Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs.

Hearts from 4 week-old weanling pigs were capable of continuous work output when perfused with Krebs-Henseleit buffer containing 11 mM glucose. Perfused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of glucose plus physiological concentrations (0.1 mM) of either palmitate or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and carnitine. When hearts were perfused with 1 mM fatty acids, the work output declined below control values. Erucic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by perfused rat hearts. In addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated perfused pig hearts used in the present study, erucic acid oxidation rates were as high as palmitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during erucic acid perfusion did not change noticeably from those during glucose perfusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to erucic acid are not replicated in the intact pig heart.

Identification of a new sphingolipid 3-O-acyl-D-erythro-sphingomyelin in newborn pig and infant plasma.

A new sphingolipid was found in newborn pig plasma at a level of 2.5 +/- 0.4% of total lipids. The compound decreased to less than half that amount by day one of age and virtually disappeared by the fourth week. On thin-layer chromatography (TLC) the new lipid migrated close to phosphatidylethanolamine. The compound was isolated by TLC from the plasma of newborn piglets and identified as a 3-O-acyl-D-erythro-sphingomyelin by chemical and chromatographic techniques, 1H- and 13C-nuclear magnetic resonance and fast-atom bombardment mass spectrometry. Mild alkaline hydrolysis at room temperature gave mainly C16 and C18 fatty acids and sphingomyelin. Subsequent reaction with Ba(OH)2 released long-chain saturated and monounsaturated fatty acids from C14 to C24, and sphingosine which was identified as the erythro configuration by gas chromatography. Less than 1% of the sphingosine was of the C20 isomer. No hydroxy fatty acids were found. The acylated sphingomyelin was only found in plasma lipids of newborn piglets and not in their red blood cell membranes or platelets of newborn piglets, or in sow plasma. This compound was tentatively identified by chromatography in trace amounts in the serum of cord blood of newborn infants, but not in the plasma lipids of adults.

Failure of dietary erucic acid to impair oxidative capacity or APT production of rat heart mitochondria isolated under controlled conditions.

1. Male, 8-week old rats were fed Purina Rat Chow for semisynthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days. 2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglyerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed. 3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rats of oxidation of different substrates and of ATP synthesis by these mitochondria were compared. 4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets. 5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals. 6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.

The separation of pyruvate-ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions.

The ferredoxin requiring cleavage of pyruvate to acetyl-CoA and CO2 is catalyzed by pyruvate ferredoxin oxidoreductase (pyruvate:ferredoxin oxidoreductase (CoA-acetylating):, EC 1.2.7.1). The same enzyme is thought to catalyze the reversal of this reaction, i.e. the synthesis of pyruvate from acetyl-CoA and CO2 in the presence of reduced ferredoxin. Evidence is presented that the forward and reverse reactions are catalyzed not by one, but by two proteins that are clearly separable by Sephadex G-200 gel filtration.

Brassica compestris var. Span: I. Fractionation of rapeseed oil by molecular distillation and adsorption chromatography.

Procedures for the large scale isolation of pure triglycerides and fractions rich in nontriglyceride components from Span rapeseed oil are described. Fractionation of Brassica campestris var. Span rapeseed oil by molecular distillation yielded 4 triglyceride fractions, all of which contained traces of sterol esters. An additional triglyceride fraction rich in free and esterified sterols and other volatile components was obtained from the oil. Separation by adsorption chromatography of Span rapeseed oil yielded three fractions; A) a pure triglyceride ffaction; B) a triglyceride fraction rich in sterol esters; and C) another fraction containing free sterols and other polar components.

Brassica campestris var. Span: II. Cardiopathogenicity of fractions isolated from span rapeseed oil when fed to male rats.

Rapeseed oils low in erucic acid caused myocardial lesions when fed to weanling male rats for 16 weeks. The cardiopathogenic properties appear to be associated with the triglycerides of the oil, and not to nontriglyceride components present in fully refined rapeseed oil. Cardiac lipid analysis confirmed that erucic acid accumulation was proportional to the concentration of this acid in the diet.

Relationship between erucic acid and myocardial changes in male rats.

