AN ABSTRACT OF THE THESIS OF. Thomas Anthony Eisele for the degree of Master of Science. in Food Science and Technology presented on June 2, 1976

Transcription

1 AN ABSTRACT OF THE THESIS OF Thmas Anthny Eisele fr the degree f Master f Science in Fd Science and Technlgy presented n June 2, 1976 Title: [9, 10-METHYLENE- 14 ClSTERCULIC ACID METABOLISM IN THE RAT: URINARY METABOLITES, LIVER TISSUE DISTRIBUTION, AND INDUCED t-hydrqxylase ACTIVITY Abstract apprved: /7 7 Jseph E. I^ixn Crn il (CO) and Sterculic fetida il (SFO) fed rats were 14 injected with [9, 10-methylene- C]sterculic acid. Less than 1% f the label was expired as carbn dixide. The majrity f the label was excreted in the urine as shrt-chain dicarbxylic acids with an intact cyclprpane ring. The majr metablites fr bth CO and SFO fed rats were cis-3, 4-methylene adipic acid and cis-3, 4- methylene suberic acid. Sterculic acid must underg p- and ^-xidatin t frm these urinary metablites, a-xidatin played a minr rle in the frmatin f cis- and trans-s* 4-methylene pimelic acid. Rats n the SFO diet culd metablize sterculic acid faster than fats n the CO diet. Hwever, bth CO and SFO fed rats prduced the same urinary metablites.

2 CO fed rats incrprated mre label frm sterculic acid int prtein and acid sluble liver fractins than SFO fed rats. Less than 0. 01% f the label frm either grup was fund in liver lipid sterl r glycerl fractins. 14 There was a tendency fr SFO fed rats t metablize n-[ 1- C] ctadecane faster than CO fed rats. This suggests that sterculic acid may induce ^-hydrxylatin f n-ctadecane.

3 14 [9, 10-methylene- C]Sterculic Acid Metablism in the Rat: "Urinary Metablites, Liver Tissue Distributin, and Induced c-hydrxylase Activity by Thmas Anthny Eisele A THESIS submitted t Oregn State University in partial fulfillment f the requirements fr the degree f Master f Science Cmpleted June 2, 1976 Cmmencement June, 1977

4 APPROVED: Pr{essj6j[ f Fd Science and Technlgy in charge f majr Head f Department f Fvbd Science and Technlgy Dean f Graduate Schl Date thesis is presented y s^j> Z-. )jjb Typed by A & S Bkkeeping fr Thmas Anthny Eisele

5 ACKNOWLEDGEMENTS I wish t thank Dr. Jseph E. Nixn and Prfessr Russell C Sinnhuber fr their guidance in planning and perfrming this wrk. I am als grateful t Dr. Nrman E. Pawlwski fr preparing the carbn-14 labelled methyl sterculate and helping interpret the spectral data, Dr. Ian J. Tinsley fr his many helpful discussins, and Dr. L. M. Libbey fr his assistance in btaining the mass spectra. The assistance f ther staff members, faculty, and graduate students is als appreciated. This wrk was supprted by Public Health Service Research Grants ES and ES The authr gratefully acknwledged this supprt. Special thanks ges t my wife, Kath, fr her technical drawings exhibited in this manuscript and fr her patience and supprt during this wrk.

6 ABBREVIATIONS a- alpha (3- beta CPFA cyclprpene fatty acids cpm cunts per minute CO crn il GC gas chrmatgraph GLC gas-liquid chrmatgraphy IG intragastric IP intraperitneal IR infrared M mlecular weight MS mass spectrum NMR nuclear magnetic resnance P parent (mlecular weight) SFO Sterculia fetida il a> mega

7 TABLE OF CONTENTS Pa g e INTRODUCTION 1 LITERATURE REVIEW 3 Cyclprpenid Fatty Acids: Structure and Occurrence 3 Reactins and Analysis 4 Bilgical Effects 5 Ccarcingenic and Carcingenic Activity 8 Cytplasmic Alteratins 9 Metablism f Cyclprpenes, Cyclprpanes, and Related Cmpunds 10 EXPERIMENT 13 Experimental Animals 13 Diet 13 Surces f Labelled Cmpunds 14 Methd f Administering Labelled Cmpunds 16 Urine Extractin 16 Gas Chrmatgraphy 17 Spectrscpy Equipment 18 Islatin f Metablites Frm Urine by Gas Chrmatgraphy Trapping 18 Liver Tissue Fractinatin 19 RESULTS AND DISCUSSION 20 Animal Health and Grwth,, Identificatin f [9, 10-methylene- C]sterculic Acid Metablites in SFO Fed Rats 20 Peak C 20 Peak B 26 Peak G 26 Peak F 33 Peaks E and D 33 Peak H 36 Peak I 40 Peak A 40 Peak J Identificatin f [9, 10-methylene- Cjsterculic Acid Urinary Metablites in Crn Oil Fed Rats 43

8 Pa g e Identificatin f Urinary Metablites in SFO Fed Rats Label [ ^C ] in Liver Fractins, Liver Lipid Fractins and Carbn Dixide frm [9, 10-methylene-^C ] sterculic Acid.. 14 Oxidative Mechanisms in [9, 10-methylene- C] sterculic Acid Metablism Prpsed Metablic Pathway fr Sterculic Acid Label [ ^C] in Liver Fractins, Liver Lipid Fractins, and Carbn Dixide frm n-[ 1-^C ] ctadecane in Rats Effects f Sterculic Acid Metablism n the Rat Tricarbxylic Acid Cycle Inhibitrs Renal Tubular Degeneratin SUMMARY AREAS OF FUTURE WORK BIBLIOGRAPHY APPENDIX I APPENDIX II APPENDIX III APPENDIX IV Rat Diet Cmpsitin 14. Spnificatin f [9, 10-methylene- C methyl Sterculate Halphen Test Preparatin f Diazmethane (93) and Fatty Acid Methyl Esters APPENDIX V Liquid Scintillatin Cunting Prcedures 78 APPENDIX VI APPENDIX VII APPENDIX VIII Prcedure fr the separatin f Liver Tissue int Acid Sluble, Lipid, and Prtein Fractin (81) 80 Prcedure fr the Separatin f Liver Lipids int Sterid, Fatty Acid, and Glycerl Fractin (28) 81 Calculated Amunt f Dicarbxylic Acid Excreted in the Urine f an SFO Fed Rat 83

9 LIST OF TABLES Table Page Bdy weight, liver weight, days fed diet, and [ C] dse given t crn il and SFO fed rats Mass fragments characteristic f methyl esters f dibasic acids (71) Urinary metablites f [9, 10-methylene- C] sterculic acid frm SFO fed rats Retentin time and percent label frm [9, 10- methylene- l^c] sterculic acid urinary metablites f CO and SFO fed rats Other urinary metablites in SFO fed rats Percent f administered [ C] dse in urine, carbn dixide, liver fractins, and liver lipid fractins f CO and SFO fed rats injected with [9, 10-methylenel^C] sterculic acid The percent f administered [ C dse in urine, carbn dixide, liver fractins, and liver lipid fractins f CO and SFO fed rats injected with n-[ C]ctadecane. 57

