Biochem. J. (1965) 95, 411

Metabolism of Propionate by Sheep LiverOXIDATION OF PROPIONATE BY HOMOGENATES

By R. M. SMITH AND W. S. OSBORNE-WHITEC.S.I.R.O. Division of BiocheMistry and General Nutrition, University Grounds, Adelaide,

South Australia

(Received 10 August 1964)

1. The rate and stability to aging of the metabolism ofpropionate by sheep-liverslices and sucrose homogenates were examined. Aging for up to 20min. at 370 inthe absence of added substrate had little effect with slices, whole homogenates or

homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose

homogenates was confined to the mitochondrial fraction, but the mitochondrialsupernatant (microsomes plus cell sap) stimulated propionate removal. 3. Therate of propionate metabolism by liver slices was higher in a high potassiumphosphate-bicarbonate medium [0-88(±s.E.M. 0-16),-tmole/mg. of N/hr.] thanin Krebs-Ringer bicarbonate medium [0-44 ( ± S.E.M. 0-13) ,umole/mg. of N/hr.].4. Metabolism ofpropionate by sucrose homogenates freed from nuclei was depend-ent on the presence of oxygen, carbon dioxide and ATP. Propionate removal wasstimulated 250% by Mg2+ ions and 670% by cytochrome c. 5. In the completemedium 2-39(±s.E.M. 0-15),umoles of propionate were consumed/mg. of N/hr.6. The ratio of oxygen consumption to propionate utilization was sufficient toaccount for the complete oxidation of half the propionate consumed. 7. The onlyproducts detected under these conditions were succinate, fumarate and malate.Propionate had no effect on the production of lactate from endogenous sources anddid not itself give rise to lactate. 8. Methylmalonate did not accumulate whenpropionate was metabolized and was not oxidized. It was detected as an inter-mediate in the conversion of propionyl-CoA into succinate. The rate of thisreaction sequence was adequate to account for the rate ofpropionate metabolism bysucrose homogenates or slices, provided that the rate offormation ofpropionyl-CoAwas not limiting. 9. The methylmalonate pathway was predominantly a mitochon-drial function. 10. The metabolism of propionate appeared to be dependent on

active oxidative phosphorylation.

During an investigation of the metabolic defectoccurring in vitamin B12 deficiency in sheep(Marston, Allen & Smith, 1961) a study was madeof the metabolism of propionate by homogenates ofnormal sheep liver. The present series of papersdescribes these findings and their subsequentextension. A preliminary note of part of thepresentworkhas appeared (Smith& Osborne-White,1961).

Short-chain fatty acids produced during ruminalfermentation of plant materials constitute in largepart the energy source of ruminants (Marston,1948). The particular importance ofpropionate as asource of glucose in ruminants was pointed out byPhillipson (1947). The ruminant absorbs negligiblequantities of free glucose from the intestinal tractand relies on gluconeogenesis, chiefly from pro-pionate, as a means ofmaintaining the carbohydrate

reserve of the body. Short-chain fatty acidsabsorbed from the rumen are carried by the portalblood to the liver before entering the generalcirculation. Annison, Hill & Lewis (1957), byexamination of arterial and portal blood in sheep,obtained evidence indicating that propionate isvery largely removed from the blood in its passagethrough the liver.

1n the present investigation the metabolism ofpropionate has been examined in sheep-liver slicesand in homogenates of sheep liver prepared in0-25M-sucrose.

MATERIALS AND METHODS

Animals. Australian Merino ewes, aged 2-5 years, wereremoved from pasture 1 week before slaughter, housed inpens and fed ad lib. on a diet of 50% of wheaten hay chaff

411

R. M. SMITH AND W. S. OSBORNE-WHITEand 50% of dried lucerne chaff (by wt.). All animals wereallowed access to food up to the time of slaughter, and werekilled by cutting the throat.

