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Metabolism of a n-Paraffin, Heptadecane, in Rats J.E. TULLI EZ and G.F. BORIES, Laboratoire de Recherches sur les Additifs Alirnentaires INRA - 180, Chemin de Tournefeuille 31300 Toulouse, France ABSTRACT 14C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed exten- sive 14CO2 excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was co-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7-10%). Heptadecanoic acid was desaturated to hepta- decenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these func- tional lipids. INTRODUCTION Hydrocarbons normally occur in foods from plant origin mostly. Increasing amounts of these substances are ingested by animals and man due to the use of mineral oil in food tech- nology and to some sources of food contami- nation such as n-alkanes from yeasts grown on paraffins~ Aliphatic hydrocarbons are absorbed well (1-6) when incorporated at low doses into the diet of mammals; absorption varies with the length of the carbon chain (7) and with the animal species (8). In 1942, Stetten (9) suggested that rat liver oxidized hexadecane. Kolattukudy and Hankin (10) observed that nonacosane was partially absorbed and oxidized to fatty acids, in parti- cular to heptadecanoic acid. More recent studies with 14C-hexadecane and octadecane have established the process of terminal oxida- tion of n-alkanes to corresponding fatty acids (11) and have localized in liver (12-15) and kid- ney (15) the microsomal enzymatic systems in- volved. Mitchell and Hubscher (16) showed that mucosa cells of the small intestine of guinea pig oxidize hexadecane also. n-Paraffins accumu- lated preferentially in the fat of rats fed diets containing low doses of eicosane (17) or green algae rich in heptadecane (18). Until now, no metabolic balance nor system- atic research on metabolites has been under- taken; and it cannot be stated a priori that oxida- tion to the corresponding saturated fatty acid constitutes the sole metabolic pathway of n- paraffin degradation. Neither is there any data on the extent of this process. This investigation attempts to fill these gaps by studying the metabolism of this class of hydrocarbons in more detail. Heptadecane was chosen because it is widespread in the marine food chain (19-23), it is one of the main components of the n-paraf- fin series used as a substrate for producing alkane yeasts, and because until now the metab- olism of odd-chain normal paraffins had not been studied. METHODS (8-14C) n-heptadecane was synthesized by the Service of Labeled Molecules at the CEA (Saclay, France) (specific activity, 27 mCi/mM). Chemical purity was checked by gas liquid chromatography (GLC) on two columns of different polarities and radiochemicaI purity was tested by thin layer chromatography (TLC) (24). Wistar male rats weighing about 200 g were fed as libitum on a semisynthetic diet (casein TABLE I Gross Distribution of 14C 7 Days after Ingestion of 14C-heptadecane Percentage of 14C fed (mean ~- SE, n = 6) Dose of heptadecane Fecesa Urine a Carcass b Total 1 rag- 10 ktCi 0.96 +-0.21 1.12 -+ 0.04 36.9 -+ 5.8 39.0 -+ 5.5 200 mg- 30 #Ci 28.0 +- 5.3 0.47 + 0.07 25.2 +- 4.2 53.7 _+2.3 aCumulative for 6 days. bResidual at day 7. llO

Metabolism of an n-paraffin, heptadecane, in rats

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Page 1: Metabolism of an n-paraffin, heptadecane, in rats

Metabolism of a n-Paraffin, Heptadecane, in Rats

J.E. TULLI EZ and G.F. BORIES, Laboratoire de Recherches sur les Addi t i fs Alirnentaires INRA - 180, Chemin de Tournefeuille 31300 Toulouse, France

ABSTRACT

14C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed exten- sive 14CO2 excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was co-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7-10%). Heptadecanoic acid was desaturated to hepta- decenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these func- tional lipids.

INTRODUCTION

Hydrocarbons normal ly occur in foods f rom plant origin most ly . Increasing amoun t s of these substances are ingested by animals and man due to the use of mineral oil in food tech- nology and to some sources of food contami- nat ion such as n-alkanes f rom yeasts grown on paraffins~ Aliphatic hyd roca rbons are absorbed well (1-6) when incorpora ted at low doses in to the diet o f mammals ; absorp t ion varies with the length of the carbon chain (7) and with the animal species (8).