10 LIST OF FIGURES Figure Page 1. Schematic f apparatus fr trapping CO' Gas chrmatgraph f methylated urine extract f a SFO fed rat injected with [9, 10-methylene- C] sterculic acid. 22 C4 3. IR (thin film) and NMR f cmpund C MS, mlecular weight, and frmula f cmpund C IR (slvent-cci ) and NMR f cmpund B MS, mlecular weight, and frmula f cmpund B IR (slvent - CS ) and NMR f cmpund G MS, mlecular weight, and frmula f cmpund G MS, mlecular weight, and frmula f cmpund F IR (slvent - CS? ) f cmpund E MS, mlecular weight, and frmula f cmpund E MS, mlecular weight, and frmula f cmpund D MS, mlecular weight, and frmula f cmpund H MS, Mlecular weight, and frmula f cmpund I MS and mlecular weight f cmpund A Gas chrmatgraph f methylated urine extract f CO fed rat injected with [9, 10-methylene- ^C] sterculic acid Gas chrmatgraph f methylated urine extract f SFO fed rat Prpsed pathway fr Sterculic Acid Metablism in the rat. 55

11 [9, 10-METHYLENE- ClSTERCULIC ACID METABOLISM IN THE RAT: URINARY METABOLITES, LIVER TISSUE DISTRIBUTION, AND INDUCED CJD -HYDROXYLASE ACTIVITY INTRODUCTION The main surce f cyclprpenids in the United States is the seed il f the cttn plant (Gssypuim hirsutum); while in sme cuntries, cyclprpenids are supplied by kapk il. Cyclprpe- nids (CPFA) are unique naturally ccurring fatty acids cntaining a highly strained and reactive unsaturated three membered ring in the center f an 18 carbn (malvalic) r 19 carbn (sterculic) chain. A review by Phelps ^t al. (62) reprted that dietary CPFA impaired reprductin in rats and chickens, delayed sexual maturity in females f these species, and caused pink disclratin in avian egg whites during strage. Other recent wrk with CPFA fed rats (46, 47) shwed fatty infiltratin and degeneratin f the liver alng with renal tubule degeneratin; and altered lipid metablism, and membrane and mitchndrial functin (50). Als, Sinnhuber and cwrkers (84) discvered that CPFA were pwerful c-carcingens when fed tgether with aflatxin B. Furthermre, Lee^taL (38) fund a higher incidence f tumrs in rats receiving a crude mixture f aflatxins B and G with cyclprpene than in thse fed the myctxins alne.

12 There is strng evidence that micrsmal activatin is required fr carcingenicity f armatic hydrcarbns. Brdie (6) and Gillette (20) suggested that the varius xidative pathways in micrsmes are clsely related and may all be assumed t be hydrxylatin reactins invlving the direct substitutin f a hydrxyl grup fr a hydrgen. Preliminary studies by Yss (97) has shwn that the frmatin f the majr urinary metablite f sterculic acid in rats required micrsmal c -xidatin. It wuld, therefre, be imprtant t knw all the actual urinary metablites frmed frm sterculic acid, and the extent the frmatin f these metablites may have n the c -hydrxylase system in the rat. Thus, the purpse f this investigatin was: 1) t identify the rat urinary metablites and 14 the distributin f [ C] in the liver tissue frm [9, 10-methylene- 14 C] sterculic acid, and Z^t determine the effect f sterculic acid 14 metablism n the inductin f the n-[ 1- Cjctadecane OD - hydrxylatin system.

13 LITERATURE REVIEW Cyclprpenid Fatty Acids: Structure and Occurrence In 1952, Nunn (53) islated the cyclprpene fatty acid, sterculic acid, frm Sterculia fetida il (SFO). Shrtly thereafter, McFarlane et al. (4) islated a secnd cyclprpene fatty acid, malvalic acid, and fllwed by Craven and Jeffery (12) with D-2- hydrxy-sterculic acid, and Mrris and Hall (48) with sterculynic acid. CH 2 ' /\ ' _ n=6 malvalic acid CH - CH -C = C- CH -COOH _ t n n=7 stercuhc acid CH^ OH A 2 CH -(CHJ -C = C-(CH.J,-CH-COOH D-2-hydrxysterculic acid CH 2 /\ 2 HCSC-(CH 0 )^-C=C-(CH^),-COOH sterculynic acid Cyclprpene acids were fund principally in seed lipids f the rder Malvales. They exist largely as triglycerides in bth the neutral and phsphlipid cmpnents (27). Gssypium hirsulum (cttn), Eridendrn anfractusum (kapk), and Sterculia fetida (java live) seed ils are the cmmn surces f cyclprpene. Cttn seed il is used extensively in the U.S. as an imprtant surce

14 f il in many fd prducts, and kapk il is used chiefly in riental cuntries. Cttn seed il cntains % malvalic and % sterculic acid (8), kapk (20) 12-14%CPFA, and Sterculia fetida il (29) 4-10% malvalic and 45-54% sterculic acid. A review by Christie (8) lists the cyclprpene fatty acid cntent f many seed ils. Reactins and Analysis Cyclprpenes are highly reactive materials due t the strain f the unsaturated ring. They xidize (2 4) with cnventinal reagents and add hydrgen as d mst lefins nly mre easily and with milder catalyst (98). Hydrgen halids in glacial acetic acid react slwly but quantitatively with cyclprpenes (7), and this has been develped int a rutine analytical technique fr determining ttal cyclprpene cntent f seed ils. Mercaptans als add readily acrss the cyclic duble bnd t give tw thil esters (31). Silver nitrate in methanl (32) reacts with cyclprpenes t frm a ket and alkyxy derivative which can be easily separated by gas chrmat- graphy. The silver nitrate methd (76) alng with the thi adducts has been used extensively fr establishing CPFA standards. Cleman (10) has reviewed many methds fr the assay f cyclprpenes. He cncluded the Halphen (22) reactin was the quickest and the simplest f the "wet" methds. A mre recent methd develped by Pawlwski (61) invlves NMR which measures

15 the rati f the cyclprpene area hydrgens t the terminal u)-methyl hydrgens. It is a quick, simple, nndestructive, direct methd that is very accurate and suitable fr samples greater than 1.0% CPFA. Other spectrscpic methds such as IR (3) which measures the in-plane wagging vibratin f the methylene grup at 1009 cm is less accurate and requires a dual cell set-up. Mass spectrmetry cupled with gas chrmatgraphy f cyclprpenes (59) and their silver nitrate in methanl derivatives seems t be the mst sensitive methd. The mlecular weight f the CPFA and the psitin f the ring can be easily determined frm the mass fragmentatin pattern (15). Bilgical Effects Cttn seed il incrprated int the diet f laying hens was bserved by Sherwd (80) t cause egg disclratin n strage, with the whites turning pink and the ylks turning a salmn-range clr. Lrenz et al.(40) fund that the fat frm the pink eggs and the cttn seed il bth gave a psitive Halphen test and suggested that the substance in bth were ne and the same. In 1952 Nunn (53) established the structure f sterculic acid, the fatty acid respnsible fr the psitive Halphen test, and fund sterculic acid in very small quantities in the glycerides f cttn seed il. The bilgical activity f sterculic acid has been shwn t

