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Rat urinary metabolites of [9,10-methylene-14C]sterculic acid
76
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 488 (1977) Biomedical Press
76-67
BBA 57023
RAT URINARY ACID *
METABOLITES
OF [ 9,10-METHYLENE-‘4C]STERCULIC
T.A. EISELE, J.K. YOSS, J.E. NIXON, R.O. SINNHUBER **
N.E. PAWLOWSKI,
Department of Food Science and Technology, 97331 (U.S.A.) (Received
February
L.M. LIBBEY
Oregon State
University,
and
Corvallis,
Oreg.
7th, 1977)
Summary 1. The metabolism of [9,10-methyZene-‘4C] sterculic acid was studied in corn oil and Stercula foetida oil fed rats. The majority of the radioactivity was excreted into the urine as short chain dicarboxylic acids. The main urinary metabolites were cis-3,4-methylene adipic acid, cis-3,4-methylene suberic acid, trans-3,4-methylene adipic acid, cis-3,4-methylene pimelic acid, and cis-3,4methylene azelic acid. 2. Formation of these urinary metabolites requires a-, p-, and w-oxidation plus reduction of the cyclopropene ring to a cyclopropane ring. Sterculic acid must be transported through both mitochondrial and microsomal systems. 3. Other non-radioactive urinary compounds were also identified. A proposed pathway for the metabolism of sterculic acid and possible detrimental effects caused by these metabolites is discussed.
Introduction Sterculic acid is a naturally occurring cyclopropenoid fatty acid containing a highly strained and reactive unsaturated three-membered ring in the center of an l&carbon chain. The main food sources of cyclopropenoid fatty acids are the cotton plant (Gossypium hirsutum) in the United States and kapok (Ericlench-an anfractuosum) in the oriental countries. These cyclopropenoid fatty acids have been held responsible for numerous physiological disorders in many animals. A review by Phelps et al. [l] reported ____
*
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State
University,
Oregon
conducted
Corvallis,
Eisele R.O. Oreg.
at
Agricultural Oregon
and
State
Doctor
Sinnhuber, 97331.
Experiment
Station.
University
in partial
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for J.K. of
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and
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77
that dietary cyclopropenoid fatty acids impaired reproduction in rats and chickens, delayed sexual maturity in females of these species and caused pink discoloration in avian egg whites during storage. Other recent work with cyclopropenoid fatty acid fed rats showed fatty infiltration and degeneration of the liver along with renal tubule degeneration [Z], altered lipid metabolism [ 31, and partial loss of membrane associated functions [4]. Sinnhuber and coworkers [ 51 discovered that sterculic acid is a liver carcinogen when fed to rainbow trout. Scarpelli et al. [6] reported that hepatoeytes from sterculic acid fed rainbow trout and rats showed induced cytoplasmic alterations. Information on the metabolism of sterculic acid would help explain its mode of action and the physiological disorders which occur. However, limited information has been published. Altenberger and co-workers [7] fed [methylene-14C]sterculic acid to fasting hens and found limited amounts of labelled carbon dioxide formed over a 24-h period. They also found more than 50% of the label in the fecal material. Yoss [B] did a time flow study on the distribution of [ “C]sterculic acid in rats. He concluded from the lack of 14C-labelled CO, that the rat did not metabolize the cyclopropene ring. The intent of this investigation was to identify the labelled sterculic acid metabolites in rat urine. Corn oil fed rats were compared to Stercutiu foetida oil fed rats to determine whether acclimation to cyclopropenoid fatty acids would produce different metabolites. Methods
and Materials
E~~erimen tal un~rn~~sand diet Four weanling Wistar male rats were fed ad libitum a Krishnaro and Draper diet [9] modified to contain 20.0% protein and 32.2% cornstarch. The diet consisted of 92.8% premix, 2.2% vitamin mix (U.S. Biochemicals), and 5.0% corn oil mixed 1 : 1 (w/v) with 3.0% agar (DIFCO) dissolved in distilled water at approximately 80°C. The hot mix was solidified at 2-5°C to a solid gel, cut into small blocks, and stored in plastic bags at -30°C until used. After feeding the 5.0% corn oil diet for two weeks, two of the four rats were transferred to a 4.0% corn oil plus 1.0% S. foetida oil diet. The cyclopropene content of the S. foetida oil diet was 0.5% of the total dry weight, or 10.0% based on total lipid composition. Rats were fed the S. foetida oil diet for at least 60 days to develop an active pathway for metabolizing cyclopropene fatty acids. Urinary metabolites from S. foetid~ oil fed rats, corn oil fed rats given labelled [9,10-methyZene-‘4C]sterculic acid, and S. foetida oil fed rats given labelled {9,10-methyZene-‘4 C] sterculic acid were compared. Method of administering and source of kabelled sterculic acid Methyl sterculate, labelled on the 9,10-methylene bridge of the cyclopropen~ ring, was synthesized by Pawlowski [lo]. The Halphen test [ll] and NMR (121 indicated 99.0 ?r 0.2% and 103.0 of:2.0% cyclupropene respectively. The free acid was prepared by saponification with 0.5 N 95% alcoholic KOH at 45-50°C for 1.5 h. Pure sterculic acid, 110 pCi/mol, was diluted to 5.0 pCi/ 0.41 g corn oil and administered intragastrically. Immediately after injection,
78
animals were placed in metabolism chambers. Urine was allowed to flow into glass tubes packed in ice. Urine extruction
The urine was diluted with an equal volume of distilled water, adjusted to pH 1.0-2.0 with HCl, and extracted twice with four volumes of ethyl ether. If necessary, combined extracts were centrifuged at 2000 rev./min to break up emulsions. The ethyl ether extract was then washed with one volume distilled water and evaporated to dryness on a vacuum rotary evaporator. The free acids were esterified with diazomethane [ 13 1. Gas chromatography Preparative gas liquid chromatography was carried out on a Varian Model 1400 flame ionization unit. The column, 14 ft X 0.085 inches internal diameter aluminum tubing packed with 10% SP-222-PS 01-1885 llOf120 mesh (Supleco, Inc., Bellefonte, Pa.), was temperature programmed at l*C/min from 120-18O’C and then held isothermally at 180°C until the end of the run. The injector temperature was 200°C and that of the detector was 220°C. Nitrogen at a flow rate of 20ml/min was used as the carrier gas. The detector was equipped with a 5 : 1 (trap : detector) split for trapping purposes. The same gas liquid chromatography conditions were used in combination with the mass