In 1942, S te t ten (9) suggested that rat liver oxidized hexadecane . Ko la t tukudy and Hankin (10) observed that nonacosane was partially absorbed and oxidized to fa t ty acids, in parti- c u l a r to hep tadecano ic acid. More recen t studies wi th 14C-hexadecane and octadecane have es tabl ished the process of terminal oxida- t ion of n-alkanes to cor responding fa t ty acids (11) and have localized in liver (12-15) and kid- ney (15) the microsomal enzymat ic systems in- volved. Mitchell and Hubscher (16) showed tha t mucosa cells of the small in tes t ine of guinea pig oxidize hexadecane also. n-Paraffins accumu- lated preferent ia l ly in the fat of rats fed diets containing low doses of eicosane (17) or green algae rich in hep tadecane (18).

Until now, no metabol ic balance nor system- atic research on metabol i tes has been under- taken; and it c anno t be stated a priori that oxida- t ion to the cor responding sa tura ted fa t ty acid const i tu tes the sole metabol ic pa thway of n- paraff in degradat ion. Neither is there any data on the ex ten t o f this process. This investigation a t t empt s to fill these gaps by studying the metabol i sm of this class of hydroca rbons in more detail. Heptadecane was chosen because it is widespread in the marine food chain (19-23), it is one of the main c o m p o n e n t s of the n-paraf- fin series used as a substrate for producing alkane yeasts, and because unti l n o w the metab- olism of odd-chain normal paraffins had not been studied.

METHODS

(8-14C) n-heptadecane was synthes ized by the Service of Labeled Molecules at the CEA ( S a c l a y , F r a n c e ) ( s p e c i f i c ac t iv i ty , 27 mCi/mM). Chemical puri ty was checked by gas liquid ch roma tog raphy (GLC) on two columns of d i f ferent polarit ies and radiochemicaI puri ty was tested by thin layer ch romatography (TLC) (24) .

Wistar male rats weighing about 200 g were fed as l ibi tum on a semisynthe t ic diet (casein

TABLE I

Gross Distribution of 14C 7 Days after Ingestion of 14C-heptadecane

Percentage of 14C fed (mean ~- SE, n = 6) Dose of

heptadecane Feces a Urine a Carcass b Total

1 rag- 10 ktCi 0.96 +- 0.21 1.12 -+ 0.04 36.9 -+ 5.8 39.0 -+ 5.5 200 mg- 30 #Ci 28.0 +- 5.3 0.47 + 0.07 25.2 +- 4.2 53.7 _+ 2.3

aCumulative for 6 days. bResidual at day 7.

l l O

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HEPTADECANE METABOLISM IN RAT 111

o

o

g

/

// 6 h 12h 2 4 h 4 8 h / 7d

T i m e a f t e r i n g e s t i o n

FIG. 1. Evolution and distribution of 14C in liver and carcass of rats fed 14C heptadecane; �9 hydrocarbon, lipids, [] nonlipids.

18%, wheat starch 39%, sucrose 24%, peanut oil 8%, cellulose 3%, minerals and vitamins 8%). (8-14C) n-Heptadecane was diluted with ana- lytical grade (99% min) n-heptadecane (Baker) and incorporated in the peanut oil of the diet. In all experiments, the animals were fed lightly the night before the experimental diet was given so that the 14C-heptadecane, admini- stered in a small amount of the feed, would be ingested within 15 rain. The animals were given the control diet ad libitum immediately after- wards. Rats were kept in metabolism cages, urine and feces were collected daily for 6 days, and the rats were killed on the 7th day. Lipid incorporation kinetics were studied in animals killed 6 hr, 12 hr, 24 hr, 48 hr, and on the 7th day, respectively, after the ingestion of labeled n-heptadecane.

Urine was measured directly by liquid scin- tillation counting with an Intertechnique SL 32 instrument, in the form of a gel stabilized by Triton X 100. Lipid fractions isolated by chromatography were measured in a PPO- dimethyl-POPOP liquid scintillation cocktail in toluene. Total radioactivity in tissues, feces, and residues of lipid extraction was measured after combustion of 500 mg samples in an auto- m a t i c a p p a r a t u s (Intertechnique Oxymat). Radioactivi ty was localized on thin layer chromatograms using a Berthold thin layer scanner.

Total lipids of tissues and feces were ex- tracted with chloroform-methanol (2:1) ac- cording to Folch's method (25) and saturated hydrocarbons, neutral and complex lipids were separated on a silica gel column (Kieselgel 60, Merck) by successive hexane, chloroform, and methanol elutions. Hydrocarbons were ana- lyzed by GLC using the technique already de- scribed (6). Neutral lipids were separated into classes on a Florisil column using Carroll's method (26). Purity of fractions was~checked by TLC on Silica Gel G using hexane-ethyl ether-formic acid, 120: 30: 1.5.