16 reside in the unsaturated ring. When the ring was destryed by mild hydrgenatin r gaseus hydrgen chlride (79) bth cttn seed il and SFO n lnger gave a psitive Halphen test nr did they cause pink egg disclratin. Furthermre, Nrdby et ajl (52) shwed the acid r ester grup was nt a requirement fr activity. Effects ther than pink egg disclratin were nted by Schneider et al. (74, 7 5, 77). They bserved that feeding SFO t hens caused embry mrtality, delay f sexual maturity in pullets, and pr egg prductin and enlarged gall bladders and livers. A cmplete review n this subject and ther bilgical effects was written by Phelps et al. (62). A mre recent finding by Jhnsn et al. (26) shwed that C sterculic acid inhibited the in vitr desaturatin f [ 14 ] stearic acid t leic acid in the hen. Allen and cwrkers (1) cnfirmed these reprts and shwed sterculic acid t be a strnger stearic fatty acyl desaturase inhibitr than malvalic acid. Shrtly thereafter, the same desaturase effect was bserved by Reiser and Raju (67) in the rat and by Lehman (35) in the muse. Nixn et al. (50) in a recent reprt shwed that dietary CPFA caused the partial lss f membrane-assciated functins in the rat. There was a nted increase in the rate f erythrcyte hemlysis, a decrease in micrsmal cdeine demethylase activity, and a cmplete inhibitin f glutathine-induced mitchndrial swelling. It was

17 suggested that the change in saturatin t unsaturatin fatty acid rati by CPFA altered the physical prperties f the membrane and thus influenced the assciated membrane functins. Other wrkers (11, 21, 66) als reprted CPFA induced alteratins in rat tissue lipids with a nted increase in lipid saturatin. Reduced grwth rates, pr feed cnversin, and elevated liver/bdy weight ratis were cmmn in female and male rats. Rainbw trut, Salm gairdneri, have prven t be very suscep- tible t CPFA with dietary levels belw 200 ppm cyclprpene prducing sme f the same effects as in mammals (68, 69). Malevski et al. (42) nted a decrease in prtein synthesis in trut fed 0. 5 mg CPFA/kg bdy wt/day while Struthers (88) fund increased liver lipids, reduced P/O ratis, and reduced cnversin f [ C]leic acid t [ CO ] in nly a few days. Taylr et al. (89) and again Malevski (42) nticed a decrease in nrmal lipid synthesis 14 frm [ C]acetate in fish fed CPFA. Histlgical abnrmalities including fatty infiltratin, the frmatin f "fibers" in liver parenchymal cells, and bile duct prliferatin were cmmn (37, 69, 84, 86, 87). Gdnight and Kemmerer (21) suggested in 1966 that CPFA fed t chickens were als respnsible fr their high serum chlest- erl levels. They demnstrated that CPFA culd indeed alter chlesterl metablism in cckerels by increasing serum

18 8 chlesterl, bile vlume, bile acid excretin, and artic ather- sclersis. Fergusn (16) nted higher plasma chlesterl levels and higher incidence f artic athersclersis using New Zealand rabbits fed CPFA when cmpared t a cntrl grup. Nixn (49) bserved that mice fed CPFA had a tendency t have higher serum chlesterl levels. Ccarcingenic and Carcingenic Activity Cyclprpene fatty acids fed as a cmpnent f Sterculia fetida il was reprted by Sinnhuber j2t al (84) t have ccarcin- genic activity tgether with aflatxin B in rainbw trut. This was cnfirmed by Lee et al (37), nt nly with aflatxin B but als with 27acetyl-aminflurene. Lee shwed that levels as lw as 20 ppm f methyl sterculate in the diet prmted the grwth f hepatma in rainbw trut. Nixn et al. (51) reprted that CPFA fed with aflatxin B t Wistar rats shwed a tendency t increase the hepatma incidence. At lw levels f aflatxin B, hepatma incidence increased 11%, while at higher aflatxin B levels, the CPFA effect was nt apparent. Friedman and Mhr (17) culd find n interactin between CPFA and aflatxin B in rats. Cnversely, Lee et al (38) fed Lng- Evans rats 220 ppm CPFA with a mixture f aflatxin B and G ver an 18-mnth perid and reprted an increased tumr incidence

19 frm 59% t 70%. The results were nt significant due t the lw numbers f animals used in the experiment. Thus, ccarcingenic activity f CPFA in species ther than rainbw trut has nt been adequately demnstrated. Recently, Sinnhuber and cwrkers (83) reprted that CPFA als were carcingens in rainbw trut. Levels as lw as 45 ppm CPFA in the diet prduced liver cancer in 11 ut f 40 fish while 405 ppm CPFA in the diet prduced even higher incidence f 10 ut f 30 fish. Cytplasmic Alteratins Scarpelli et al. (73) reprted that hepatcytes frm rainbw trut and rats fed 2 00 ppm CPFA fr fur weeks shwed induced cytplasmic alteratins. These alteratins, cleft-like striatins, parallel arrays, and whrled membrane prfiles f rugh-surfaced endplasmic reticulum, became prgressively mre marked with cntinued feeding. A fllw-up paper by the same authr (72) shwed CPFA at 500 ppm in the diet fr tw weeks f male Sprauge- Dawley rats significantly increased mittic activity in the pancreas as well as in liver helatcytes. The significant pint made in Scarpelli's paper was that CPFA were acting as a mitgen. Cell divisin usually ccurs nly in the presence f adjacent cell death

20 and active grwth (yung animals). CPFA was stimulating cell 10 divisin f the rat pancreas withut regards t death r active grwth. Metablism f Cyclprpenes, Cyclprpanes, and Related Cmpunds Wd and Reiser (96) studied the metablism f cis- and trans- 9, 10-methylene ctadecanic acid in weanling rat adipse tissue. The main metablites were cis- and trans-3, 4-methylene ddecanic acid. The prducts were apparently the result f the inability f the (3-xidatin enzyme system t cntinue dwn the fatty acid chain. This was supprted by the fact that large amunts f the material accumulated in the adipse tissue. Chung (9) carried this ne step further by using the [methylene- 14 C]cis-9, 10-methylene hexadecanic acid and cis-9, 10-methylene ctadecanic acids incubated with rat liver mitchndria. He shwed that the cyclprpane lng chain fatty acids were cnverted t 14 shrt chain cyclprpane fatty acids, and that the [methylene- C] 14 labelled ring was nt cnverted t [ CO ]. In a review n lipids (13), Deuel reprted that in branched chain fatty acid metablism in mammals, whenever there is a hinderence f p-xidatin, that c-xidatin plays a much larger rle in metabliz- ing these fatty acids. Hence, a dicarbxylic acid f shrter chain length is frmed then eliminated thrugh the urine as a cnjugate r a free dicarbxylic acid.