Homogenates. For manometric work, precooled pieces ofthe livers of freshly killed animals were extruded throughcircles of 16-mesh stainless-steel gauze supported in asyringe barrel. A 1:7 homogenate ( 0g. of liver mash plus6-Oml. of 0-25M-sucrose) was prepared at 00 with a Potter-Elvehjem homogenizer (Potter & Elvehjem, 1936). Thehomogenate was centrifuged for lOmin. at 600g in anInternational refrigerated centrifuge, and the supernatantfluid that was employed for manometric work contained anaverage of 3-5mg. of N/ml. Microscopic examination aftervital staining with Janus Green B showed that the 600gsupernatant, comprising a suspension of mitochondriaand microsomes, was essentially free from cell fragmentsand nuclei, and that the 600g sediment consisted mostlyof unbroken cells together with cell fragments, nuclei(clearly visible as unstained voids), erythrocytes and somesedimented mitochondria.In large-scale experiments one part of liver to five parts

of 0-25M-sucrose was used with a homogenizer of 150ml.capacity fitted with a polyethylene pestle manufactured asdescribed by Kamphausen & Morton (1956). The nuclear-free homogenate (600g supernatant) in these experimentscontained an average of 4-3mg. of N/ml., and (Table 3)about 31% of the nitrogen of the whole homogenate, and16% of the total activity with propionate was rejected inthe 600g sediment. Centrifugal fractionation of homo-g3nates was performed by the method of Hogeboom,Schneider & Pallade (1948).The temperature was maintained between O° and 30

throughout the preparation.Disrupted mitochondria (Tables 6 and 7) were prepared

by homogenization of whole liver or isolated mitochondriafor 3-5min. at top speed at 00 in a Servall Omnimixer(Ivan Sorvall Inc., Norwalk, Conn., U.S.A.) in 0-04M-tris-HCI buffer, pH8.

Manometric procedure. Rates of consumption of 02 andevolution of CO2 were measured in an atmosphere of02+ CO2 (95:5) by Warburg's indirect method (Dixon,1951, p. 76; Umbreit, 1949).In early experiments with nuclear-free homogenates it

was found that reproducible rates could be obtained withpropionate as substrate only when stringent precautionswere taken to ensure gas saturation of the medium at allstages where homogenate was present. This led to ratherprolonged gassing (9min.) and equilibration (10 min.)stages in the bath, and it was found that, when substratehad been present from zero time, only relatively briefperiods (15-20min.) of respiration at linear rates could beobserved. It was then found that if propionate was addedat the conclusion of the gassing and equilibration times(i.e. after l9min. in the bath, this period being determinedin part by manipulative considerations, in handling twobanks of six flasks) linear rates of consumption of 02 couldbe measured for the final 30min. of a 36min. incubation.This procedure was adopted, but, since it involved incuba-tion of the nuclear-free homogenate for 19 min. in theabsence of substrate before measurement of respiration, astudy was made of the stability of various centrifugalfractions of the homogenate to such incubation. Table 2shows that only a small fraction ofthe capacity to metabolizepropionate is lost during l9min. incubation both in the

whole homogenate (average loss 8%) and in the nuclear-freehomogenate (average loss 5%1. Table 1 shows that suchstability to incubation in the absence of substrate is alsofound in sheep-liver slices metabolizing propionate inKrebs-Ringer bicarbonate medium.The standard medium in which gas exchange and

consumption of substrate was measured had the followingfinal composition: K2HPO4 (16-4mM), KH2PO4 (3-6mM),NaHCO3 (25-0mM), KCI (90mM), MgCl2 (5-0mM), sucrose(46-3mM), ATP (1-3mM), cytochrome c (11M), substrate(5-0mM; added after 19min. in the bath) and liver proteincontributing 2-2-5-5mg. of N to a final volume of 6-Oml.in equilibrium at 370 with 02+CO2 (95:5) (final pH7-4).The medium was kept saturated with gas mixture at

room temperature before and during addition of thenuclear-free homogenate. After a 9min. gassing period inthe bath flasks were equilibrated for lOmin. and substratein buffer was added from the side arm. Gas exchange wasmeasured in pairs of flasks at 6min. intervals for 36min.,the first reading taken 6min. after adding substrate beingtaken as the zero point of the measurement. The valuesquoted for rates of consumption of 02 thus represent theperiod from 6 to 36min. after the addition of substrate;rates for consumption of substrate were measured over theentire incubation period.Measurements of gas exchange were made in conventional