spectrometer. Isolation of metabolites from urine by gas chromatography trapping Glass capillary tubes packed in solid CO* (of a size slightly larger than the internal diameter of the splitter, to prevent back pressure) were attached to the splitter with Teflon tubing. A heat gun was used to keep the sample from condensing near the splitter-capillary interface and to drive the condensed material to the cold section of the capillary tubing. All peaks and the base line area between the peaks were trapped separately. The capillary tubes were sealed at the ends with a microflame and stored in the freezer until analyzed. To determine which peaks were radioactive, the capillary tubes were washed with 1.0 ml of PPO-POPOP-Toluene (PPO (2,5diphenyloxazole) Sigma Chem. Co., POPOP (1,4-bis(2,5-phenyloxazole)benzene), Nuclear Chicago) into vials to be counted. Radioactive peaks and the more abundant non-radioactive peaks from several runs were combined to obtain sufficient amounts of each compound for identification. Spectroscopy equipment Infrared spectra were obtained on a Beckman Model IR-18A using a beam condenser. A micro salt cell with path length of 0.1 mm or a salt plate was used to hold the sample. Spectra were run in carbon tetrachloride or carbon disulfide. NMR spectra were run on a Varian HA-100 with a time-averaging computer, using carbon tetrachloride as solvent and benzene as a lock signal. Mass spectra were obtained with a Finnigan Model 1015C quadrupole mass spectrometer interfaced to a Varian Model 1400 gas chromatograph oven with a Gholke glass jet molecular separator. The ion source pressure was 10e6 mmHg;
79
ionizing current 400 PIA; ionizing potential 70 eV; and manifold temperature 150°C. Mass spectral data was acquired and processed by a System Industries System 250 data system. Results Identification of [9,1 0-methylene-‘4C]sterculic acid urinary metabolites in S. foetida oil fed rats A typical gas chromatogram of methylated urine extract from a S. foetida oil fed rat given [9,10-methylene- *4C]sterculic acid is illustrated in Fig. 1. Peaks labelled A-J contained radioactivity. The scale on the left of the gas chromatogram indicates counts per min representative of each peak. Peak C contained the largest amount of carbon-14 label followed by peak G. As shown in Table I, mass spectra were obtained on each peak, followed by NMR and infrared when sufficient material was available. The infrared spectrum of peak C of Fig. 1 shows an intense carbonyl absorption at 1740 cm-‘, indicative of an aliphatic ester [14] ; strong 2860 cm-‘, due to carbon-hydrogen methyl stretching; strong 2930 cm-’ of methylene carbonmethylene bendhydrogen stretching; moderate 1465 cm -’ of carbon-hydrogen ing; moderate 1440 cm-’ of methylene cyclopropane bending; moderate-toweak 1260 cm-‘, 1195 cm-‘, and 1175 cm-’ for carbon-oxygen stretching indicative of a methyl ester; and a medium 1020 cm-’ absorption from skeletal vibration of the cyclopropane ring [15,16]. All of the infrared spectra (Table I) for each of the labelled peaks exhibited these absorptions varying somewhat in intensity . The mass spectra (Table I) of these labelled metabolites show fragments characteristic of methyl esters of dibasic acids. Ryhage and Stenhagen [17] observed that the most intense peak in the high mass range was m/e (P-31) or m/e (P-32), that m/e (P-64) was also very intense, and that the parent ion (P) was very small or nonexistent. Thus spectrum C exhibits P-32 (m/e = 154), P-64 (122), and also P-59 (127), and P-73 (113). Compound C has no parent ion present, but based on the above data, the parent would be at m/e = 186. The NMR spectrum of compound C (Table I) shows four different types of protons: methoxyl of an ester, methylene adjacent to a carbonyl, methine of I
0.3
2.000
5
% 0.6 2
I :
it
fi 0.4 0
1,000
6 0.2 s LT
0 70
60
50
40
30
20
IO
0
MINUTES
Fig. 1. Gas chromatogram of methylated urine extract of a S. foefida oil fed rat injected with [9,10methylene-14C1stemlic acid. Reading right to left: A. unknown; B. fmwmethyl-3.4~methylene adfpate: C. cis-methyl-3,4-methylene adipate; D. trans-methyl-3.4-methylene pimelate; E. cis-methyl-3.4-methylene pimelate; F, trans-methyl-3,4-methylene suberate; G. cis-methyl-3.4-methylene suberate; H, cismethyl-3,4-methylene azelate; I, cis-methyl-3.4-methylene sebacate; J. unknown.
80 TABLE I NUCLEAR MAGNETIC RESONANCE, 14C1 STERCULIC ACID METHYLATED Peak
INFRARED, AND MASS SPECTRA RAT URINE METABOLITES
OF [9,10-METHYLENE-
Compound structure and name
Spectral data
Unknown
MS *: 27(7), 29(7). 41(20), 43(100), 55(12). 59(g), 69(25). 72(2l), 87(2l), 95(9), 98(6). 123(2). 129(l), 140(l), 172(0.2), [M = 1721.
trans-Methyl-3,4-methylene 0
H
adipate
H
II
CH30C-CH2
H if H
CH,-COCH,
cis-Methyl-3.4-methylene
adipate
NMR: (CC14) 6.40@,6H.OCH3),
7.74(d,4H,J = 6, O=CCH2).8.72 (m,2H,methine cyclopropane), 9.08 (m,lH,cis-methylenecyclopropane).9.94 (m,lH. J = 5,cismethylene cyclopropane). IR: (liq. film)2930(s),2860(m),1740(s),1465(m), 1440(m),1260(w),1195(m),ll75(m), 1020(m). MS: 27(38).29(23),39(67),41(85), 55(51),59(93), 67(98), 71(67),84(76).85(100),95(27),99(23). 100(18),113(53),122(56).127(47),137(4),154(10), 155(7),171(2).[M = 1861.
CH, -COCH3
trans-Methyl-3,4-methylene
NMR **: (CC14) 6.40(s,6H,OCH3), 7.74(d,4H. J = 6. O=CCHz), 8.72 (m, 2H. methine cyclopropane), 9.49(t, 2H, J = 5, cis-methylene cyclopropane). IR ***: (CC14) 2930(m), 2860(m), 1740(s), 1465(w), 1440(m), 1260(m). 1195(m), 1175(m). 1020(m). MS: 27(22). 29(14), 39(40). 41(69), 55(28), 59(100), 67(83), 71(57). 84(53), 85(60), 95(17), 99(22), 112(g). 113(43), 122(28), 127(38), 154(6), 155(2), CM = 1861.
pimelate
MS: 27(33),29(19),39(36).41(39),55(100). 59(49), 67(32),74(16),80(32),81(70).94(17).95(15). 108(40).109(24),125(3),127(3),136(59), 140(26),153(1),168(2),169(5). [M = 2001.
3
cis-Methyl-3.4-methylene
’ CH30C-CH2
pimelate
.-&H+iOCH
?
3
?
trans-Methyl-3.4-methylene
H
suberate
0 3
cis-Methyl-3,4-methylene
suberate
IR: (CS2) 2930(s),2860(m),1740(s).1260(m). 1195(m),1175(s).1020(w). MS: 27(39),29(24).39(44),41(61). 55(100). 59(79), 67(49),71(20),80(30).81(75),94(17),95(14). 108(35),109(29),126(6),127(10),136(70).140(31), 141(10),168(1),169(1), [M = 2001. IR:(CS~)1740(m).1260(w).ll95(w),ll75(w). MS: 27(31),29(20),39(47),41(99), 55(88).59(100), 67(61).74(48).80(57),81(88),94(22).95(28), 109(21).114(11),122(28).123(29).140(17), 141(12).150(60).151(27),128(2),183(1), [M = 2141. NMR: (CC14) 6.40 (s,GH,OCH3), 7.70 (t,4H,J= 6, 0=CCH2), 8.3 (m,2H, J = 6, CHz), 8.5-8.7 (m, 4H,CHz plus methine cyclopropane), 9.07 (m, lH, cis-methylene cyclopropane), 10.06 (m. lH J = 5,ciwnethylene cyclopropane).