21 11 Altenburger and cwrkers (2) intravenusly administered 14 [methylene- C]sterculic acid t fasting hens. They shwed that limited amunts f 14 CO-> frmed ver a 24-hur perid, and that mre than 50% f the label was fund in the fecal material (feces and urine). This indicated that the cmpund was being metablized t a certain extent and then excreted in the fecal material with an intact methylene grup, much the same as a branched fatty acid wuld be. The same bservatins were made by Berry (5) alng with the fact that methyl sterculate was absrbed thrugh the gut at a slwer rate then methyl leate. 14 Yss (97) did a cmparative study n the metablism f C- sterculic acid in the rat and rainbw trut. He fund that the rat culd excrete apprximately 48% f the administered dse in 16 hurs while the fish required five days. The distributin f the label in subcellular fractins in bth species shwed that radiactivity peaked first in the micrsmes and then in the mitchndria. He suggested that CPFA metablism by mitchndria may first require an initiatin step by the micrsmes. The majr urinary metablite frmed in the rat was cis-3, 4-methylene adipic acid. Frmatin f this metablite requires (3- and CD -xidatin plus reductin f the cyclprpene ring t a cyclprpane ring. N metablites were identified in the rainbw trut. In summary, CPFA in rats, trut, chickens, and rabbits alter

22 12 lipid metablism, mdify certain enzymic activities, and are hepattxic. CPFA are ptential ccarcingens in mammals and als carcingens in rainbw trut. Humans may cnsume CPFA in small quantities alng with ther carcingenic nitrsamines and myctxins. Therefre, it is f great imprtance that the metabl- ism f CPFA be studied in mammalian systems t help evaluate the ptential health hazard. It is the intent f this investigatin that the results will aid in the understanding f the mde f actin f CPFA n mammalian systems, especially in regards t its actin as a ccarcingen.

23 13 EXPERIMENTAL Experimental Animals Eight weanling Wistar strain male rats (30 days ld) frm a clsed clny maintained by the Department f Agricultural Chemistry at Oregn State University were hused in plastic bx cages cntain- ing grund crn cbs. The animals were fed water ad libitum and a slid agar semipurified diet. Fd cnsumptin and individual weight was taken n a weekly basis as an index f verall grwth and health. Diet The cmpsitin f the semipurified diet (33) is shwn in Appendix I. The ttal diet cnsisted f 92. 8% premix, 2,2% vitamin mix, and 5. 0% crn il mixed 1: 1 (w/v) with 3. 0% agar (DIFCO) disslved in distilled water at apprximately 80 C. The ht mix was slidified at tw t five degree Centigrade, cut int small blcks, and stred in plastic bags at -30 C until used. After tw weeks n 5. 0% crn il diet, fur f the eight rats were transferred t a 4. 0% crn il plus 1.0% Sterculia fetida il diet n a dry weight basis. The diets were labelled CO fr crn il cntrl and SFO fr cyclprpene experimental. The SFO diet

24 14 cntained 0. 5% cyclprpene (malvalic and sterculic acid) r 10% f the lipid n a dry weight basis. Surces f Labelled Cmpunds Sterculic acid, labelled n the 9, 10-methylene bridge f the cyclprpene ring, was synthesized by Pawlwski (60). The Halphen test (Appendix III) and NMR (61) indicated % and % cyclprpene respectively. The free acid was prepared by sapnificatin with alchlic KOH (Appendix II). Pure methyl sterculate, 110 fj.ci/mmle, was diluted t 4.0 j.ci/0.41 grams 14 crn il. n-[l- C] -ctadecane was purchased frm Amersham/ Searle mci/mmle was diluted t 10 j.ci/0. 4 grams cld n-ctadecane. The mde f administratin t the rats was intragastric (IG) fr sterculic kcid and intraperitneal (IP) fr n-ctadecane. Immedi- ately after injectin, animals were placed in metablism chambers (Figure 1). Air was pulled thrugh several KOH traps t cllect 14 CO, and ten ml. samples were taken every hur and cunted in scintillatin flur (Appendix V). Urine was allwed t flw int glass tubes packed in ice. After the alltted time, animals were decapi- tated and the livers were remved and frzen n dry ice, then stred in a freezer until fractinated.

25 15 C~} Aspiratr 3-way valve 180 ml 20% KOH trap 900 ml 20% KOH trap Metablism chamber with urine cllectr tube. 180 ml 20% KOH trap A Air Flw /h Figure 1. Schematic f apparatus fr trapping CO.

26 16 Methd f Administering Labelled Cmpunds 14 Rats numbered 1 thrugh 4 were given [9, 10-methylene- C] sterculic acid by IG. This rute was cnsidered the "natural" way animals cnsume fd ils. Animals were given fd up t the time f injectin f label, but nly water was prvided in the metablism chamber. The experiment was terminated at the end f ten hurs fr the CO and SFO fed rats. Yss (97) determined that the rat n 14 CO diet excreted the maximum amunt f label frm C- sterculic acid between eight and 16 hurs. Hence, ten hurs seemed an apprpriate time. 14 Rats numbered 5 thrugh 8 were given n-[ 1- C]ctadecane by IP. The experiment was terminated at the end f eight hurs fr CO and SFO rats. Table 1 shws the dse in (j.ci f label given t each animal. Each experiment was run with ne CO and ne SFO fed raj;, then repeated a secnd time t crrect fr bilgical variability. Urine Extractin An equal vlume f distilled water was added t the urine. ph was adjusted t with HC1, and extracted with fur vlumes f ethyl ether (64). The cmbined extract was centrifuged at 2000 rpm t break up emulsins, if frmed. The cmbined ethyl

27 17 ether extract was washed with ne vlume distilled water and the slvent evaprated ff n a vacuum rtary evapratr. The free acids were esterified with diazmethane (Appendix IV). The label in the rat urine culd be extracted nly under acid 14 cnditins. The maximum extractable [ C] was 88 t 90% fr bth CO and SFO fed rats. Extracting the urine with additinal ethyl ether did nt imprve the yield. Therefre, abut 10% f the label was prbably excreted as the glucurnide (30) r sme ther cnjugate. Gas Chrmatgraphy Preparative GLC was carried ut n a Varian Mdel 1400 flame inizatin unit. The clumn, 14 ft by in I. D. aluminum tubing packed with 10% SP-222-PS /120 mesh (Supelc, Inc., Bellefnte, PA), was temperature prgrammed at 1 C/minfr 120 t 180 C and then held isthermally at 180 C until the end f the run. The injectr temperature was 200 C and the detectr 220 C. Nitrgen at a flw rate f 20 ml/min was used as the carrier gas. The detectr was equipped with a 5:1 split fr trapping purpses: 1 part detectr and 5 parts trap. The same GLC cnditins were used in cmbinatin with the mass spectrmeter.