Warburg flasks (20-25 ml. volume) in final volumes of6-0 and 3-Oml. (or 2-Oml.) containing 1-0 and 0-5ml. (or0-33ml.) of the nuclear-free homogenate respectively.(Flasks containing 6-Oml. volumes were without centrewells.) Shaking rates of 130-140 7cm.-oscillation cycles/min. were employed, and measured respiration was pro-portional to the amount of nuclear-free homogenate.Measurements of consumption of 02 with propionate assubstrate were regarded as valid only when it had beenshown that the rates of consumption of substrate (per ml.of nuclear-free homogenate) were identical in the twoflasks constituting a measurement.Under these conditions replicate measurements of

propionate oxidation indicated errors (95% confidenceinterval of the mean of three replicates) of + 6% for 02and +9% for CO2. The validity of the method of measure-ment was tested by a study of the oxidative decarboxylationof p-hydroxyphenylpyruvate by the enzyme p-hydroxy-phenylpyruvate hydroxylase (EC 1.99.1.14), preparedfrom pig liver by the method of Hager, Gregerman &Knox (1957). Satisfactory agreement with the directmethod for 02 was obtained.

Rates quoted represent the total exchanges of 02measured in pairs of flasks. No corrections have beenapplied for gas exchange in the absence of substrate,although these rates were measured. The conditions of theexperiments were chosen to obtain linear rates of consump-tion of 02 over the 30min. period from 6 to 36min. afteradding substrate. Rates in the absence of substrate,however, were generally not linear, but fell off withtime.

Incubation of homogenate fractions without manometricmeasurement8. In the experiments listed in Tables 2 and 3,where multiple measurements of propionate consumptionby homogenate fractions were made, and in the experimentdescribed in Table 4, where measurements were made inthree different gas phases, centrifugal fractions of sucrosehomogenates were incubated in the reaction vessels and

1965412

OXIDATION OF PROPIONATE BY SHEEP LIVERunder the conditions described by Smith, Osborne-White &Russell (1965). Reaction tubes in these experiments were

supported in racks to permit simultaneous incubation ofup to 50 tubes. The only difference in the conditions ofthese experiments from those performed in Warburgflasks was that all vessels were continually flushed withgas mixture (200ml./min. to each flask) throughout theentire incubation. Similar precautions with regard to theinitial saturation of the medium with gas mixture were

employed as in experiments where gas exchange wasmeasured. /

Reactions were terminated by the addition of 1 ml. of10% (wfv) sodium tungstate dihydrate and 1 ml. of0-66N-H2SO4. Water was added to give a volume of20ml. and the tube contents were centrifuged to remove

protein. Consumption of volatile acid was estimated bydifference from initial values determined at the time oftipping. Values quoted are the means of duplicate incuba-tions.

Preparation and incubation of liver slices. Liver sliceswere cut and incubation begun within lhr. of killing theanimals. Plugs of liver for preparation of slices were cutfrom the liver immediately after death, with a steel corkborer of 1-0cm.2 cross-section, and were plunged into ice-cold0-25m-sucrose to chill. After draining, slices (0-4-0-6mm.thick) were cut by hand in a cold room at 0-1° by the methodof Deutsch (1936), weighed and placed directly in 5-4ml.of pregassed medium at 0-1°. Four slices (total weightabout 200mg.) were added to each vessel. Vessels were

flushed continuously with 02+CO2 (95:5) gas mixtureboth during the preparation of slices and throughout theincubation period. The reaction vessels and the method ofincubation were as described by Smith et al. (1965).