Fatty acid methyl esters, prepared by trans- methylation, were purified on silica gel plates (elution with hexane-ethyl ether, 90: 10), then analyzed by GLC on 1/8 in. x 3 m stainless- steel column packed with 10% DEGS-PS on 80/100 Supelcoport. Separation according to the number of double bonds was performed by TLC on silica impregnated with 20% silver ni- trate (27). Saturated and unsaturated fatty acid methyl esters were separated according to the number of carbon atoms by reversed phase chromatography on a Lipidex 5000 column ( P a c k a r d ) ( e l u t i o n with methanol-water- methylene chloride, 80:20:10). Squalene and cholesterol, in the nonsaponifiable fraction, were identified by GLC on a 1/4 in. x 1.5 m glass column packed with 3% Dexsil 300 on 80/100 W AW DMCS Chromosorb. Phospholipids were

LIPIDS, VOL. 13, NO. 2

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112 J.E. TULLIEZ AND G~ BORIES

TABLE II

Distribution of 14C in Liver Neutral Lipids 24 hr after Ingestion of 20 gCi 14C-heptadecane

Percent of a Fraction total 14C Specific activity b

Monoglycerides 1.2 880 Diglycerides

+ 25.8 5510 Cholesterol Free fatty acids 20.4 12440 Triglycerides 34.6 1250 Cholesterol esters

+ 18.0 3980 Squalene

aNeutral lipids separated from saturated hydrocarbons. bdpm/mg.

TABLE III

Radioactivity of Fatty Acids and Total Cholesterol in Liver Neutral Lipids

Specific activity a

Time after ingestion 6 hr 24 hr 7 days

Fatty acids 2810 1280 81 Cholesterol 500 1050 190

adpm/mg.

separa ted in to f rac t ions on a silicic acid c o l u m n (Mal l inkrod t ) (28). Pur i ty of f rac t ions was tes ted by TLC on Silica Gel G (e lu t ion wi th c h l o r o f o r m - m e t h a n o l - a c e t i c a c i d - w a t e r , 100 :55 : 16:4) . Fa t t y acids were ana lyzed af te r t r a n s m e t h y l a t i o n using the t e c h n i q u e descr ibed above.

R E S U L T S

The me tabo l i c ba lance was measu red af te r a single admin i s t r a t i on to a rat of t w o doses of 14C-hep tadecane , and was ca lcula ted f rom the rad ioac t iv i ty excre ted in u r ine and feces for 6 days, and f rom the res idual rad ioac t iv i ty mea- sured in the whole carcass at the end of the expe r imen t . Table I ind ica tes t ha t h e p t a d e c a n e was h ighly absorbed . The rad ioac t iv i ty mea- sured in the feces was on ly in the form of hep- tadecane . In this and a previous s tudy (6) in- volving an i n t e r m e d i a t e dose (15 mg), the a b s o r p t i o n level was closely re la ted to the admin i s t e r ed dose~

In b o t h s tudies (6), n o h e p t a d e c a n e was f o u n d in urine~ The high a m o u n t of unac- c o u n t e d rad ioac t iv i ty (61% and 46.3%, respec- t ively) mus t be due to 14CO2 excre t ion , wh ich i m p l i e s t h a t h e p t a d e c a n e was c o m p l e t e l y me tabo l i zed . Fif ty-f ive pe rcen t of the inges ted 14C was e l imina ted in the f irst 6 hr as 14CO 2. A small a m o u n t of the n o n r e c o v e r e d 14C could

be exp la ined by h y d r o c a r b o n excre t ion in sweat (29-30) and sebum lipids (31) .

Figure 1 shows the d i s t r ibu t ion of radio- act ivi ty in the l iver and in the remain ing car- cass. Tota l r ad ioac t iv i ty decreased rapidly in liver; it leveled off wi th in 6 hr, t hen changed l i t t le over a 7 day per iod in the carcass. 14C was main ly i n c o r p o r a t e d in the l ipid f rac t ion of the liver, whereas label ing of the nonl ip id frac- t ion of the r ema in ing carcass was already signi- f icant at 6 h r and the rea f t e r (33 .7% to 44 .4% of to ta l rad ioac t iv i ty) .