28 18 Spectrscpy Equipment IR spectra were btained n a Beckman Mdel IR-18A using a beam cndenser. A micr salt cell with path length f 0. 1 mm r a salt plate was used t hld the sample. Spectra were run in carbn techtrachlride r carbn disulfide. NMR spectra were run n a Varian HA-100 with a time averaging cmputer using carbn tetrachlride as a slvent and benzene as a lck signal. Mass spectra were btained with a Finnigan Mdel 1015C Quadruple mass spectrmeter interfaced t a Varian Mdel 1400 gas chrmatgraph ven with a Ghlke glass jet mlecular separatr. The in surce pressure was 10 mm Hg; inizing current, 400 (xa; inizing ptential, 70 ev; and manifld temperature 150 C. Islatin f Metablites Frm Urine by Gas Chrmatgraphy Trapping Glass capillary tubes packed in dry ice (size slightly larger than internal diameter f splitter t prevent back pressure) were attached t the splitter f the GC with tefln tubing. A heat gun was used t keep the sample frm cndensing near the splitter-capillary interface and t drive the cndensed material t the cld sectin f the capillary tubing. All peaks and the base line area between the peaks were trapped separately. The capillary tubes were sealed

29 19 at the ends with micrflame and stred in the freezer until needed. T determine which peaks were labelled, the capillary tubes were washed with 1. 0 ml f apprpriate scintillatin slutin 14 (Appendix V) int vials t be cunted fr [ C]. Radiactive peaks and the mre abundant nn-radiactive peaks were trapped several times t btain sufficient amunts f each cmpund fr identificatin. Liver Tissue Fractinatin Livers were fractinated accrding t the methd f Shibk and cwrkers (81) int three fractins (Appendix VI): glycgen fractin (plar water sluble cmpunds), lipid fractin, and prtein fractin. These were cunted in the apprpriate scintillatin slu- tin. The lipids in turn were fractinated int three mre fractins (Appendix VII): sterid fractin (hexane nn-plar sluble), free fatty acid fractin (ethyl ether sluble), and glycerl fractin (water-alchl sluble). These were als cunted fr activity.

30 20 RESULTS AND DISCUSSION Animal Health and Grwth Animals were fed the SFO diet fr at least tw mnths t enable them t develp a sufficiently active pathway fr metablizing CPFA. Weekly bdy weights and feed cnsumptin shwed CO and SFO fed rats t be healthy. Table 1 indicates the perspective bdy weight, liver weight, days n diet, and the dse f labelled cmpund given t each animal. 14 Identificatin f [9, 10-methylene- Clsterculic Acid Metablites in SFO Fed Rats A typical gas chrmatgraph f methylated urine extract frm 14 a SFO fed rat given [9, 10-methylene- C]sterculic acid is illustrated in Figure 2. radiactivity. Peaks labelled with the letters A thrugh J cntain The scale n the left f the GC indicates the dpm 1 s representative f each peak. Since peak C cntains the largest amunt f material and label, it was identified first. Peak C The IR (Figure 3) f the largest peak C (Figure 2) shws an intense carbnyl absrptin at 1740 cm indicative f an aliphatic ester (82); strng 2860 cm due t carbn-hydrgen methyl

31 14 Table 1. Bdy weight, liver weight, days fed diet, and [ C] dse given t crn il and SFO fed rats Animal # Diet Bdy Weight Liver Weight Days n Dse (grh) '(gm) Diet JJICI 14 C-cmpund CO sterculic acid SFO sterculic acid CO sterculic acid SFO sterculic acid 5 CO n-ctadecane 6 SFO n-ctadecane 7 CO n-ctadecane 8 SFO n-ctadecane r

32 O 2,000-1,000 - X2 0.8 en HO.6 tr tr H0.4S Q: MINUTES 10 0 Figure 2. Gas chrmatgraph f methylated urine extract f a SFO fed rat injected with [9, 10-methylene- 4 C]sterculic acid.

33 23 stretching; strng 2930 cm f methylene carbn-hydrgen stretch- ing; mderate 1465 cm f carbn-hydrgen methylene bending; mderate 1440 cm f methylene cyclprpane bending; a mderate t weak 1260 cm, 1195 cm, and 1175 cm fr carbn-xygen stretching indicative f a methyl ester; and a medium 1020 cm absrptin frm skeletal vibratin f the cyclprpane ring (9, 96). The MS (Figure 4) f peak C (Figure 2) shws fragments characteristic f methyl esters f dibasic acids (Table 2). P-32 (m/e 154), P-59(m/e 127), P-64(m/e 122), and P-73(m/e 113) are present. The series m/e 53, 67, 81 and 95 crrespnding t the empirical frmulae f C H Ol and the series m/e 71, 85, 99, n 2n-5 113, and 127 crrespnding t [ C H O?! are als present. Ryhage and Stenhagen (71) bserved in the mass spectra f methyl- ated dibasic acids that the mst intense peak in the high mass range was m/e(p-31) r m/e(p-32), that the m/e(p-64) was als very intense, and that the parent in was very small r nnexistent. Based n these bservatins, the mlecular weight f cmpund C wuld be m/e(l ) = 186. The parent peak is nt present in the MS f cmpund C (Figure 4). The NMR (Figure 3) f C indicates a shrt chain cyclprpane dibasic ester. The large singlet at 6. 40T (14) indicates six methxyl prtns f a methyl ester and the dublet at 7.74T is assigned t fur methylene prtns adjacent t a carbnyl grup. Therefre, there

34 WAVELENGTH, MICRONS 6 I ' I ' I < E 20 h 4,000 3,000 2,000 WAVENUMBER, CM -I 1,000 Figure 3. IR (thin film) and NMR f cmpund C. t I*-

35 Table 2. Mass fragments characteristic f methyl esters f dibasic acids (71). m/e Fragment lst Remarks P-31 -OCH. Characteristic f all methyl esters f dibasic acids. P-32 -OCH + H P-60 -COOCH + H This peak is prminent nly fr shrt chain esters. P-63 (-OCH 3 ) 2 +H Prminent fr all esters except methyl adipate. P-64 (-OCH 3 ) 2 +2H P-73 -CH -COOCH Characteristic f all esters. P-91 -COOCH + -OCH H P-92 is higher than P-91 frm methyl azelate upwards. P-92 COOCH + -OCH + 2H P-105 CH -COOCH + -OCH + H CJ J J Lw fr methyl adipate, high fr higher esters. P-106 CH -COOCH +-OCH +2H Marked nly fr very lng chain esters.