The back and belly fat of pigs fed a diet containing 20% by wt rapeseed oil (22% erucic acid) for 16 weeks was rendered into oil. This rendered pig fat, which contained 5.6% erucic acid, was fed to male rats in three separate experiments at 20% by wt of the diet for 16 weeks. In experiment I rendered pig fat was compared only to Brassica campestris var. Span rapeseed oil containing 4.8% erucic acid. In experiments II and III, rendered pig fat was compared to commercial lard containing 0.2% docosenoic acid, commercial lard to which 5.4% free erucic acid was added, and Span rapeseed oil. There was no significant (P less than 0.01) differences observed in the level of erucic acid in the hearts of rats fed diets of rendered pig fat, Span rapeseed oil, or commercial lard plus erucic acid. However, the incidence (P less than 0.001) and severity (P less than 0.01) of cardiac lesions were significantly higher in Span rapeseed oil fed rats compared to rats fed control diets. The number of rats affected or the severity of lesions in the rendered pig fat fed group was not significantly different from controls. The results of this study indicate that the myocardial lesions associated with feeding 20% rapeseed oil diets are not related to the content of erucic acid per se. The possible reasons why rapeseed oil causes cardiac lesions in rats are discussed. It is suggested that a triglyceride imbalance in the oil might play an important role in causing these lesions in rats.

Effects of dietary saturated fat on erucic acid induced myocardial lipidosis in rats.

Male Sprague-Dawley rats were fed for one week diets containing 20% by weight fat/oil mixtures with different levels of erucic acid (22:1n-9) (approximately 2.5 or 9%) and total saturated fatty acids (approximately 8 or 35%). Corn oil and high erucic acid rapeseed (HEAR) oil were fed as controls. The same hearts were evaluated histologically using oil red O staining and chemically for cardiac triacylglycerol (TAG) and 22:1n-9 content in cardiac TAG to compare the three methods for assessing lipid accumulation in rat hearts. Rats fed corn oil showed trace myocardial lipidosis by staining, and a cardiac TAG content of 3.6 mg/g wet weight in the absence of dietary 22:1n-9. An increase in dietary 22:1n-9 resulted in significantly increased myocardial lipidosis as assessed histologically and by an accumulation of 22:1n-9 in heart lipids; there was no increase in cardiac TAG except when HEAR oil was fed. An increase in saturated fatty acids showed no changes in myocardial lipid content assessed histologically, the content of cardiac TAG or the 22:1n-9 content of TAG at either 2.5 or 9% dietary 22:1n-9. The histological staining method was more significantly correlated to 22:1n-9 in cardiac TAG (r = 0.49; P less than 0.001) than to total cardiac TAG (r = 0.40; P less than 0.05). The 22:1n-9 content was highest in cardiac TAG and free fatty acids. Among the cardiac phospholipids, the highest incorporation was observed into phosphatidylserine, followed by sphingomyelin. With the addition of saturated fat, the fatty acid composition showed decreased accumulation of 22:1n-9 and increased levels of arachidonic and docosahexaenoic acids in most cardiac phospholipids, despite decreased dietary concentrations of their precursor fatty acids, linoleic and linolenic acids.

Hematological and lipid changes in newborn piglets fed milk-replacer diets containing erucic acid.

Canola oil is not presently permitted in infant formulations in the United States because of lack of information concerning the effects of feeding canola oil to the newborn. We have previously reported a transient decrease in platelet counts and an increase in platelet size in newborn piglets fed canola oil for 4 wk, and have confirmed this in the present study. In canola oil-fed piglets, changes in platelet size and number were overcome by adding either long-chain saturated fatty acids from cocoa butter (16:0 and 18:0), or shorter-chain saturates from coconut oil (12:0 and 14:0). Feeding a high erucic acid rape-seed (HEAR) oil, with 20% 22:1n-9, led to an even greater platelet reduction and increased platelet size throughout the 4-wk trial. Bleeding times were longer in piglets fed canola oil or HEAR oil compared to sow-reared and soybean oil-fed piglets. There were no other diet-related changes. Diet-induced platelet changes were not related to platelet lipid class composition, but there were fatty acid changes. The incorporation of 22:1n-9 into platelet phospholipids of piglets fed canola oil was low (0.2-1.2%), and even for the HEAR oil group ranged from only 0.2% in phosphatidylinositol to 2.4% in phosphatidylserine. A much greater change was observed in the concentration of 24:1n-9 and in the 24:1n-9/24:0 ratio in platelet sphingomyelin (SM). The 24:1n-9 increased to 49% in the HEAR oil group compared to about 12% in animals fed the control diets (sow-reared piglets and soybean oil-fed group), while the 24:1n-9/24:0 ratio increased from about 1 to 12. Even feeding canola oil, prepared to contain 2% 22:1n-9, led to a marked increase in 24:1n-9 to 29% and had a 24:1n-9/24:0 ratio of 5. The canola oil/cocoa butter group, which also contained 2% 22:1n-9, showed a lower level of 24:1n-9 (20%) and the 24:1n-9/24:0 ratio (3) compared to the canola oil group. The results suggest that the diet-related platelet changes in newborn piglets may be related to an increase in 24:1n-9 in platelet SM, resulting from chain elongation of 22:1n-9. The inclusion of canola oil as the sole source of fat in the milk-replacer diets of newborn piglets resulted in significant platelet and lipid changes.