After the stated period of incubation at 370 in the absenceofsubstrate, buffered substrate (0-6 ml. containing 30,umolesof potassium propionate) was added from the side arm andincubated for 36min. with shaking. Reactions wereterminated by the addition of 2ml. of 2N-H2SO4, waterwas added to give a volume of 20ml. and the vessel contentswere centrifuged to remove slices and fragments.

Residual volatile acid was determined in samples of thesupernatant, and consumption determined from controlflasks to which acid was added at the time of tipping. Allvalues quoted for consumption of propionate represent themeans of duplicate incubations.Two media were employed to measure slice metabolism:

Krebs-Ringer bicarbonate medium (Cohen, 1949), and thestandard medium employed for measurement ofhomogenatemetabolism, but from which ATP and cytochrome c were

omitted.Incubation of Omnimixer homogenates with 14CO2 and

isolation of products. Animals were killed and sections ofliver homogenized in a Servall Omnimixer at 0-2° in 9vol.of 0-04M-tris-HCl buffer, pH8-0, containing GSH (lmm),for 3-5min. at top speed. For enzyme assay two dilutionsof the homogenate were prepared in 0-04M-tris-HClbuffer, pH7-4, containing GSH (1mm).

Incubations were carried out in short tubes (internaldiam. 15mm.) of total volume about 6ml., sealed withrubber serum caps. The composition of the medium was

as follows: tris-HCl buffer, pH7-4 (50mM), GSH (5mm),MgC12 (3mm), ATP (3mM), propionyl-CoA (1 mM), Na214CO3(10mM; specific activity 200m,tc/,umole) and liver homo-genate contributing 0-425-3-16mg. of protein to a final

volume of 1 ml. Incubation in a gas phase of air was for20min. at 30°, and reactions were started by the addition ofNa214CO3 by syringe through the serum cap.

The reactions were terminated by the addition of 0-2 ml.of 2w-KOH, and tubes were allowed to stand for hr. atroom temperature to hydrolyse CoA thio esters. Carrieracids (125,ug. each of methylmalonic acid, succinic acid,fumaric acid, malic acid, malonic acid and citric acid) were

then added, the solution was acidified with 0-5ml. of4N-HCI and 14CO2 was discharged in a fume hood for lhr.Residual 14CO2 was removed by leaving the tubes overnightin an evacuated desiccator over 2N-NaOH. Tubes were

then heated for 1 min. in a boiling-water bath and thecontents transferred quantitatively in 0-1 N-HCl to taredcentrifuged tubes and weighed (final volume about 8ml.).Tubes were centrifuged to remove protein, and, afterremoval of small weighed samples (about 0-2g.) for deter-mination of total fixation of 14CO2 by combustion, most ofthe remainder was extracted for 24hr. with freshly distilledperoxide-free diethyl ether in a liquid-liquid extractor.The ether was evaporated on a rotary evaporator, and

the residue taken up in water and transferred with washingto 15ml. conical centrifuge tubes. These solutions were

evaporated to dryness by heating in a water bath (800)under a stream of N2. The residue was taken up in a smallvolume of water (0-2 ml.), and samples (501l.) were spottedon Whatman no. 1 paper and chromatographed for 17hr.at 250 in 3-methylbutan-1-ol saturated with 4N-formicacid (Flavin & Ochoa, 1957).

After spraying lightly with bromophenol blue, radioactivespots were located with a strip scanner, cut out and burntto 14CO2 for determination of 14C as described below.Recovery of 14C by combustion of paper as compared withthe initial determination of total fixation was 95%.