36 26 are tw methxyl grups and tw methylene grups. A prtn upfield at and anther at 9. 08T indicate a methylene grup f a cyclprpane (2 5, 39) f the cis-cnfiguratin. The tw methine prtns f the ring absrb at 8. 72T. Absrptins at 6. 65T 7.29T 8. 18T, 8.25T, and 8. 97T are frm slvent cntaminatin. The abve data suggests a cmpund with the structure shwn in Figure 4. Peak B Peak B (Figure 2) exhibits the same IR (Figure 5) and MS (Figure 6) as cmpund C (Figures 3 and 4). Als, the NMR (Figure 5) shws the same methxyl (6. 40T) and methylene (7. 74T) prtns. The difference between the tw cmpunds is the dwnf.ield shift f the methylene cyclprpane prtns (9. 49T) in the NMR. Wd and Reiser (96) assigned the trans cnfiguratin t this absrptin. Furthermre, cmpund B has a lwer retentin time n a GC clumn (Figure 2), versus fr cmpund C, a character- istic expected f a trans ismer n the GC clumn used. ing NMR absrptins are due t slvent cntaminatin. ple was scanned 208 times and averaged n a cmputer). The remain- (This sam- Therefre, the data suggests a structure fr cmpund B shwn in Figure 6. Peak G The IR (Figure 7) f peak G exhibits the same absrptin

37 > UJ 60 - i- < u 20 tr M/E T r -1 <*- ~i r (f) z < {- t- H3CO-C-CH2 II CH2-C-QCH3 M=186 cis-methyl-s^-methylene adipate Figure 4. MS, mlecular weight, and frmula f cmpund C. -0

38 28 pattern as cmpunds B (Figure 5) and C (Figure 3), nly less intense. Again, there is a carbnyl stretch at 1740 cm ; carbn- hydrgen methyl stretch at 2860 cm ; carbn-hydrgen methylene stretch at 2930 cm ; carbn-xygen stretch frm the methyl ester at 1260 cm, 1195 cm, and 1175 cm ; and a weak cyclprpane ring skeletal vibratin at 1020 cm The MS (Figure 8) fragments characteristic f dibasic methyl- ated acids (Table 2): P-32 (m/e 182), P-60 (m/e 154), and P-64 (m/e 150) and the series [C H ^O] and [C H,, CL1 are present. L n 2n-5 L n 2n-3 2 The parent peak is absent, but crrespnds t m/e This is 28 mass units r tw methylene grups larger than cmpund B (Figure 6). Als, the m/e 74 representative f the McLafferty (45) rearrangement f typical methyl esters is mderately intense (40%), This wuld suggest that at least three saturated carbns exist adja- cent t ne f the ester ends. The NMR (Figure 7) shws a singlet fr tw methxyl grups at 6. 39T an d what appears t be a dublet superimpsed n a triplet at 7. 8T. The dublet indicates that there is ne methylene grup between the cyclprpene ring and the ester. The ther methylene prtns, equal t fur, appear in a cmplex pattern at 8. 2T. A prtn at T and ne at 9. 16T indicate a methylene grup f a cyclprpane with cis cnfiguratin. The abve data suggests a structure fr G shwn in Figure 8.

39 WAVELENGTH, MICRONS i l 1 ' 1 ' 1 ' _ ~ A s* ^/V^ V ^*jr*\ik. r^^\^r-^r ^^ ''"^^"'X vv, - ^^>* A^^- B 80 z - - \r^v^ ^ \ /^ ) A A - X 60-1 P ] \ ^ A f \ \ I \ A - ^\ / z 40 - V \l \ / V \ - r\. < - V V V \ / \ ^ 20 y \ - / 0 1 i i i i i 1 V-J i 4,000 3,000 2,000 1,000 WAVENUMBER, CM' 8 10 PPM (T) Figure 5. IR (slvent-ccl ) and NMR f cmpund B.

40 *1 P RELATIVE INTENSITY, % r 00 O O O O " i i 1 i 1 ' ' ' _ " h ~ r - ->i _ - UI z Ol =- -^1 ->1 4>. Ul a>_ r ~ CO : Ol w - 0) n (D P 3" P < H 1 (A) Si OQ =? " ^ 3 - (" < 3 CD CL 3 t-h CD 0 H U) a. 3 u i 03 fu <-? CD O Hh n 3 T) O P 3 a. td m (0 ^ {~\ ~ «> fvi- O r - PO r r.&- L r - r fc. _i\> -s w DO r en - O Ol TOTAL IONS, % e

41 WAVELENGTH, MICRONS 6 4,000 3,000 2,000 1,000 WAVENUMBER, CM" G 8 PPM (T) Figure 7. IR (slvent-cs ) and NMR f cmpund G.

42 ^ CO 5 60 H 2 w LU Jll Hii imi JljJ JJl lllll., J I I Ju 182. "T r^ r -i r M/E 150 r* z < H3CO-C-CH2 H \H H II CH2-(CH2)2-C-OCH3 M=214 cis-methyl-3,4-methylene suberate Figure 8. MS, mlecular weight, and frmula f cmpund G. 00

43 33 Peak F Peak F (Figure 2) has an MS (Figure 9) identical t cmpund G (Figure 8). There was an insufficient amunt f sample fr an NMR, and the IR was very weak. The IR did indicate a carbnyl stretch at 1740 cm and the carbn-xygen stretch at 1260 cm, cm, and 1175 cm fr a methyl ester. Cmpund F has a lwer retentin time n the GC (Figure 2) than cmpund G, versus Since bth have the same mlecular weight and MS pattern, cmpund F is prbably the trans ismer f cmpund G (Figure 8). Thus, a prbable structure fr F is shwn in Figure 9. Peaks E and D An NMR culd nt be btained fr peak E. The IR (Figure 10) absrptins were very weak shwing nly carbnyl stretch at 1740 cm, carbn-xygen stretch f methyl esters at 1260 cm, 1195 cm, and 1175 cm, and als a pssible very weak 1020 cm fr cyclprpane skeletal vibratin. Cmpund E shws the typical MS (Figure 11) fragmentatin pattern f methylated dibasic acids: P-31 (m/e 169), P-60 (m/e 140), P-64 (m/e 136), and P-92 (m/e 108) and the series [ C EL Ol + and n 2n-5 [C H.O 1. There is very little m/e 74 (14%) frm the n 2n-3 2 McLafferty rearrangement, implying that there are less than three

44 - RELATIVE INTENSITY, % ^3 O O O O O 1 ' *] (TQ i m -x> X -^ fl) % O CO i 1 :0 ri ' CD <- fl> n <_ p ^ N) P CO hj V^1 0) OP IT v- 1 h 1 *- L 1" / 3 (D ri- ^_ I "* < r P CD I 3 CD O X W INO c P i > CD 1 i-t CO i-n n 3 T) p 3 a = 1 CO 2 m r_.&_ (^_ r - IV) p r r >) Ol r cn : " : - -s ^- ^ =r = : - V -«-00 r O <D 01 ->j tn O ^e PO -n r <7> O Ol TOTAL IONS, %

45 WAVELENGTH, MICRONS ,000 3,000 2,000 WAVENUMBER, CM" 1,000 Figure 10. IR (slvent-cs ) f cmpund E. CO

46 36 saturated carbns between the ring and the ester grup. The parent at m/e 200 is absent. This is 14 mass units r ne methylene grup larger than cmpund C (Figure 4). Cmpund D (Figure 2) has an MS (Figure 12) identical t cm- pund E (Figure 11). Infrared r NMR spectra culd nt be btained. Cmpund D has a lwer retentin time, versus 2. 04, than cmpund E. Since bth have the same mlecular weight, the data weakly suggests a structure fr E as shwn in Figure 11 and fr D as shwn in Figure 12. Peak H An NMR culd nt be btained fr cmpunds H r I. The IR's were very weak due t insufficient material and indicated nly carbnyl stretch at 1740 cm The MS (Figure 13) f cmpund H (Figure 2) shws the typical fragments f dibasic acid methyl esters: P-32 (m/e 196), P-60 (m/e 168), P-64 (m/e 164), and P-92 (m/e 136). The parent peak is absent, but wuld crrespnd t a mlecular weight f m/e 228. This is 14 mass units r ne methylene grup larger than cmpund G (Figure 8). The same series [C H O], m/e 53, 67, 81, and n Zn- 5 95, and [C H O ], m/e 71, 85, 99, 113, and 127 are present. Als, m/e 74 (88%) is very intense indicating a carbn length f three r mre frm the ester. Since the MS f H is very similar t