Rad ioac t iv i ty in the form of h e p t a d e c a n e de- creased rap id ly in the liver bu t r emained a lmos t u n c h a n g e d in the carcass b e t w e e n the 12 th h o u r and 7 th day, showing r e t e n t i o n of hepta- decane and o t h e r n-paraf f ins (C2o, C21, C24) in rat (6). The c o n c o m i t a n t increase of the rad ioac t iv i ty of the l ipids in the carcass, and decrease of labeled h e p t a d e c a n e and lipids in the liver over a 7 day per iod, conf i rm the t rans- f o r m a t i o n of h e p t a d e c a n e in to lipid compo- nents .

Neutral Lipids

In liver and adipose tissue, 65% of the radio- act ivi ty was b o u n d to sa tu ra ted acids and the rest to m o n o u n s a t u r a t e d acids. The d i s t r ibu t ion of r ad ioac t iv i ty according to l eng th of f a t ty acids was es tabl i shed on ly in the mos t radio-

LIPIDS, VOL. 13, NO. 2

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HEPTADECANE METABOLISM IN RAT

TABLE IV

Distribution of 14C in Saturated and Monounsaturated Fatty Acids from Neutral Lipids of Liver and Adipose Tissue

113

Fatty acids

Liver a

Radioactivity Composition

Adipose tissue b

Radioactivity Composition

<15 1.5 c 1.8 ND 2.1 15:0 6.0 ND d 11.3 ND 16:0 3.7 27.3 16.1 23.0 16:1 4.8 2.7 6.3 4.7 17:0 48.5 ND 37.1 ND 17:1 22.8 ND 19.3 ND 18:0 5.1 5.0 2.1 3.7 18:1 5.6 46.0 7.8 44.0

> 1 8 2 .0 1%2 N D 22 .5

a6 Hr after ingestion. b7 Days after ingestion. CMainly C13:0 dND = non detectable.

active samples, liver af ter 6 hr and adipose tis- sue after 7 days.

Table II indicates that hep tadecano ic acid and hep tadeceno ic are s t rongly labeled. This in- dicates the ox ida t ion of hep tadecane to hepta- decanoic acid and an active desa tura t ion pro- cess. The presence of labeled 15:0 and 13:0 in the liver shows tha t hep tadecano ic acid under- goes the usual fl-oxidation. Radioact ive satu- ra ted and unsa tura ted even fat ty acids mus t result f rom resynthesis involving 14C-acetate. Al though neutral l ipids of liver and adipose tissue exhibi t similar fa t ty acid compos i t ions , a d i f ference in the d is t r ibu t ion of the radio- activity was not iced (Table II). Six hr af ter 14C-heptadecane adminis t ra t ion , about 80% of the 14C was in liver odd fa t ty acids, whereas 7 days later, the even fa t ty acid resynthesis in adipose tissue, especially of palmitic acid, was substantial~

Neutral lipids of liver and adipose tissue f reed f rom h y d r o c a r b o n s were separated in to their d i f fe rent classes. Radioact ivi ty in adipose tissue was mainly (90%) in the tr iglycerides, whereas it was widespread in the lipid classes of the liver (Table Ill) . Specific activity of free fa t ty acids was about 7 t imes higher than tha t

TABLE V

Distribution of 14C in Saturated and Monounsaturated Fatty Acids from Liver

Phospholipids 24 br after Ingestion of 14C-beptadecane

Fatty acids Radioactivity a Composition b

14:0 0.5 0.3 15:0 9.3 0.2 16:0 37.1 35.0 16:1 e 0.6 17:0 31.9 0.6 17:1 9.6 ND 18:0 11.2 52.9 18:1 0 .4 10 .4

ap. 100 of 14C in fatty acids. bp. 100 of saturated + monounsaturated fatty acids.

of t r iglyceride fa t ty acids. This preferent ia l labeling mus t co r re spond to the initial meta- bolic stage of hep tadecane w-oxida t ion , to hep tadecano ic acid. Separat ion of cholesterol , diglycerides on one hand, squalene and choles- terol esters on the o ther , es tabl ished tha t choles terol was s t rongly labeled and 14C-squa- lene was present .