H
H
IR:(CS~)2930(m).2860(w).1740(s),126O(w). 1195(w).1175(m), 1020(w). MS: 27(33),29(30).41(86),43(52). 55(82).59(100), 67(57).74(40),80(57),81(87),94(22). 95(29). 109(20).114(12),122(30),123(32), 140(16). 141(12),150(62),151(23) 161(2),171(l). 182(2). 183(2),197(l), [M= 2141.
81 TABLE I (continued) NUCLEAR
MAGNETIC
14Cl STERCULIC
RESONANCE,
ACID METHYLATED
INFRARED,
AND
MASS SPECTRA
OF [9,10-METHYLENE-
RAT URINE METABOLITES
Peak
Compound structure and name
Spectral data
H
cis-Methyl-3.4~methylene
IR: (CS2) 1740(w). MS: 27(18), 39(42), 41(88), 55(100). 59(87), 67(58), 74(87), 79(29). 81(50), 94(34), 96(47), 108(11), 109(12), 119(31), 123(23). 136(23), 137(21), 154(8), 155(7), 164(26). 165(10),178(l), 196(2), IN = 2281.
f;
HH
aselate
(u-i,)4-
CH3OC-CH2
k,
3
l?H
H
cis-Methyl-3,4-methylene
I
CH 3.i-CH
2
H
H
-?r H
H
sebacate
KH,,,&H
3
MS: 27(28), 29(34), 41(97), 43(46), 55(98), 59(100), 67(70), 74(65), 81(58), 82(38f, 93(29), 95(45), 109(25). llO(24). 119(B), 122(8), 133(22), 137(19), 150(15), 151(12). 160(4), 168(3), 178(10). 179(6), 210(2), 211(l). [M = 2421.
MS, mass spectrometry: m/e (% base peak); IM = molecular ion]. Spectra were abbreviated by Iisting the two largest ions every 14 a.m.u. beginning at m/e = 20. Complete spectra can be obtained by writing to principal author. ** NMR: (solvent) ?‘ (splittii pattern, number protons, functionaf. group): s = singlet, d = doublet, f = triplet, m = muhtiplet. *** IR. infrared: (solvent) principtibands in cm-*: s = strong, m = medium, w = weak. *
a cyclopropane, and methylene of a cyclopropane. A large singlet at 6.40 r represents the methoxyl groups of an ester f143, and six of the total 14 protons present. The diesters’ four a-methylene protons appear as a doublet at 7.74 T. The remaining four protons show a pattern expected for cis-distributed cyclopropane. The two methine protons of the small ring absorb at 8.72 T while the sypzand anti methylene protons absorb at 9.94 7 and 9.08 r [X3,19]. The data suggests a structure for C as shown in Table I. The mass spectrum of B (Table I) is identical to that of C. Compound B differs from C in the downfield shift of the methylene cyclopropane protons (9.497) in the NMR (Table I) to which Wood and Reiser 1163 assigned the TABLE II RETENTION TIME AND 46 LABEL FROM [9,10-i%fETHYLENE-14C] METABOLITES OF CORN OIL AND S. FOETIDA OIL FED RATS Compound
A B C D E
F G II I J
S. foetido
oil fed rat
STERCULIC
ACID
URINARY
Corn oil fed rat
Rt *
%cpm
Rt *
% cpm
1.30 1.55 1.80 1.86 2.04 2,09 2.35 2.64 2.98 3.21
1.42 7.37 47.51 1.42 8.75 2.50 18.06 5.89 5.36 1.73
1.31 1.59 1.85 1.91 2.09 2.16 2.44 2.81 3.20 3.53
1.61 5.83 46.79 3.37 *+ 2.32 9.57 11.88 3.35 1.11
* Relative retention time was crdculated using methyl phenyl acetate as a reference, peak 6 (Fig. 3). * * Compound E radioactivity is summed with compound F.
82
trans configuration. A suggested structure for B is shown in Table I. Thus, B and C differ only in their orientation around the cyclopropane ring. B, being the trans compound and more of a “straight” molecule, would be eluted from a gas liquid chromatography column before C, as shown in Fig. 1. Using the same techniques, a structure was assigned to all of the labelled peaks except A and J. The amount of material was much too small to obtain any data except for a mass spectrum on A, and this did not show any fragments analogous to the other spectra. The spectra, suggested structure, and names of each metabolite are shown in Table I. Identification oil fed rats
of [9,1 O-methylene-
‘%‘]sterculic
acid urinary metabolites
in corn
The purpose of this section was to determine if corn oil fed rats given steracid produced the same urinary metabolites as S. foetida oil fed rats. A typical gas chromatogram of methylated corn oil fed, sterculic injected, rat urine is illustrated in Fig. 2. Peaks that contained radioactivity were analyzed by mass spectrometry and found to have identical mass spectra as the corresponding peaks of S. foetida oil fed rats. Table II shows the relative retention times of each peak from both groups of animals, and the total percentage of activity for each labelled peak. Peak E in the corn oil fed rat could not be detected due to its close proximity to peak F. The small dashed line in the gas chromatogram (Fig. 2) shows where it would be, The data indicates that corn oil fed rats can metabolize cyclopropenoid fatty acids the same as S. foetida oil fed rats even though they had not been acclimated to the cyclopropene diet. The main metabolites for both groups were: cis-methyl-3,4-methylene adipate, compound C (46-47%); and cis-methyl-3,4methylene suberate, compound G (g-18%). The gas chromatographic elution pattern of Fig. 2 differs from that of Fig. 1 because the relative amount of label and metabolites in the corn oil fed rat urine (20%) was lower than the amount of label and material in the S. foetida oil fed rat urine (51%). cubic
Identification
of urinary metabolites
in S. foetida
oil fed rats
The purpose of this experiment was to determine whether S. foetida oil fed rats excreted the same urinary metabolites as S. foetida oil fed rats injected with a large single dose of labelled sterculic acid. Fig. 3 shows a typical gas chromatogram of methylated rat urine from a S.
MINUTES Fig. 2. Gas chromatogram of methylated urine extract of a corn oil fed rat injected with [9.10-mefhyb eru~-~~C] stenxlic acid. Names of lettered peaks are as Fig. 1.
83
IO
4
I
6
C
”
%
0.8 5 . % -0.6 ki -
1
A
- 0.4 2 G
.:,::c_il~~~,~~~
I
H
s F II ._:..