47 z -I < H > H H U / H3CO-C-CH2 CH2-CH2-C-OCH3 M = 200 cis-methyl-3,4-methylene pimelate H O ii Figure 11. MS, mlecular weight, and frmula f cmpund E. 00

48 M/E II CH2-CH2-C-OCH3 H3CO-C-CH2 H M=200 trans-methyl-s^-methylene pimelate Figure 12. MS, mlecular weight, and frmula f cmpund D. 00

49 RELATIVE INTENSITY, % r 0) O O O O i i 1 1 ' *.._ r Ol CD O l-t w a e OP P t * Mi O 3 TS O P (^ X O CO I 3 CD =r CO I 3 CD i 3 - ><_ CD CD ct- ** CU p N 3 CD CL &> M-i CD OI O" $ Ol.0> _ ^ 00_ = - ^=- _ O- = r- O O O 00_ O r - r r- O r " zr- ^.5 0) i i 0) r OI m I ^ TOTAL IONS, % Ol 6

50 40 that f G, the structure f H is prbably like G. Als, cnsidering that the amunt f cis ismers are prevalent ver the trans ismers, a prbable structure fr cmpund H is shwn in Figure 13. Peak I Peak I (Figure 2) has an MS (Figure 14) fragmentatin pattern similar t G (Figure 8) and H (Figure 13). The mass spectra f the cyclprpane urinary metablites all appear t have the same basic fragments. The typical fragments f methylated dibasic esters are present: P-32 (m/e 210), P-60 (m/e 182), P-64 (m/e 178), and P-92 (m/e 150), alng with the tw series [C H^ 0] + and 0 L n 2n-5 [C H O-1. The parent peak is absent, but wuld crrespnd t L n2n-32 J ^ ^ ^ m/e 242. This is 14 mass units r ne methylene grup larger than cmpund H. Again, based n the same cnsideratins given cmpund H, a prbable structure f I is shwn in Figure 14. Peak A The MS (Figure 15) f peak A (Figure 2) shws a parent f m/e 172. This is 14 mass units less than cmpund C (Figure 4), cis- methyl-3, 4-methylene adipate. The fragmentatin pattern is dif- ferent frm the ther cmpunds f this series. The IR and NMR culd nt be btained, thus n attempt was made t assign a structure t A.

51 >_- 80 h CO UJ 60 H Z ^ ^ CO* z 133 UJ ,182 illlliilh illi llll, II niilllllli I i " i ""'" "I ' I i I I ' I I '"" I-., { ' I- 'I I T" >- i 11/ H i 1 r M/E H3CO-C-CH2 H H H \JL H < 0 11 CH2-(CH2)4-C-OCH3 M = 242 cis-nnethyl-3,4-methylene sebacate Figure 14. MS, mlecular -weight, and frmula f cmpund I. *-

52 cvl % 'SNOI g 1 i ivii O CVJ 42 < CM O CM CM O -O CM -i c d ex s u VI -M 00 O -< O 5; CvJ c c 3 u n) t i d HI <J> fo CJ O CM_= r 8- - t^- - - CM int - -= "CD ^ - -. a) d CV1. O O 1 i i (-> O (0 % 'AiiSNaiNi 3Aiivn3a

53 43 Peak J A clser lk at the GC (Figure 2) indicates anther pssible labelled metablite numbered J. N spectral data was btained n this cmpund due t insufficient amunts. Hwever, being the last cmpund t cme ff the clumn, it is prbably an analg f the methylated dibasic cyclprpane esters. In summary, eight metablites were tentatively identified ut f ten labelled peaks shwn n the GC (Figure 2). They are listed in Table 3 giving the mlecular weight and name f each cmpund. 14 Identificatin f [9, 10-methylene- Clsterculic Acid Urinary Metablites in Crn Oil Fed Rats The purpse f this sectin was t test if crn il fed rats given sterculic acid (IG) prduced the same urinary metablites as SFO fed rats. A typical gas chrmatgraph f methylated CO fed rat urine is illustrated in Figure 16. Peaks that cntained label 14 [ C] were analyzed by MS and fund t have identical mass spectra as the crrespnding peaks f SFO fed rats. Table 4 shws the relative retentin times f each peak frm bth grups f animals, and the ttal percentage f activity fr each labelled peak. Peak E in the CO fed rat culd nt be detected due t its clse similarity t peak F. The dashed line in the GC (Figure 16) shws where it wuld be present.

54 a h MINUTES Figure 16. Gas chrmatgraph f methylated urine extract f CO fed rat injected with [9, 10-methylene- 14 C] sterculic acid.

55 14 Table 3. Urinary metablites f [9, 10-methylene- C]sterculic acid frm SFO fed rats Peak Mlecular Weight Name (Methyl Ester) A B C D E F G H I J Unknwn Trans-methyl-3, 4-methylene adipate C is-methyl-3, 4-methylene adipate Trans-methyl-3, 4-methylene pimelate Cis-methyl-3, 4-methylene pimelate Trans-methyl-3, 4-methylene suberate Cis-methyl-3, 4-methylene suberate Cis-methyl-3, 4-methylene azelate Cis-methyl-3, 4-methylene sebacate Unknwn

56 46 Table 4. Retentin time and percent label frm [9, 10-methylene- C]sterculic acid urinary metablites f C O and SFO fed rats. SFO Fed Rat CO Fed Rat Cmpund # a/ a/ R - %CPM R"- %CPM A B C D E F G H I J a/ ~ Retentin time was calculated using methyl phenyl acetate as a reference, peak 6 (Figure 17). b/ CmpundE CPM's are summed with cmpund F.