Cholesterol specific activity increased af ter

TABLE VI

Distribution of 14C in Liver Phospholipids 6 hr after Ingestion of 14C-heptadecane

Palmitic + stearic acids Percent of Specific total 14C activity a total fatty acids

Phosphatidic acid 1.9 292 Phosphatidyl ethanolamine 27.1 815 Phosphatidyl serine 3.7 807 Phosphatidyl choline 64.2 1050 Sphingomyelin 3.1 620

0.08 0.49 0.49 0.52 0.30

adpm/mg.

LIPIDS, VOL. 13, NO. 2

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114 J.E. TULLIEZ AND G.F. BORIES

the 6th hour, whereas that of fa t ty acids de- creased rapidly (Table IV). This t ime lag is due to the successive stages of co-oxidation to heptadecanoic acid, /3-oxidation, 14C-acetate product ion , and synthesis of squalene and cholesterol .

Phospholipids

The incorpora t ion of 14C in hepatic phos- pholipids was studied 24 hr after ingestion of heptadecane. Radioact ivi ty was entirely in fa t ty acids. The separat ion of fa t ty acid methyl esters according to the degree of unsatura t ion showed that 90% of the radioact ivi ty was associated with saturated acids and the rest with mono- unsaturated acids. The dis t r ibut ion by chain length (Table V) indicates major 14C incor- pora t ion into heptadecanoic and pentadecanoic acids (about 3200 dpm//~g for bo th acids), and a strong labeling of nonmeasurable quanti t ies of heptadecenoic acid. Twenty- four hr after in- gestion of heptadecane, resynthesized 14 C even fat ty acids, especially palmit ic acid, were evi- dent.

Analysis of phosphol ipid classes (Table VI) shows that most of radioact ivi ty was in phos- phat idylchol ine, but specific activities indicate a more homogeneous dis t r ibut ion closely re- lated to saturated fat ty acids contents of each fraction.

14C-heptadecane but very lit t le labeled lipids even after 48 hr. Pokrovskii (33), in similar ex- per imental condit ions, found that 94% of fat radioact ivi ty was still bound to 14C-hepta- decane 15 days after ingestion. Under the food contamina t ion condi t ions of our experiments , heptadecane metabol ism is more rapid.

Our exper iments clearly show that the first stage of heptadecane metabol ism is co-oxidation to heptadecanoic acid, which undergoes the same fate as normal fa t ty acids (34). The pre- sence of the homologous 15:0 and 13:0 odd acids indicate the first stages of H-oxidation. Squalene and cholesterol labeling imply the availability of 14C-acetyl CoA. Due to label on carbon 8 of heptadecane, 14C-acetate is only produced after four successive steps, which means that heptadecanoic acid is thoroughly me tabo l i zed . The ex ten t and intensi ty of 14CO2 produc t ion show that heptadecane is well used by the rat as an energetic source.

P r e f e r e n t i a l incorpora t ion of 14C-octa- decane radioact ivi ty into lecithin fatty acids has been repor ted (35) based on total counts per fraction. Measurement of specific activities shows that the odd fat ty acids resulting f rom the ox ida t ion of heptadecane are distributed in phospholipid classes the same as the other fa t ty acids. Thus, no modi f ica t ion of structural lipids role should be expected.

DISCUSSION

Metabolism of heptadecane was studied at low levels of the diet encountered in food con- taminat ion. This is in contras t to most experi- ments on n-paraffins which have been carried out with high doses given ei ther by gastric intu- bat ion or intravenous injections, with or with- o u t p r e v i o u s emulsif icat ion. Considerable variations in the absorpt ion rates result f rom the nonnutr i t ional condi t ions (7,32). Our re- sults show decreased absorpt ion with increased dose, over a 200-fold range, indicating that low doses of n-paraffins incorpora ted in the diet are largely absorbed.

Heptadecane accumulates during the first 6 hr and then levels off, whereas labeled lipids accumulate progressively. Heptadecane prob- ably follows the lymphat ic route and reaches the tissues, especially adipose tissue, where it accumulates due to its l ipophil ic nature (5,7). Because of the l imited ox ida t ion capaci ty of the liver, the propor t ions of heptadecane and labeled lipids are related to extent and rate of 14C-heptadecane absorpt ion. To ensure a fast absorpt ion Savary and Constant in (5) used a gastic probe of 200 mg emulsif ied octadecane and found that rat adipose tissue retained

ACKNOWLEDGMENTS

This work was supported in part by research grant (75-136) from Ministere de la Qualite de la Vie. S. Bressolles provided technical assistance.

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HEPTADECANE METABOLISM IN RAT 115

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[Received April 4, 1977]

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