.. .R
5
6
67 A
0.2 s
I .#,:
E
30 IO 0 40 20 MINUTES Fig. 3. Gas chromatogram of methylated urine extract of a S. foetido oil fed rat. Names of lettered peaks are as in Fig. 1. The numbered peaks are: 1. methyl lactate; 2, dimethyl oxalate: 3, dimethyl malonate: 4. methyl benzoate; 5. dimethyl succinate; 6. methyl phenyl acetate; I, dimethyl-g-methyl glutarate(?); 8, dimethyl adipate: 9, dimethyl pimelate; 10. p-cresol: 11, dimethyl octadecen(?)ioate. 70
60
50
foetida oil fed rat. The lettered peaks A-J correspond to the same sterculic acid metabolites as shown in Fig. 1. Compounds D and E, cis- and truns-methyl3,4-methylene pimelate, could not be detected by mass spectrometry. The dashed line indicates where they would be on the chromatogram. The peaks numbered l-11 were also identified by mass spectrometry [203 infrared, and gas liquid chroma~~aphic retention time (Fig. 3). Compound 6, methyl phenyl acetate, which was the largest in amount, is formed by the normal metabolism of phenylalanine to phenylpyruvic by transamination, and further decarboxylation to phenyl acetic acid [21]. The compound giving the other large peak, No. 10, pcresol, is formed in the gut by bacterial action on tyrosine, followed by absorption into the intestinal tract and then excretion through the urine [22J. The other, lesser, compounds are found in various amounts in the urine of most mammals [ 23-251. Discussion
~x~atiu~ mech~n~ms in [9,1 ~-meth~~e~e-i4C]ste~cu~icacid rneta~o~~rn Formation of the metabolites would require a combination of (Y-, &, and o oxidative mechanisms. P-Oxidation occurs in the mitochondria [26] and CY-and o-oxidation takes place in the microsomes [27]. Normal straightchain fatty acids undergo p-oxidation; however, it has been shown that in certain cases straight chain as well as branched chain fatty acids do undergo woxidation 1281. Bergstrom et al. [29] studied the metabolism of 2,2~l-14C~dimethyls~~c acid in the rat. They found very little carbon dioxide and recovered 90% of the label in the urine as 2,2[1-14C]dimethyladipic acid. In another study on (Ysubstituted alkyl myristates and stearates in dogs, Weitzel [ 30 ] observed that when the side chain was greater than an ethyl group large amounts of the corresponding ~-substituted adipic acid was excreted in the urine. The metabolism of [9,10-me thyleneJ4C] sterculic acid @educes cis- and truns-3,4-methylene adipic and cis- and tram+3,4-methylene suberic acid as the main products. Therefore, sterculic acid metabolism seems to be a special case of branched chain fatty acid metabolism with the 9,10-methylene of the ring acting as a branch.
84
Another alternative pathway, a-oxidation, was demonstrated by Stokke et al. [31] to be prominent in certain types of branched chain fatty acid metabolism. After the ingestion of 3,6-dimethy1[8-‘4C]octanoic acid by man, labelled carbon dioxide was expired and 2,5~imethylhep~noic acid was found in the urine. They suggested that this pathway would account for about l-2% of all fatty acid oxidation. The total amount of cis- and trans-3,4-methylene pimelic acid, the products of a- p- and w-oxidation, isolated in the urine from corn oil and S. foet~da oil fed rats injected with labelled sterculic acid, was approx. 4% and 10% respectively (Table II). However, the S. foetida oil fed rat that was not given label (Fig. 3) did not form these two products indicative of cu-oxidation. Thus e-oxidation seems to occur only when an animal is given a large single dose of oil. During moderate, regular exposure to S. foetida oil and/or corn oil, this pathway was less evident. Proposed metabolic puth~ay for sterculic acid Yoss [32] observed in a time distribution study of [9,10-methyZene-‘4C]sterculic acid in intragastrically injected rats that during the first hour the 12 000 X g mitochondrial fraction contained more label than the 105 000 X g microsamal fraction. From 2-4 h the activity was higher in the microsomes. After 4 h the microsomal counts dropped below the mitochondrial and remained there for the rest of the 26-h study period. Based on those observations, the urinary metabolites identified, and the fact the ring was hydrogenated, the following pathway (Fig. 4) is proposed for stercuhc acid. Sterculic acid probably is activated to a CoA ester in the soluble cytosol and subsequently transported to the mitochondria where it is P-oxidized to within two carbons of the ring. Then transport to the extramitochondrial cytoplasm (microsomes) leads to reduction of the eyclopropene ring to a cyclopropane ring plus w-oxidation of the methyl group to an acid. It is probably during reduction that the two isomers, cis and trans, are formed. Evidence is lacking as to the order in which reduction and w-oxidation take place and also why the trans is formed. Some of the dicarboxylic acid is further shortened one carbon by cr-oxidation. The dicarboxylic acids are then transported back to the mitochondria and further @-oxidized to the corresponding urinary metabolites. Whether this is the preferred pathway has yet to be established, but to form the dicarboxylic acids and to reduce the ring, sterculic acid must be transported through both oxidative systems. Of particular interest is the identification of 3,4-methylene adipic acid by Lindstedt [33] and co-workers in all 50 human urine samples they examined. Since animals have been shown to degrade cyclopropane and cyclopropene fatty acids into shorter chain cyclopropane fatty acids, this raises the possibility that the source of the human urine metabolite may be dietary cyclopropene fatty acids. Effects of sterculic acid metabolism on the rat Triearboxylic acid cycle inhibitors. The major urinary metabolite of sterculic acid is 3,4-methylene adipic acid which resembles the tricarboxylic acid cycle intermediates. These metabolites may be acting as inhibitors of the tricarboxylic acid cycle, thus causing some of the observed physiological effects of cyclo-
85
STERCULIC
ACID
HH Ctiy(CH&
_2L
1
tCH,),-COOH MITOCHONDRIA HH
URINE
w-OXIDATiON
MICROSOMES Fig. 4. Proposed pathway for sterculic acid metabolism in the rat.
propenoid fatty acids [4,34]. Also, Pettersen et al. [35] demonstrated that dicarboxylic acids compete with monocarboxylic acids for the same ATP-aetivation enzymes that form CoA derivatives, and consequently, may inhibit the over-all lipid energy balance of the organism. Renal tubular degeneration. Weitzel et -al. 1361, when feeding &substituted short chain dicarboxylic acids to dogs, observed the following “tolerances” (g/ kg body wt. per day) for the following acid derivatives: n-adipic acid, (6.27); P-methyl adipic acid, (0.16); and p-ethyl adipic acid, (0.15). The animals could “tolerate” higher levels of straight chain dicarboxylic acids. Also, Rose [37]
86
tested many dicarboxylic short chain fatty acids on rabbits. He noticed a failure in renal function, marked retention of nitrogenous waste products, and nephrosis, which is the degeneration of the renal tubules without inflammation. Thus, the evidence suggests that the excessive amounts of branched chain dicarboxylic acids, such as the sterculic acid metabolites, can cause physiological changes in animal species. A rough calculation of the amount of 3,4-methylene adipic acid excreted from a rat fed the chylopropenoid fatty acid diet in this paper amounts to approximately 0.15 g/kg body wt. per day. Nixon et al. [4] reported that focal degeneration of the kidney tubules was common in cyclopropenoid fatty acid fed rats; hence cyclopropenoid metabolites may cause renal damage. Acknowledgements We wish vestigation University, Division of
to thank Kath Eisele for was supported in part by and Public Health Service the Environmental Health
the excellent technical drawings. This inthe General Research Fund, Oregon State Grants ES 00550 and ES 00263 from the Service.
References 1
Phelps.