57 47 The data indicates that CO fed rats can metablize CPFA the same as SFO fed rats even thugh they had nt been acclimated t the CPFA diet. The main metablites fr bth grups were cis- methyl-3, 4-methylene adipate, cmpund C (46-47%), and cis- methyl-3, 4-methylene suberate, cmpund G (9-18%). These are the same tw metablites that Yss (97) identified in his crn il fed rats given labelled sterculic acid. The GC elutin pattern f Figure 16 differs frm that f Figure 2 because the relative amunt f label and metablites in the CO fed rat urine (1-40%) was lwer than the amunt f label and material in the SFO fed rat urine (46-57%) (Table 6). Identificatin f Urinary Metablites in SFO Fed Rats The purpse f this sectin was t determine if SFO fed rats excreted the same urinary metablites as SFO fed rats injected (IG) with labelled sterculic acid. Figure 17 shws a typical GC f methylated rat urine frm a SFO fed rat. The lettered peaks A thrugh J crrespnd t the sterculic acid metablites. Cmpund D and E, cis and trans methyl- 3, 4-methylene pimelate, culd nt be detected by MS. The chpped line indicates where they wuld be n the chrmatgraph. The numbered peaks 1 thrugh 11 were als identified by

58 MINUTES 20 Figure 17. Gas chrmatgraph f methylated urine extract f SFO fed rat.

59 49 cmbinated MS, IR, and GC retentin time (Table 5). Cm- pund 6, methyl phenyl acetate which was the largest amunt, is frmed by the nrmal metablism f pehnylalanine t phenylpyruvic by by transaminatin and further decarbxylatin t phenyl acetic acid (56). The ther large peak 10, p-cresl, is frmed in the gut by bacterial actin n tyrsine, fllwed by absrptin in the intes- tinal tract, and then excretin thrugh the urine (57). The ther lesser cmpunds are fund in varius amunts in the urine f mst mammals (43, 58, 64). Label f 14 C 1 in Liver Fractins, Liver Lipid Fractins and Carbn Dixide frm [9, 10-methylene-^C ] sterculic Acid The primary purpse f fractinating the liver and cllecting carbn dixide was t investigate the pssibility f [9, 10-methylene- 14 C] sterculic acid being incrprated int ther prducts. Table 6 shws the distributin f label in the varius fractins expressed as a percent f the administered labelled dse. The mst bvius bservatin is that the majrity f the label was lcated in the urine ver the ten hur perid. The labelled carbn dixide accunted fr abut 1. 0% r less f the activity. Hence, very little f the ring was metablized. In the liver, the majrity f the label is in the lipid fractin with mst f this in the fatty acid material. A clser lk at the liver fractins shws a

60 Table 5. Other urinary metablites in SFO fed rats. Identificatin Methd Peak # Cmpund Name GC (R^- 7 MS^ mf Surce f Reference Cmpund Methyl lactate Dimethyl xalate Dimethyl malnate Methyl benzate Dimethyl succinate Methyl phenyl acetate Dimethyl-2-methyl glutarate? Dimethyl adipate Dimethyl pimelate P-cresl Dimethyl ctadecen (? ) iate 0.21 X Eastman Org. Chem 0.42 X MCB X Sigma X X MCB X Eastman 1.00 X X Aldrich X 1.23 X Sigma X Sigma 1.89 X X Aldrich X a/ Methyl phenyl acetate was used as a reference standard. b/ MS -were cmpared t knwn cmpund (92). c/ IR were cmpared t knwn cmpunds listed in surce.

61 14 Table 6. Percent f administered [ C] dse in urine, carbn dixide, liver fractins, and liver lipid fractins f CO and SFO fed rats injected with [9, 10-methylene- i4 C ] sterculic acid. Activity Expressed as Percent f Administered Dse Liver Fractins Liver Lipid Fractins Rat # Diet Liver Urine CO Prtein Lipid Acid Sl. Sterid 1 CO SFO FA Glycerl 3.78 ± a/ 3 CO / 4 SFO a/ */ a/ The amunt f activity in these fractins was less than 0. 01% f dse.

62 52 significant amunt f label in the prtein fractin fr CO and SFO fed rats. Kircher (31) suggested that sulfhydryl grups f prteins may react with cyclprpenes, thus causing the physilgical effects bserved frm feeding CPFA. The prcedure used in the fractina- tin f the liver shuld remve all lipid frm the prtein. Cnse- quently, this adds mre evidence that Raju and Reiser (65) may be crrect in implying that CPFA binds irreversibly t enzymatic sulfhydryl grups. In summary, a large percentage f sterculic acid is being metablized t dicarbxylic acids and excreted in the urine. Crn il fed rats can metablize CPFA almst as effectively as SFO fed rats with the difference being nly the time required. 14 Oxidative Mechanisms in [9, 10-methylene- C] sterculic Acid Metablism The metablites indicate a cmbinatin f a, (3, and c - xidative mechanisms taking place. (3-xidatin ccurs in the mitchndria (34) and r and t-xidatin takes place in the micr- smes (54, 55). Nrmal straight chain fatty acids underg p-xidatin, hwever, it has been shwn that in certain cases, straight chain as well as branched chain (13) fatty acids d underg CJD -xidatin. Bergstrm et al, (4) studied the metablism f

63 14 [ 1- C] -2, 2-dimethylstearic acid in the rat. They fund very little labelled carbn dixide and recvered 90% f the label in the urine as 14 [ 1- C] -2, 2-dimethyladipic acid. In anther study n Q-- substituted alkyl myristates and stearates in dgs, Wetzel (95) bserved that when the side chain was greater than an ethyl grup, large amunts f the crrespnding a-substituted adipic acid was excreted in the 14 urine. The metablism f [9, 10-methylene- C]sterculic acid prduces cis and trans-3, 4-methylene adipic and cis and trans- 3, 4-methylene suberic acid as the main prducts. Therefre, sterculic acid metablism seems t be a special case f branched chain fatty acid metablism with the 9, 10-methylene f the ring acting as a branch. Anther laternate pathway, a-xidatin, was demnstrated by Stkke et al (85) t be prminent in certain types f branched chain 14 fatty acid metablism. After the ingestin f 3, 6-dimethyl[ 8- C] ctanic acid by man, labelled carbn dixide was fund in the expiratry air, and 2, 5-dimethylheptaniic acid was demnstrated 53 t be present in the urine. He suggested that this pathway wuld accunt fr abut 1-2% f all fatty acid xidatin. The ttal amunt f cis- and trans-3, 4-methylene pimelic acid, the prducts f a, (3, and CD -xidatin, islated in the urine frm CO and SFO fed rats injected with labelled sterculic acid, was apprximately 4% and 10% respectively (Table 4). Hwever, the SFO fed rat that was nt given

64 54 label (Figure 17), did nt frm these tw prducts indicative f a- xidatin. Thus r-xidatin seems t ccur nly when an animal is given a large dse f il at ne time. During mderate, regular expsure t SFO and/r CO, this pathway was less evident. Prpsed Metablic Pathway fr Sterculic Acid Yss (97) bserved in a time distributin study f [9, methylene- C] sterculic acid in IG injected rats, that initially, 0-1 hur, the 12, 000 Xg mitchndrial fractin cntained mre label than the 105, 000 Xg micrsmal fractin. the activity was higher in the micrsmes. Frm tw t fur hurs After fur hurs the micrsmal cunts drpped belw the mitchndrial and remained there fr 26 hur study perid. Based n the abve bservatins, the urinary metablites identified, and the fact the ring in hydrgenated, the fllwing pathway (Figure 18) is prpsed fr sterculic acid. Sterculic acid is transprted t the mitchndria where it is (3-xidized t within tw carbns f the ring. Further transprt t the micrsmes leads t reductin f the cyclprpene ring t a cyclprpane ring plus OD -xidatin f the methyl grup t an acid. It is prbably during reductin that the tw ismers, cis and trans, are frmed. Evidence is lacking as t the rder that reductin and OD-xidatin take place and als why the cis is frmed in preference