Effect of cold stress on rapeseed oil fed rats.

No mortality was observed in 6 week old male Sprague-Dawley rats subjected to cold at 4 C for 3 weeks and fed either a control diet (Chow) or a semisynthetic diet containing 20% by wt rapeseed oil high in erucic acid (23.6%). All rats fed the Chow diet and 17 of 20 rats fed the rapeseed oil-containing diet survived 4 weeks in the same environment. Three rats on the latter diet died of self-mutilation. Marked myocardial lipidosis as well as a large acumulation of 20:1 and 22:1 was observed in the hearts of rats fed the rapeseed oil-containing diet. Five of 20 rats on the Chow diet and 2 of 20 rats on the rapeseed oil-containing diet had focal necrotic areas in the myocardium.

Hematological and lipid changes in newborn piglets fed milk replacer diets containing vegetable oils with different levels of n-3 fatty acids.

To test if linolenic acid (18:3n-3) from vegetable oils would affect bleeding times and platelet counts in newborns, piglets were used as a model fed milk replacer diets containing 25% (by wt) vegetable oils or oil mixtures for 28 d and compared to sow-reared piglets. The oils tested included soybean, canola, olive, high oleic sunflower (HOAS), a canola/coconut mixture and a mixture of oils mimicking canola in fatty acid composition. All piglets fed the milk replacer diets showed normal growth. Bleeding times increased after birth from 4-6 min to 7-10 min by week 4 (P < 0.001), and were higher in pigs fed diets containing 18:3n-3, as well as in sow-reared piglets receiving n-3 polyunsaturated fatty acids (PUFA) in the milk, as compared to diets low in 18:3n-3. Platelet numbers increased within the first week in newborn piglets from 300 to 550 x 10(9)/L, and remained high thereafter. Milk replacer diets, containing vegetable oils, generally showed a transient delay in the rise of platelet numbers, which was partially associated with an increased platelet volume. The oils showed differences in the length of delay, but by the third week of age, all platelet counts were > 500 x 10(9)/L. The delay in rise in platelet counts appeared to be related to the fatty acid composition of the oil, as the effect was reproduced by a mixture of oils with a certain fatty acid profile, and disappeared upon the addition of saturated fatty acids to the vegetable oil. There were no alterations in the coagulation factors due to the dietary oils. Blood plasma, platelets and red blood cell membranes showed increased levels of 18:3n-3 and long-chain n-3 PUFA in response to dietary 18:3n-3. The level of saturated fatty acids in blood lipids was generally lower in canola and HOAS oil-fed piglets as compared to piglets fed soybean oil or reared with the sow. The results suggest that consumption of milk replacer diets containing vegetable oils rich in 18:3n-3 does not represent a bleeding risk, and that the transient lower platelet count can be counterbalanced by the addition of saturated fatty acids to the vegetable oils.

Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids.

Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (18:2) purported to be anticarcinogenic, low levels of essential fatty acids, and trans fatty acids that increase when essential fatty acids are increased in dairy rations. Milk and rumen fatty acid methyl esters (FAME) were prepared using several acid- (HCl, BF3, acetyl chloride, H2SO4) or base-catalysts (NaOCH3, tetramethylguanidine, diazomethane), or combinations thereof. All acid-catalyzed procedures resulted in decreased cis/trans (delta 9c,11t-18:2) and increased trans/trans (delta 9t,11t-18:2) conjugated dienes and the production of allylic methoxy artifacts. The methoxy artifacts were identified by gas-liquid chromatography (Gl.C)-mass spectroscopy. The base-catalyzed procedures gave no isomerization of conjugated dienes and no methoxy artifacts, but they did not transesterify N-acyl lipids such as sphingomyelin, and NaOCH3 did not methylate free fatty acids. In addition, reaction with tetramethylguanidine coextracted material with hexane that interfered with the determination of the short-chain FAME by GLC. Acid-catalyzed methylation resulted in the loss of about 12% total conjugated dienes, 42% recovery of the delta 9c,11t-18:2 isomer, a fourfold increase in delta 9t,11t-18:2, and the formation of methoxy artifacts, compared with the base-catalyzed reactions. Total milk FAME showed significant infrared (IR) absorption due to conjugated dienes at 985 and 948 cm-1. The IR determination of total trans content of milk FAME was not fully satisfactory because the 966 cm-1 trans band overlapped with the conjugated diene bands. IR accuracy was limited by the fact that the absorptivity of methyl elaidate, used as calibration standard, was different from those of the other minor trans fatty acids (e.g., dienes) found in milk. In addition, acid-catalyzed reactions produced interfering material that absorbed extensively in the trans IR region. No single method or combination of methods could adequately prepare FAME from all lipid classes in milk or rumen lipids, and not affect the conjugated dienes. The best compromise for milk fatty acids was obtained with NaOCH3 followed by HCl or BF3, or diazomethane followed by NaOCH3, being aware that sphingomyelins are ignored. For rumen samples, the best method was diazomethane followed by NaOCH3.