Incubation of Omnimixer homogenates with [2-14C]-propionyl-CoA and isolation of products. A homogenate(1: 10) in 0-25m-sucrose was prepared and the mitochondriawere isolated and washed twice as described above. Portionsof the mitochondria were then suspended in equal volumesof 0-08M-tris-HCl buffer, pH 8-0, containing GSH (2mM),and either 0-25 M-sucrose or the mitochondrial supernatant,and homogenized for 3-5min. at top speed in a ServallOmnimixer at 0-2°. A further portion of the mitochondrialsupernatant was mixed with an equal volume of the samebuffer and similarly homogenized for 3-5min.Samples (0-4ml.) ofthe three homogenates were incubated

with [2-14C]propionyl-CoA in a medium of the followingfinal composition: [2-14C]propionyl-CoA (1 mM; specificactivity 330m,uc/,mole), tris-HCl buffer, pH7-5 (50mM),MgCl2 (3mm), MnCl2 (1-3mM), NADP (0-3mM), GSH(5mM) and where appropriate NaHCO3 (3mM) and ATP(3mM) in final volumes of 1 ml.

Incubations in a gas phase of air were carried out for20min. at 300 in short tubes (internal diam. 15mm.) ofabout 6ml. volume, sealed with rubber serum caps. Thereaction was started by the addition ofNaHCO3 by syringethrough the serum cap and terminated by the addition of1 drop of 8N-NaOH (final pH> 12). Flasks were thenallowed to stand for 1 hr. at room temperature to hydrolyseCoA thio esters.

Carrier acids (125,ug. each of methylmalonic acid,succinic acid, fumaric acid, malic acid, malonic acid andcitric acid) were added in 0-1 ml. and tube contents acidified(pH < 2) with 1 drop of 50% (v/v) H2SO4. After standing

Vol. 95 413

R. M. SMITH AND W. S. OSBORNE-WHITEfor 1 hr. in a fume hood tubes were placed overnight in anevacuated desiccator over 2N-NaOH to remove 14CO2.The acidified mixture was mixed intimately with 2g. of

dry Celite (Celite 545; Johns-Manville Co., New York,N.Y., U.S.A.) suspended in chloroform and applied as acap to a column of Celite. The column (1 cm. diam.),consisted of 5g. of Celite containing 2ml. of 0-2N-H2SO4and was equilibrated with chloroform saturated with0-2N-H2S04. The last traces of the reaction mixture inCelite were transferred to the column with the aid of glasswool moistened with chloroform and the cap was packedtightly.

Radioactive propionate was eluted from the column in125 ml. ofchloroform previously saturated with 0-2 N-H2SO4.The mixed carrier acids were then eluted with 150ml. ofbutan-l-ol-chloroform (1:1, v/v) previously saturatedwith 0-2N-H2SO4. This procedure was based on the methodof Swim & Krampitz (1954) for the chromatographicseparation of organic acids and was found to achieve acomplete separation of propionic acid from the non-volatilecarrier acids listed above.The eluted carrier acids were extracted from the organic

solvent into an excess of NaOH (5ml. of 2N) with the aidof a vibrating emulsifier, and the aqueous phase wasrecovered after centrifugation. The organic phase waswashed twice in a similar manner with 2ml. of0-02N-NaOH,and the aqueous phases were combined. The aqueoussolution containing the carrier acids was then acidified(pH < 2) with H2SO4 and extracted for 24 hr. with freshlydistilled peroxide-free diethyl ether in a liquid-liquidextractor.The ether was evaporated, the residue transferred

quantitatively with ether to a 15ml. centrifuge tube andthe ether again evaporated. The residue was concentratedin the tip of the tube and dissolved in 0-2 ml. of water.Samples (501zl.) of this solution were spotted on Whatmanno. 1 paper and chromatographed for 17hr. at 250 in3-methylbutan-1-ol saturated with 4N-formic acid. Afterbeing dried, papers were sprayed and radioactive spotsdetected with a strip scanner. Spots were cut out and burntto C02 to determine 14C.