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Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 488 (1977) Biomedical Press
76-67
BBA 57023
RAT URINARY ACID *
METABOLITES
OF [ 9,10-METHYLENE-‘4C]STERCULIC
T.A. EISELE, J.K. YOSS, J.E. NIXON, R.O. SINNHUBER **
N.E. PAWLOWSKI,
Department of Food Science and Technology, 97331 (U.S.A.) (Received
February
L.M. LIBBEY
Oregon State
University,
and
Corvallis,
Oreg.
7th, 1977)
Summary 1. The metabolism of [9,10-methyZene-‘4C] sterculic acid was studied in corn oil and Stercula foetida oil fed rats. The majority of the radioactivity was excreted into the urine as short chain dicarboxylic acids. The main urinary metabolites were cis-3,4-methylene adipic acid, cis-3,4-methylene suberic acid, trans-3,4-methylene adipic acid, cis-3,4-methylene pimelic acid, and cis-3,4methylene azelic acid. 2. Formation of these urinary metabolites requires a-, p-, and w-oxidation plus reduction of the cyclopropene ring to a cyclopropane ring. Sterculic acid must be transported through both mitochondrial and microsomal systems. 3. Other non-radioactive urinary compounds were also identified. A proposed pathway for the metabolism of sterculic acid and possible detrimental effects caused by these metabolites is discussed.
Introduction Sterculic acid is a naturally occurring cyclopropenoid fatty acid containing a highly strained and reactive unsaturated three-membered ring in the center of an l&carbon chain. The main food sources of cyclopropenoid fatty acids are the cotton plant (Gossypium hirsutum) in the United States and kapok (Ericlench-an anfractuosum) in the oriental countries. These cyclopropenoid fatty acids have been held responsible for numerous physiological disorders in many animals. A review by Phelps et al. [l] reported ____
*
Technical
Paper
the
research
thesis
Master **
of Science
No.
4451.
for
T.A.
requests
to:
Send
reprint
State
University,
Oregon
conducted
Corvallis,
Eisele R.O. Oreg.
at
Agricultural Oregon
and
State
Doctor
Sinnhuber, 97331.
Experiment
Station.
University
in partial
of Philosophy Department
U.S.A.
for J.K. of
Food
This
paper
fulfillment
represents for
the
part degree
of of
Yoss.
Science
and
Technology,
Oregon
77
that dietary cyclopropenoid fatty acids impaired reproduction in rats and chickens, delayed sexual maturity in females of these species and caused pink discoloration in avian egg whites during storage. Other recent work with cyclopropenoid fatty acid fed rats showed fatty infiltration and degeneration of the liver along with renal tubule degeneration [Z], altered lipid metabolism [ 31, and partial loss of membrane associated functions [4]. Sinnhuber and coworkers [ 51 discovered that sterculic acid is a liver carcinogen when fed to rainbow trout. Scarpelli et al. [6] reported that hepatoeytes from sterculic acid fed rainbow trout and rats showed induced cytoplasmic alterations. Information on the metabolism of sterculic acid would help explain its mode of action and the physiological disorders which occur. However, limited information has been published. Altenberger and co-workers [7] fed [methylene-14C]sterculic acid to fasting hens and found limited amounts of labelled carbon dioxide formed over a 24-h period. They also found more than 50% of the label in the fecal material. Yoss [B] did a time flow study on the distribution of [ “C]sterculic acid in rats. He concluded from the lack of 14C-labelled CO, that the rat did not metabolize the cyclopropene ring. The intent of this investigation was to identify the labelled sterculic acid metabolites in rat urine. Corn oil fed rats were compared to Stercutiu foetida oil fed rats to determine whether acclimation to cyclopropenoid fatty acids would produce different metabolites. Methods
and Materials
E~~erimen tal un~rn~~sand diet Four weanling Wistar male rats were fed ad libitum a Krishnaro and Draper diet [9] modified to contain 20.0% protein and 32.2% cornstarch. The diet consisted of 92.8% premix, 2.2% vitamin mix (U.S. Biochemicals), and 5.0% corn oil mixed 1 : 1 (w/v) with 3.0% agar (DIFCO) dissolved in distilled water at approximately 80°C. The hot mix was solidified at 2-5°C to a solid gel, cut into small blocks, and stored in plastic bags at -30°C until used. After feeding the 5.0% corn oil diet for two weeks, two of the four rats were transferred to a 4.0% corn oil plus 1.0% S. foetida oil diet. The cyclopropene content of the S. foetida oil diet was 0.5% of the total dry weight, or 10.0% based on total lipid composition. Rats were fed the S. foetida oil diet for at least 60 days to develop an active pathway for metabolizing cyclopropene fatty acids. Urinary metabolites from S. foetid~ oil fed rats, corn oil fed rats given labelled [9,10-methyZene-‘4C]sterculic acid, and S. foetida oil fed rats given labelled {9,10-methyZene-‘4 C] sterculic acid were compared. Method of administering and source of kabelled sterculic acid Methyl sterculate, labelled on the 9,10-methylene bridge of the cyclopropen~ ring, was synthesized by Pawlowski [lo]. The Halphen test [ll] and NMR (121 indicated 99.0 ?r 0.2% and 103.0 of:2.0% cyclupropene respectively. The free acid was prepared by saponification with 0.5 N 95% alcoholic KOH at 45-50°C for 1.5 h. Pure sterculic acid, 110 pCi/mol, was diluted to 5.0 pCi/ 0.41 g corn oil and administered intragastrically. Immediately after injection,
78
animals were placed in metabolism chambers. Urine was allowed to flow into glass tubes packed in ice. Urine extruction
The urine was diluted with an equal volume of distilled water, adjusted to pH 1.0-2.0 with HCl, and extracted twice with four volumes of ethyl ether. If necessary, combined extracts were centrifuged at 2000 rev./min to break up emulsions. The ethyl ether extract was then washed with one volume distilled water and evaporated to dryness on a vacuum rotary evaporator. The free acids were esterified with diazomethane [ 13 1. Gas chromatography Preparative gas liquid chromatography was carried out on a Varian Model 1400 flame ionization unit. The column, 14 ft X 0.085 inches internal diameter aluminum tubing packed with 10% SP-222-PS 01-1885 llOf120 mesh (Supleco, Inc., Bellefonte, Pa.), was temperature programmed at l*C/min from 120-18O’C and then held isothermally at 180°C until the end of the run. The injector temperature was 200°C and that of the detector was 220°C. Nitrogen at a flow rate of 20ml/min was used as the carrier gas. The detector was equipped with a 5 : 1 (trap : detector) split for trapping purposes. The same gas liquid chromatography conditions were used in combination with the mass spectrometer. Isolation