Myocardial changes in newborn piglets fed sow milk or milk replacer diets containing different levels of erucic acid.

This study was undertaken to determine whether the neonate was more susceptible to the effects of dietary erucic acid (22:1n-9) than the adult. Newborn piglets were used to assess the safety of different levels of 22:1n-9 on lipid and histological changes in the heart. Newborn piglets showed no myocardial lipidosis as assessed by oil red 0 staining, but lipidosis appeared with consumption of sow milk and disappeared by seven days of age. Milk replacer diets containing soybean oil, or rapeseed oil mixtures with up to 5% 22:1n-9 in the oil, or 1.25% in the diet, gave trace myocardial lipidosis. Rapeseed oil mixtures with 7 to 42.9% 22:1n-9 showed definite myocardial lipidosis in newborn piglets, which correlated to dietary 22:1n-9, showing a maximum after one week on diet. The severity of the lipidosis was greater than observed previously with weaned pigs. There were no significant differences among diets in cardiac lipid classes except for triacylglycerol (TAG), which increased in piglets fed a rapeseed oil with 42.9% 22:1n-9. TAG showed the highest incorporation of 22:1n-9, the concentration of 22:1n-9 in TAG was similar to that present in the dietary oil. Among the cardiac phospholipids, sphingomyelin and phosphatidylserine had the highest, and diphosphatidylglycerol (DPG) the lowest level of 22:1n-9. The low content of 22:1n-9 in DPG of newborn piglets is not observed in weaned pigs and rats fed high erucic acid rapeseed oil. The relative concentration of saturated fatty acids was lowered in all cardiac phospholipids of piglets fed rapeseed oils, possibly due to the low content of saturated fatty acids in rapeseed oils. The results suggest that piglets fed up to 750 mg 22:1n-9/kg body weight/day showed no adverse nutritional or cardiac effects.

Degradation of soluble and insoluble proteins by Bacteroides amylophilus protease and by rumen microorganisms.

Various soluble and insoluble proteins (6.25 mg) were incubated at 37 C with partially purified protease from Bacteroides amylophilus (156 micrograms) in 2.0 ml of .1 M potassium phosphate buffer, pH 7.6, for 2, 4, 6 and 18 hr, and the liberated amino acids were determined by the ninhydrin method. Results showed that (1) although soluble, serum albumin and ribonuclease A were resistant to hydrolysis; (2) soluble and insoluble proteins of soybean meal were hydrolyzed at almost identical rates; (3) soluble proteins from soybean meal, rapeseed meal and casein were hydrolyzed at different rates, and (4) treatment of resistant proteins (serum albumin, ribonuclease A and insoluble fish meal and rapeseed meal proteins) with mercaptoethanol in 8 M urea or oxidation with performic acid rendered these proteins susceptible to hydrolysis. It is concluded that (1) solubility or insolubility of a protein is not by itself an indication of the protein's resistance or susceptibility to hydrolysis by rumen bacterial protease; (2) structural characteristics of the properties which renders feed protein resistant to degradation is the presence of crosslinking disulfide bonds.

Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added to the diet.

We measured effects of continuous vs twice-daily feeding, the addition of unsaturated fat to the diet, and monensin on milk production, milk composition, feed intake, and CO2-methane production in four experiments in a herd of 88 to 109 milking Holsteins. Methane and CO2 production increased with twice-daily feeding, but the CO2:CH4 ratio remained unchanged. Soybean oil did not affect the milkfat percentages, but fatty acid composition was changed. All saturated fatty acids up to and including 16:0 decreased (P < .01), whereas 18:0 and trans 18:1 increased (P < .001). The 18:2 conjugated dienes also increased (P < .01) when the cows were fed soybean oil. Monensin addition to the diet at 24 ppm decreased methane production (P < .01); the CO2:CH4 ratios reached 15, milk production increased (P < .01), and milkfat percentage and total milkfat output decreased (P < .01), as did feed consumption, compared with cows fed diets without monensin (P < .05). Milk fatty acid composition showed evidence of depressed ruminal biohydrogenation: saturated fatty acids (P < .05) decreased and 18:1 increased (P < .001); most of the increase was seen in the trans 18:1 isomer. As with soybean oil feeding, addition of monensin also increased (P < .05) the concentration of conjugated dienes. The monensin feeding trial was repeated 161 d later with 88 cows, of which 67 received monensin in the diet in the first trial and 21 cows were newly freshened and had never received monensin. Methane production again decreased (P < .05), but this time the CO2:CH4 ratio did not change and all other monensin-related effects were absent. The ruminal microflora in the cows that had previously received monensin seemed to have undergone some adaptive changes and no longer responded as before.