Incubation of nuclear-free homogenates with propionate andCoA, and detection of hydroxamic acids. The medium wassimilar to the standard medium, but contained a higherconcentration of ATP (2-6mM) and where appropriateadditional CoA (1 mM) and neutralized hydroxylamine.Final volumes of 2ml. were employed containing 0-5ml.of nuclear-free homogenate (2.07mg. of N) and potassiumpropionate (5 mM). All components except the nuclear-freehomogenate were added to the main chamber and pregassedat room temperature with N2+CO2 (95:5). Reactionswere started by the addition of the nuclear-free homogenateand the flasks were attached to manometers, placed in thebath and regassed for 9min. with shaking. After a further28min. neutralized hydroxylamine (final concn. 0-67M or1 M) or tungstic acid was added to terminate the reactions.After standing at room temperature for 15 min. the

contents of the flasks containing hydroxylamine wereadded to 25vol. of ethanol and centrifuged to removeprotein. The ethanol extract was evaporated to dryness atroom temperature. The crystalline residue was trituratedthree times with 0-7ml. portions of ethanol and filteredthrough small plugs of glass wool in capillary tubes. Thecombined filtrates were evaporated to dryness at room

temperature under a stream of N2, taken up in 0-2 ml. ofethanol, and 50,IL. samples were spotted on Whatmanno. 1 paper with and without standard acetyl-, propionyl-and succinyl-hydroxamic acid. The standards wereprepared from acid anhydrides by the method of Lipmann& Tuttle (1945).Chromatograms were developed with butan-l-ol-acetic

acid-water (4:1:5, by vol.), as described by Thompson(1951), and sprayed with the FeCl3 reagent described byStadtman & Barker (1950).

Materials. The NaHCO3 (A.R.) was obtained fromStandard Laboratories (Pty) Ltd., Melbourne, Victoria,Australia; K2HPO4 (pure grade) was obtained fromHopkin and Williams (Pty) Ltd., Chadwell Heath, Essex.2,4-Dinitrophenol, propionic anhydride, malonic acid,succinic acid and fumaric acid were laboratory-reagent-grade chemicals obtained from British Drug Houses Ltd.,Poole, Dorset; succinic acid, fumaric acid and malonicacid were recrystallized from water and dried before use.L-Malic acid, GSH and crystalline bovine albumin wereobtained from Nutritional Biochemicals Corp., Cleveland,Ohio, U.S.A. Disodium ATP (from muscle), CoA andNADPwere obtained from the Sigma Chemical Co., St Louis, Mo.,U.S.A.

Propionic acid (laboratory-reagent grade) was obtainedfrom British Drug Houses Ltd. and fractionally distilledtwice. The final material was collected over the boilingrange 141-142-5° and was demonstrated to be free (lessthan 1%) of homologous short-chain fatty acids by thegas-liquid chromatographic method of James & Martin(1952).

Other chemicals were of analytical-reagent grade.Solvents were redistilled.Cytochrome c was prepared from horse heart by the

method of Keilin & Hartree (1945) or purchased from theSigma Chemical Co. (horse heart, type III) and was estim-ated by the spectrophotometric method of Potter (1949).Methylmalonic acid was prepared by saponification,

extraction into ether after acidification, and recrystallizationofa sample ofthe diethyl ester obtained through the courtesyof Dr J. A. Mills of this Laboratory. The ester had beenprepared from diethyl malonate and methyl iodide by themethod of Weiner (1943).Propionyl-CoA was prepared from propionic anhydride

and CoA by the method of Simon & Shemin (1953). Excessof anhydride and free propionic acid were removed afteracidification with HCI by extraction with three successivelots of 3 vol. of diethyl ether. The solution was then broughtto pH5 with NaHCO3 and stored at -20°. Propionyl-CoAwas estimated by the method of Grunert & Phillips (1949).