of metabolites from urine by gas chromatography trapping Glass capillary tubes packed in solid CO* (of a size slightly larger than the internal diameter of the splitter, to prevent back pressure) were attached to the splitter with Teflon tubing. A heat gun was used to keep the sample from condensing near the splitter-capillary interface and to drive the condensed material to the cold section of the capillary tubing. All peaks and the base line area between the peaks were trapped separately. The capillary tubes were sealed at the ends with a microflame and stored in the freezer until analyzed. To determine which peaks were radioactive, the capillary tubes were washed with 1.0 ml of PPO-POPOP-Toluene (PPO (2,5diphenyloxazole) Sigma Chem. Co., POPOP (1,4-bis(2,5-phenyloxazole)benzene), Nuclear Chicago) into vials to be counted. Radioactive peaks and the more abundant non-radioactive peaks from several runs were combined to obtain sufficient amounts of each compound for identification. Spectroscopy equipment Infrared spectra were obtained on a Beckman Model IR-18A using a beam condenser. A micro salt cell with path length of 0.1 mm or a salt plate was used to hold the sample. Spectra were run in carbon tetrachloride or carbon disulfide. NMR spectra were run on a Varian HA-100 with a time-averaging computer, using carbon tetrachloride as solvent and benzene as a lock signal. Mass spectra were obtained with a Finnigan Model 1015C quadrupole mass spectrometer interfaced to a Varian Model 1400 gas chromatograph oven with a Gholke glass jet molecular separator. The ion source pressure was 10e6 mmHg;
79
ionizing current 400 PIA; ionizing potential 70 eV; and manifold temperature 150°C. Mass spectral data was acquired and processed by a System Industries System 250 data system. Results Identification of [9,1 0-methylene-‘4C]sterculic acid urinary metabolites in S. foetida oil fed rats A typical gas chromatogram of methylated urine extract from a S. foetida oil fed rat given [9,10-methylene- *4C]sterculic acid is illustrated in Fig. 1. Peaks labelled A-J contained radioactivity. The scale on the left of the gas chromatogram indicates counts per min representative of each peak. Peak C contained the largest amount of carbon-14 label followed by peak G. As shown in Table I, mass spectra were obtained on each peak, followed by NMR and infrared when sufficient material was available. The infrared spectrum of peak C of Fig. 1 shows an intense carbonyl absorption at 1740 cm-‘, indicative of an aliphatic ester [14] ; strong 2860 cm-‘, due to carbon-hydrogen methyl stretching; strong 2930 cm-’ of methylene carbonmethylene bendhydrogen stretching; moderate 1465 cm -’ of carbon-hydrogen ing; moderate 1440 cm-’ of methylene cyclopropane bending; moderate-toweak 1260 cm-‘, 1195 cm-‘, and 1175 cm-’ for carbon-oxygen stretching indicative of a methyl ester; and a medium 1020 cm-’ absorption from skeletal vibration of the cyclopropane ring [15,16]. All of the infrared spectra (Table I) for each of the labelled peaks exhibited these absorptions varying somewhat in intensity . The mass spectra (Table I) of these labelled metabolites show fragments characteristic of methyl esters of dibasic acids. Ryhage and Stenhagen [17] observed that the most intense peak in the high mass range was m/e (P-31) or m/e (P-32), that m/e (P-64) was also very intense, and that the parent ion (P) was very small or nonexistent. Thus spectrum C exhibits P-32 (m/e = 154), P-64 (122), and also P-59 (127), and P-73 (113). Compound C has no parent ion present, but based on the above data, the parent would be at m/e = 186. The NMR spectrum of compound C (Table I) shows four different types of protons: methoxyl of an ester, methylene adjacent to a carbonyl, methine of I
0.3
2.000
5
% 0.6 2
I :
it
fi 0.4 0
1,000
6 0.2 s LT
0 70
60
50
40
30
20
IO
0
MINUTES
Fig. 1. Gas chromatogram of methylated urine extract of a S. foefida oil fed rat injected with [9,10methylene-14C1stemlic acid. Reading right to left: A. unknown; B. fmwmethyl-3.4~methylene adfpate: C. cis-methyl-3,4-methylene adipate; D. trans-methyl-3.4-methylene pimelate; E. cis-methyl-3.4-methylene pimelate; F, trans-methyl-3,4-methylene suberate; G. cis-methyl-3.4-methylene suberate; H, cismethyl-3,4-methylene azelate; I, cis-methyl-3.4-methylene sebacate; J. unknown.
80 TABLE I NUCLEAR MAGNETIC RESONANCE, 14C1 STERCULIC ACID METHYLATED Peak
INFRARED, AND MASS SPECTRA RAT URINE METABOLITES
OF [9,10-METHYLENE-
Compound structure and name
Spectral data
Unknown
MS *: 27(7), 29(7). 41(20), 43(100), 55(12). 59(g), 69(25). 72(2l), 87(2l), 95(9), 98(6). 123(2). 129(l), 140(l), 172(0.2), [M = 1721.
trans-Methyl-3,4-methylene 0
H
adipate
H
II
CH30C-CH2
H if H
CH,-COCH,
cis-Methyl-3.4-methylene
adipate
NMR: (CC14) 6.40@,6H.OCH3),
7.74(d,4H,J = 6, O=CCH2).8.72 (m,2H,methine cyclopropane), 9.08 (m,lH,cis-methylenecyclopropane).9.94 (m,lH. J = 5,cismethylene cyclopropane). IR: (liq. film)2930(s),2860(m),1740(s),1465(m), 1440(m),1260(w),1195(m),ll75(m), 1020(m). MS: 27(38).29(23),39(67),41(85), 55(51),59(93), 67(98), 71(67),84(76).85(100),95(27),99(23). 100(18),113(53),122(56).127(47),137(4),154(10), 155(7),171(2).[M = 1861.
CH, -COCH3
trans-Methyl-3,4-methylene
NMR **: (CC14) 6.40(s,6H,OCH3), 7.74(d,4H. J = 6. O=CCHz), 8.72 (m, 2H. methine cyclopropane), 9.49(t, 2H, J = 5, cis-methylene cyclopropane). IR ***: (CC14) 2930(m), 2860(m), 1740(s), 1465(w), 1440(m), 1260(m). 1195(m), 1175(m). 1020(m). MS: 27(22). 29(14), 39(40). 41(69), 55(28), 59(100), 67(83), 71(57). 84(53), 85(60), 95(17), 99(22), 112(g). 113(43), 122(28), 127(38), 154(6), 155(2), CM = 1861.
pimelate
MS: 27(33),29(19),39(36).41(39),55(100). 59(49), 67(32),74(16),80(32),81(70).94(17).95(15). 108(40).109(24),125(3),127(3),136(59), 140(26),153(1),168(2),169(5). [M = 2001.
3
cis-Methyl-3.4-methylene
’ CH30C-CH2
pimelate
.-&H+iOCH
?
3
?
trans-Methyl-3.4-methylene
H
suberate
0 3
cis-Methyl-3,4-methylene
suberate
IR: (CS2) 2930(s),2860(m),1740(s).1260(m). 1195(m),1175(s).1020(w). MS: 27(39),29(24).39(44),41(61). 55(100). 59(79), 67(49),71(20),80(30).81(75),94(17),95(14). 108(35),109(29),126(6),127(10),136(70).140(31), 141(10),168(1),169(1), [M = 2001. IR:(CS~)1740(m).1260(w).ll95(w),ll75(w). MS: 27(31),29(20),39(47),41(99), 55(88).59(100), 67(61).74(48).80(57),81(88),94(22).95(28), 109(21).114(11),122(28).123(29).140(17), 141(12).150(60).151(27),128(2),183(1), [M = 2141. NMR: (CC14) 6.40 (s,GH,OCH3), 7.70 (t,4H,J= 6, 0=CCH2), 8.3 (m,2H, J = 6, CHz), 8.5-8.7 (m, 4H,CHz plus methine cyclopropane), 9.07 (m, lH, cis-methylene cyclopropane), 10.06 (m. lH J = 5,ciwnethylene cyclopropane).