Inhibition of methylcoenzyme M methylreductase by a uridine 5'-diphospho-N-acetylglucosamine derivative.

Uridine-5'-diphospho-N-acetylglucosamine, when oxidized with periodate to the corresponding aldehyde (o-UDP-GlcNAc), was a potent inhibitor of the methylcoenzyme M methylreductase reaction which catalyzes the reductive demethylation of methylcoenzyme M to methane. The oxidation product, o-UDP-GlcNAc, appears to bind to the UDP-GlcNAc site of the enzyme and inhibits the reduction of methylcoenzyme M both by MRF or its active hydrolytic fragment HS-HTP. The kinetic patterns indicate that o-UDP-GlcNAc inhibition is noncompetitive with HS-HTP or MRF, and the Hill coefficient indicated that there was cooperativity between the UDP and HS-HTP binding sites. The methylreductase enzyme was protected from o-UDP-GlcNAc inhibition by prior exposure to low concentrations of MRF. HS-HTP, at the same concentration as MRF, was not effective in protecting the enzyme from inhibition by o-UDP-GlcNAc.

Tetrahydromethanopterin methyltransferase, a component of the methane synthesizing complex of Methanobacterium thermoautotrophicum.

A new enzyme, tetrahydromethanopterin methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate, has been found in the methane synthesizing complex of Methanobacterium thermoautotrophicum. The enzyme is oxygen sensitive and has a well defined pH optimum at pH 6.7. There was no methyl group transfer when the enzyme was heated to 100 degrees for 5 min. The product of the forward reaction, methyl-CoM, was positively identified by TLC and high voltage paper electrophoresis. The demethylation of methyl-CoM, in the absence of methane synthesis, was dependent on the addition of H4MPT which suggests that the enzyme reaction is reversible.

Changes in oxidation reduction potentials and volatile fatty acid production by rumen bacteria when methane synthesis is inhibited.

Rumen inoculum was cultured in specially designed fermenters that allowed simultaneous measurement of pH, oxidation-reduction potentials, and gas production. The cultures were maintained at pH 6.8 by addition of 1 M NaHCO3 and continuous infusion of artificial saliva. Gas flow was maintained at 20.0 ml/min with a stream of O2-free N2. Monensin at 7.0 micrograms/ml inhibited CH4 production 49% below control concentrations. The sodium salt of 2-bromoethanesulfonic acid added at an initial concentration of 5 x 10(-5) M inhibited CH4 production by 86% and increased H2 production from less than .5 mumol/min in the control to 24.5 mumol/min in the inhibited fermenter. The redox potentials in the control fermenter remained above -.20 V and did not change with the addition of monensin. Bromoethanesulfonic acid rapidly decreased the redox potential in the fermenter to -.33 V. Volatile fatty acid production was not significantly altered by the addition of 2-bromoethanesulfonic acid. The addition of monensin gave the expected decrease in acetate:propionate ratios, decreased acetate and butyrate production, and increased valerate (but not propionate) production.

Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.

Evidence for separate enzymes of pyruvate decarboxylation and pyruvate synthesis in soluble extracts of Clostridium pasteurianum.

Additional evidence to that already presented (Sauer, F. D., Bush, R. S., and Stevenson, I. L. (1976) Biochim. Biophys. Acta 445, 518-520) suggests that pyruvate-ferredoxin oxidoreductase isolated from Clostridium pasteurianum consists of two separate enzymes: (a) pyruvate lyase, which catalyzes the CoA and electron acceptor-dependent decarboxylation of pyruvate, and (b) pyruvate synthase, which catalyzes the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate. The enzymes separated on Sephadex G-200 and with acrylamide gel electrophoresis but complete separation of one enzyme free of the other was not achieved. Extensive purification procedures were not used because both enzymes are unstable. The results confirm published reports that pyruvate lyase contains thiamin and a chromophore which participates in electron transfer. Pyruvate synthase, however, did not appear to be a thiamin enzyme and there was no evidence to indicate participation of an enzyme chromophore in the pyruvate synthase reaction.