0.14 Ž0.080. 0.99 Ž0.513. 0.09 Ž0.004. 0.23 Ž0.112. 0.03 Ž0.002.
29.59 Ž2.864. 0.18 Ž0.014. 0.06 Ž0.012. 0.13 Ž0.024. 0.14 Ž0.016. 0.51 Ž0.407. 0.06 Ž0.009. 0.27 Ž0.030. 0.93 Ž0.813. 0.11 Ž0.012. 0.12 Ž0.076. 0.03 Ž0.001.
35.09 Ž2.052. 0.17 Ž0.076. 0.04 Ž0.024. 0.11 Ž0.079. 0.15 Ž0.044. 0.12 Ž0.061. 0.03 Ž0.006. 0.28 Ž0.050. 2.02 Ž1.146. 0.07 Ž0.010. 0.25 Ž0.044. 0.02 Ž0.003.
34.30 Ž1.886. 0.11 Ž0.020. 0.05 Ž0.006. 0.06 Ž0.008. 0.17 Ž0.015. 0.13 Ž0.020. 0.05 Ž0.014. 0.18 Ž0.054. 0.85 Ž0.343. 0.08 Ž0.007. 0.15 Ž0.049. 0.03 Ž0.002.
32.75 Ž3.588. 0.11 Ž0.007. 0.05 Ž0.006. 0.07 Ž0.009. 0.19 Ž0.031. 0.11 Ž0.028. 0.03 Ž0.006. 0.36 Ž0.049. 0.19 Ž0.057. 0.07 Ž0.002. 0.19 Ž0.055. 0.02 Ž0.009.
Total activity recovery
94.9 Ž2.66.
93.8 Ž3.10.
96.6 Ž1.72.
99.4 Ž8.26.
95.1 Ž3.1.
96.3 Ž3.11.
Results are expressed as means of the percentage of the radioactivity administered per gram of tissue Ž"SD. from three animals Ž9cis,12 cis-18:2: 18:2cc; 9cis,12 trans-18:2: 18:2ct; 9trans,12 cis-18:2: 18:2tc; 9cis,12 cis,15cis-18:3: 18:3ccc; 9cis,12 cis,15trans-18:3: 18:3cct; 9trans,12 cis,15cis-18:3: 18:3tcc.. GIT: gastrointestinal tract. a Obtained by dividing the value of 14CO 2 production by the weight of the rat at t s 0. b Significantly different from the corresponding values 24 h after administration of both other octadecadienoic acids at P - 0.05. c Significantly different from the corresponding values 24 h after administration of both other octadecadienoic acids at P - 0.01.
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Fig. 2. 14CO 2 recovery per 100 g of rat after oral administration to the fasting rat of: ŽA. w1-14Cx radiolabelled linoleic acid and of its D9trans and D12 trans isomers; ŽB. w1-14Cx radiolabelled a-linolenic acid and of its D9trans and D15trans isomers. Data have been obtained from three male Wistar rats for each fatty acid. Results are expressed as means of the radioactivity administered recovered as 14 CO 2 per 100 g of rat "SD. ) and ) ) significantly different from the corresponding values after administration of both other octadecadienoic acids at P - 0.05 and 0.01, respectively.
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dose was recovered. The three octadecatrienoic acids were similarly oxidized Ž P ) 0.05.. On the other hand, 9cis,12 trans-18:2 was significantly more catabolized into 14CO 2 than were linoleic acid and its D9trans isomer Ž P s 0.019.. The 14C activities in the liver 24 h after administration of 9trans,12 cis-18:2 and of linoleic acid were similar but it was significantly higher than the 14C retention after administration of 9cis,12 trans-18:2 Ž P s 0.0025.. The radioactivity per gram of heart was significantly lower after administration of 9cis,12 trans-18:2 when compared to what was observed after ingestion of 9trans,12 cis18:2 and to linoleic acid Ž P s 0.04. . In the gastrocnemian muscles, the radioactivity recovered was significantly higher 24 h after oral administration of linoleic acid than after feeding its D9trans or D12 trans isomers Ž P s 0.01.. No difference of radioactivity per gram of tissue was found in any compartment of the fasting rat after administration of the three octadecatrienoic acids Ž P ) 0.05.. 3.2.2.
14
CO 2 recoÕery
3.2.2.1. Metabolic oxidation of linoleic acid and of its mono-trans isomers. Fig. 2ŽA. presents the 14CO 2 recovery after administration of linoleic acid and of its D9 and D12 trans isomers. The three curves presented a similar pattern with asymptotic profiles.