H
H
IR:(CS~)2930(m).2860(w).1740(s),126O(w). 1195(w).1175(m), 1020(w). MS: 27(33),29(30).41(86),43(52). 55(82).59(100), 67(57).74(40),80(57),81(87),94(22). 95(29). 109(20).114(12),122(30),123(32), 140(16). 141(12),150(62),151(23) 161(2),171(l). 182(2). 183(2),197(l), [M= 2141.
81 TABLE I (continued) NUCLEAR
MAGNETIC
14Cl STERCULIC
RESONANCE,
ACID METHYLATED
INFRARED,
AND
MASS SPECTRA
OF [9,10-METHYLENE-
RAT URINE METABOLITES
Peak
Compound structure and name
Spectral data
H
cis-Methyl-3.4~methylene
IR: (CS2) 1740(w). MS: 27(18), 39(42), 41(88), 55(100). 59(87), 67(58), 74(87), 79(29). 81(50), 94(34), 96(47), 108(11), 109(12), 119(31), 123(23). 136(23), 137(21), 154(8), 155(7), 164(26). 165(10),178(l), 196(2), IN = 2281.
f;
HH
aselate
(u-i,)4-
CH3OC-CH2
k,
3
l?H
H
cis-Methyl-3,4-methylene
I
CH 3.i-CH
2
H
H
-?r H
H
sebacate
KH,,,&H
3
MS: 27(28), 29(34), 41(97), 43(46), 55(98), 59(100), 67(70), 74(65), 81(58), 82(38f, 93(29), 95(45), 109(25). llO(24). 119(B), 122(8), 133(22), 137(19), 150(15), 151(12). 160(4), 168(3), 178(10). 179(6), 210(2), 211(l). [M = 2421.
MS, mass spectrometry: m/e (% base peak); IM = molecular ion]. Spectra were abbreviated by Iisting the two largest ions every 14 a.m.u. beginning at m/e = 20. Complete spectra can be obtained by writing to principal author. ** NMR: (solvent) ?‘ (splittii pattern, number protons, functionaf. group): s = singlet, d = doublet, f = triplet, m = muhtiplet. *** IR. infrared: (solvent) principtibands in cm-*: s = strong, m = medium, w = weak. *
a cyclopropane, and methylene of a cyclopropane. A large singlet at 6.40 r represents the methoxyl groups of an ester f143, and six of the total 14 protons present. The diesters’ four a-methylene protons appear as a doublet at 7.74 T. The remaining four protons show a pattern expected for cis-distributed cyclopropane. The two methine protons of the small ring absorb at 8.72 T while the sypzand anti methylene protons absorb at 9.94 7 and 9.08 r [X3,19]. The data suggests a structure for C as shown in Table I. The mass spectrum of B (Table I) is identical to that of C. Compound B differs from C in the downfield shift of the methylene cyclopropane protons (9.497) in the NMR (Table I) to which Wood and Reiser 1163 assigned the TABLE II RETENTION TIME AND 46 LABEL FROM [9,10-i%fETHYLENE-14C] METABOLITES OF CORN OIL AND S. FOETIDA OIL FED RATS Compound
A B C D E
F G II I J
S. foetido
oil fed rat
STERCULIC
ACID
URINARY
Corn oil fed rat
Rt *
%cpm
Rt *
% cpm
1.30 1.55 1.80 1.86 2.04 2,09 2.35 2.64 2.98 3.21
1.42 7.37 47.51 1.42 8.75 2.50 18.06 5.89 5.36 1.73
1.31 1.59 1.85 1.91 2.09 2.16 2.44 2.81 3.20 3.53
1.61 5.83 46.79 3.37 *+ 2.32 9.57 11.88 3.35 1.11
* Relative retention time was crdculated using methyl phenyl acetate as a reference, peak 6 (Fig. 3). * * Compound E radioactivity is summed with compound F.
82
trans configuration. A suggested structure for B is shown in Table I. Thus, B and C differ only in their orientation around the cyclopropane ring. B, being the trans compound and more of a “straight” molecule, would be eluted from a gas liquid chromatography column before C, as shown in Fig. 1. Using the same techniques, a structure was assigned to all of the labelled peaks except A and J. The amount of material was much too small to obtain any data except for a mass spectrum on A, and this did not show any fragments analogous to the other spectra. The spectra, suggested structure, and names of each metabolite are shown in Table I. Identification oil fed rats
of [9,1 O-methylene-
‘%‘]sterculic
acid urinary metabolites
in corn
The purpose of this section was to determine if corn oil fed rats given steracid produced the same urinary metabolites as S. foetida oil fed rats. A typical gas chromatogram of methylated corn oil fed, sterculic injected, rat urine is illustrated in Fig. 2. Peaks that contained radioactivity were analyzed by mass spectrometry and found to have identical mass spectra as the corresponding peaks of S. foetida oil fed rats. Table II shows the relative retention times of each peak from both groups of animals, and the total percentage of activity for each labelled peak. Peak E in the corn oil fed rat could not be detected due to its close proximity to peak F. The small dashed line in the gas chromatogram (Fig. 2) shows where it would be, The data indicates that corn oil fed rats can metabolize cyclopropenoid fatty acids the same as S. foetida oil fed rats even though they had not been acclimated to the cyclopropene diet. The main metabolites for both groups were: cis-methyl-3,4-methylene adipate, compound C (46-47%); and cis-methyl-3,4methylene suberate, compound G (g-18%). The gas chromatographic elution pattern of Fig. 2 differs from that of Fig. 1 because the relative amount of label and metabolites in the corn oil fed rat urine (20%) was lower than the amount of label and material in the S. foetida oil fed rat urine (51%). cubic
Identification
of urinary metabolites
in S. foetida
oil fed rats
The purpose of this experiment was to determine whether S. foetida oil fed rats excreted the same urinary metabolites as S. foetida oil fed rats injected with a large single dose of labelled sterculic acid. Fig. 3 shows a typical gas chromatogram of methylated rat urine from a S.
MINUTES Fig. 2. Gas chromatogram of methylated urine extract of a corn oil fed rat injected with [9.10-mefhyb eru~-~~C] stenxlic acid. Names of lettered peaks are as Fig. 1.
83
IO
4
I
6
C
”
%
0.8 5 . % -0.6 ki -
1
A
- 0.4 2 G
.:,::c_il~~~,~~~
I
H
s F II ._:..
.. .R
5
6
67 A
0.2 s
I .#,:
E
30 IO 0 40 20 MINUTES Fig. 3. Gas chromatogram of methylated urine extract of a S. foetido oil fed rat. Names of lettered peaks are as in Fig. 1. The numbered peaks are: 1. methyl lactate; 2, dimethyl oxalate: 3, dimethyl malonate: 4. methyl benzoate; 5. dimethyl succinate; 6. methyl phenyl acetate; I, dimethyl-g-methyl glutarate(?); 8, dimethyl adipate: 9, dimethyl pimelate; 10. p-cresol: 11, dimethyl octadecen(?)ioate. 70
60
50
foetida oil fed rat. The lettered peaks A-J correspond to the same sterculic acid metabolites as shown in Fig. 1. Compounds D and E, cis- and truns-methyl3,4-methylene pimelate, could not be detected by mass spectrometry. The dashed line indicates where they would be on the chromatogram. The peaks numbered l-11 were also identified by mass spectrometry [203 infrared, and gas liquid chroma~~aphic retention time (Fig. 3). Compound 6, methyl phenyl acetate, which was the largest in amount, is formed by the normal metabolism of phenylalanine to phenylpyruvic by transamination, and further decarboxylation to phenyl acetic acid [21]. The compound giving the other large peak, No. 10, pcresol, is formed in the gut by bacterial action on tyrosine, followed by absorption into the intestinal tract and then excretion through the urine [22J. The other, lesser, compounds are found in various amounts in the urine of most mammals [ 23-251. Discussion
~x~atiu~ mech~n~ms in [9,1 ~-meth~~e~e-i4C]ste~cu~icacid rneta~o~~rn Formation of the metabolites would require a combination of (Y-, &, and o oxidative mechanisms. P-Oxidation occurs in the mitochondria [26] and CY-and o-oxidation takes place in the microsomes [27]. Normal straightchain fatty acids undergo p-oxidation; however, it has been shown that in certain cases straight chain as well as branched chain fatty acids do undergo woxidation 1281. Bergstrom et al. [29] studied the metabolism of 2,2~l-14C~dimethyls~~c acid in the rat. They found very little carbon dioxide and recovered 90% of the label in the urine as 2,2[1-14C]dimethyladipic acid. In another study on (Ysubstituted alkyl myristates and stearates in dogs, Weitzel [ 30 ] observed that when the side chain was greater than an ethyl group large amounts of the corresponding ~-substituted adipic acid was excreted in the urine. The metabolism of [9,10-me thyleneJ4C] sterculic acid @educes cis- and truns-3,4-methylene adipic and cis- and tram+3,4-methylene suberic acid as the main products. Therefore, sterculic acid metabolism seems to be a special case of branched chain fatty acid metabolism with the 9,10-methylene of the ring acting as a branch.
84
Another alternative pathway, a-oxidation, was demonstrated by Stokke et al. [31] to be prominent in certain types of branched chain fatty acid metabolism. After the ingestion of 3,6-dimethy1[8-‘4C]octanoic acid by man, labelled carbon dioxide was expired and 2,5~imethylhep~noic acid was found in the urine. They suggested that this pathway would account for about l-2% of all fatty acid oxidation. The total amount of cis- and trans-3,4-methylene pimelic acid, the products of a- p- and w-oxidation, isolated in the urine from corn oil and S. foet~da oil fed rats injected with labelled sterculic acid, was approx. 4% and 10% respectively (Table II). However, the S. foetida oil fed rat that was not given label (Fig. 3) did not form these two products indicative of cu-oxidation. Thus e-oxidation seems to occur only when an animal is given a large single dose of oil. During moderate, regular exposure to S. foetida oil and/or corn oil, this pathway was less evident. Proposed metabolic puth~ay for sterculic acid Yoss [32] observed in a time distribution study of [9,10-methyZene-‘4C]sterculic acid in intragastrically injected rats that during the first hour the 12 000 X g mitochondrial fraction contained more label than the 105 000 X g microsamal fraction. From 2-4 h the activity was higher in the microsomes. After 4 h the microsomal counts dropped below the mitochondrial and remained there for the rest of the 26-h study period. Based on those observations, the urinary metabolites identified, and the fact the ring was hydrogenated, the following pathway (Fig. 4) is proposed for stercuhc acid. Sterculic acid probably is activated to a CoA ester in the soluble cytosol and subsequently transported to the mitochondria where it is P-oxidized to within two carbons of the ring. Then transport to the extramitochondrial cytoplasm (microsomes) leads to reduction of the eyclopropene ring to a cyclopropane ring plus w-oxidation of the methyl group to an acid. It is probably during reduction that the two isomers, cis and trans, are formed. Evidence is lacking as to the order in which reduction and w-oxidation take place and also why the trans is formed. Some of the dicarboxylic acid is further shortened one carbon by cr-oxidation. The dicarboxylic acids are then transported back to the mitochondria and further @-oxidized to the corresponding urinary metabolites. Whether this is the preferred pathway has yet to be established, but to form the dicarboxylic acids and to reduce the ring, sterculic acid must be transported through both oxidative systems. Of particular interest is the identification of 3,4-methylene adipic acid by Lindstedt [33] and co-workers in all 50 human urine samples they examined. Since animals have been shown to degrade cyclopropane and cyclopropene fatty acids into shorter chain cyclopropane fatty acids, this raises the possibility that the source of the human urine metabolite may be dietary cyclopropene fatty acids. Effects of sterculic acid metabolism on the rat Triearboxylic acid cycle inhibitors. The major urinary metabolite of sterculic acid is 3,4-methylene adipic acid which resembles the tricarboxylic acid cycle intermediates. These metabolites may be acting as inhibitors of the tricarboxylic acid cycle, thus causing some of the observed physiological effects of cyclo-
85
STERCULIC
ACID
HH Ctiy(CH&
_2L
1
tCH,),-COOH MITOCHONDRIA HH
URINE
w-OXIDATiON
MICROSOMES Fig. 4. Proposed pathway for sterculic acid metabolism in the rat.
propenoid fatty acids [4,34]. Also, Pettersen et al. [35] demonstrated that dicarboxylic acids compete with monocarboxylic acids for the same ATP-aetivation enzymes that form CoA derivatives, and consequently, may inhibit the over-all lipid energy balance of the organism. Renal tubular degeneration. Weitzel et -al. 1361, when feeding &substituted short chain dicarboxylic acids to dogs, observed the following “tolerances” (g/ kg body wt. per day) for the following acid derivatives: n-adipic acid, (6.27); P-methyl adipic acid, (0.16); and p-ethyl adipic acid, (0.15). The animals could “tolerate” higher levels of straight chain dicarboxylic acids. Also, Rose [37]
86
tested many dicarboxylic short chain fatty acids on rabbits. He noticed a failure in renal function, marked retention of nitrogenous waste products, and nephrosis, which is the degeneration of the renal tubules without inflammation. Thus, the evidence suggests that the excessive amounts of branched chain dicarboxylic acids, such as the sterculic acid metabolites, can cause physiological changes in animal species. A rough calculation of the amount of 3,4-methylene adipic acid excreted from a rat fed the chylopropenoid fatty acid diet in this paper amounts to approximately 0.15 g/kg body wt. per day. Nixon et al. [4] reported that focal degeneration of the kidney tubules was common in cyclopropenoid fatty acid fed rats; hence cyclopropenoid metabolites may cause renal damage. Acknowledgements We wish vestigation University, Division of
to thank Kath Eisele for was supported in part by and Public Health Service the Environmental Health
the excellent technical drawings. This inthe General Research Fund, Oregon State Grants ES 00550 and ES 00263 from the Service.
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