They reached a plateau 12 h after the oral administration. The values of 14CO 2 recovery from 6 to 24 h were significantly higher after administration of 9cis,12 trans-18:2 compared to the other octadecadienoic acids Ž P - 0.05. . 3.2.2.2. Metabolic oxidation of a-linolenic acid and of its D 9trans and D15trans isomers. The catabolic profiles of the octadecatrienoic acids exhibited an asymptotic pattern Ž Fig. 2Ž B.. with a plateau 12 h after the oral administration, as observed for the octadecadienoic acids. However, no significant differences were found at any time between the three curves Ž P ) 0.05..
4. Discussion Most part of the radioactivity administered has been recovered as 14CO 2 Žfrom 55.5% to 73.5% of the radioactivity administered, data not shown. . Even if these data have to be related to the fasting status of the rat, the differences observed between the fatty acids regarding the values of 14CO 2 production reflect some differences about the ability of the animal to oxidize the different isomers studied. a-linolenic acid was more oxidized than linoleic acid Ž P s 0.0086, data 24 h after oral administration not shown. . This result was consistent with previous data reported
Fig. 3. 14CO 2 recovery per 100 g of rat per 3 h during 24 h after oral administration to the fasting rat of w1-14Cx radiolabelled linoleic acid and of its D9trans and D12 trans isomers. Data have been obtained from three male Wistar rats for each fatty acid. Results are expressed as means of the percentage of the radioactivity administered recovered as 14CO 2 per 100 g of rat "SD. ) significantly different from the corresponding values after administration of 9trans,12 cis-18:2 and linoleic acid at P - 0.05.
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with 21-day-old rats w21x. Along the 24 h period, CO 2 was monitored more often in our study Ž hourly during the first 12 h, at 15, 18, 21, 22, 23 and 24 h. than what was previously done w17,21x. The main part of the fatty acids was oxidized in the first 12 h Ž Fig. 2A and B.. Moreover, our results indicate that the catabolism of linoleic acid occurred mainly during the first 3 h ŽFig. 3., as observed for a-linolenic acid and 9cis,12 cis,15trans-18:3 Ž data not shown. . 9cis,12 trans-18:2 and 9trans,12 cis-18:2 exhibited a maximum 14CO 2 recovery between 3 and 6 h after oral administration Fig. 3. . A similar trend was observed for 9trans,12 cis,15cis-18:3 Ždata not shown.. This delay in maximum 14CO 2 recovery suggested some differences in bioavailability of these trans fatty acids. A lower specificity of the intestinal fatty acid binding protein towards 9cis,12 trans-18:2, 9trans,12 cis-18:2 and 9trans,12 cis,15cis-18:3, compared to linoleic and a-linolenic acids, may be explained by the above observation. Moreover, the catabolism of 9cis,12 trans-18:2 and of 9trans,12 cis18:2 seemed to be slightly different: the 14CO 2 production 6 h after administration of 9cis,12 trans-18:2 was higher than after ingestion of 9trans,12 cis-18:2 ŽFig. 2ŽA... During the b-oxidation of polyunsaturated fatty acids Že.g. linoleic acid. a trans intermediate is formed Žstep catalyzed by the D3-D2 trans-enoylCoA isomerase. . This trans compound may occur during the b-oxidation of 9cis,12 trans-18:2, without action of the enzyme. Thus, it was previously suggested that the catabolism of 9cis,12 trans-18:2 was higher than that of linoleic acid as this step does not occur w16x. However, this hypothesis cannot explain our results. Indeed, as the radioactivity recovered as 14 CO 2 was only due to the first cycle of b-oxidation, the D12 trans double bond was not able to influence the 14CO 2 recovery. Another hypothesis might be the specificity of the enzymes involved in the transport through the outer and the inner mitochondrial membranes and in the activation of the fatty acids ŽAcylCoA synthase, Carnitine Palmitoyltransferase I and II, 3-ketoacyl-CoA thiolase, etc.. . 14 C retention in the liver, in the heart and in the gastrocnemian muscle was lower after administration of 9cis,12 trans-18:2 than after ingestion of linoleic acid ŽTable 1.. These organs are the major sites of b-oxidation and are rich in mitochondria. A higher 14
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activity of the enzymes involved in fatty acid acylation or activation towards 9cis,12 trans-18:2 than towards linoleic acid may explain the differences observed in 14C retention and in 14CO 2 recovery. Consequently, further studies in rat mitochondria would be useful to ascertain the specificity of the Carnitine Palmitoyltransferase I towards each trans fatty acid as this enzyme is known to regulate the uptake of the fatty acids. In order to fully elucidate the partitioning of the trans fatty acid between oxidation, conversion in higher metabolites and lipid neosynthesis, additional studies using perfused rat liver will be carried out.
Acknowledgements This study was supported by a grant from the European Community Žcontract FAIR CT95-0594.. Lionel Bretillon was funded by an INRA and LESIEUR fellowship.
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