[2-14C]Propionyl-CoA was prepared from sodium [2-14C]-propionate, obtained from The Radiochemical Centre,Amersham, Bucks. The sodium [2-14C]propionate (100,uain 23-5,umoles) was diluted with a solution of unlabelledsodium propionate to a specific activity of 0-33ptcf/,umole.The water was evaporated and the residue dried in a smallglass-stoppered reaction tube at 1900 for 2hr. in a stream ofdry N2. The material was ground with a glass rod andredried before conversion into the anhydride with sulphurand bromine by a semi-micro modification of the method ofOrshansky & Bograchov (1944). The thoroughly groundand dried sodium [2-14C]propionate (300 tmoles) wasmixed by means of a glass bead with 6jul. (17.7mg.; density2.945) of a solution of 0-272g. of resublimed sulphur in

414 1965

OXIDATION OF PROPIONATE BY SHEEP LIVER2-99g. of pure bromine. The tube was stoppered andsealed heavily with wax before heating at 50° with con-tinuous mechanical vibration for 2hr. The propionicanhydride was distilled by the 'cold-finger' technique underhigh vacuum (10-5mm. Hg) with liquid N2. The yield ofanhydride was 69% as estimated by the hydroxamic acidmethod of Lipmann & Tuttle (1945). The anhydride wasthen converted into [2-14C]propionyl-CoA as describedabove, and estimated as thiol group appearing on alkalinehydrolysis. The overall yield from [2-14C]propionate was23%.The Na214CO3 was obtained from The Radiochemical

Centre and diluted with unlabelled Na2CO3 to a specificactivity of 0-2,uc/,umole.

Chemical methods. Reactions in Warburg flasks wereterminated by the addition of l ml. of 10%o (w/v) sodiumtungstate dihydrate, and 1 ml. of 0-66N-H2SO4/6ml. offlask contents. After dilution with water to 20ml. andcentrifugation to remove protein, the supernatant wasfrozen in glass-stoppered containers before assay. Inexperiments involving the detection of ether-solubleproducts, tungstic acid was replaced with Zn(OH)2 asprotein precipitant. In this case 6ml. of flask contents wastreated with 0-5 ml. of25% (w/v) ZnSO4,7H20 and equiva-lent NaOH (phenolphthalein end point) in 1-5ml. Thistreatment eliminated contamination of the supernatantwith ether-soluble lipids, but allowed complete recovery ofpropionic acid, estimated as described below.

Volatile acid in protein-free supernatants was estimatedafter steam-distillation by titration under N2. Samplescontaining 4-10jtmoles of propionic acid were distilled atconstant volume from 20ml. of 50% (w/v) MgSO4,7H20adjusted to pH2 (Congo red paper) with H2SO4. Distillate(100ml.) was collected over about 40min. and 50ml.samples were titrated. The distillation flask was fitted withan efficient Cyclone-type centrifugal splash head designedby Mr V. A. Stephen of this Laboratory. Distillation vapourentered the lagged 7 cm.-diam. hollow glass disk tangentiallyand left through a take-off normal to the centre of the disk.Centrifugal force in the rapidly spinning vapour streamprevented the carry over of spray droplets to the take-off.With selected samples of MgSO4, blank titrations of3-4wul. of 001 N-NaOH/50ml. of distillate were regularlyobtained.

Titrations were performed with 0 01 N-NaOH (carbonate-free) delivered from an Agla micrometer syringe. Titrationvessels were treated with silicone (Desicote) and wereloosely covered during titration. Nitrogen (freed fromCO2 and NH3) was bubbled constantly through the solutionduring titration and for 3min. beforehand. The course ofthe titration was followed with a glass electrode and aCambridge portable pH-meter, the end point being takenas pH7-0.Recovery of acetic acid and propionic acid was within

+3% over the range 4-10,umoles distilled.In experiments where manometric measurements were

made, consumption of propionate over 36min. was estim-ated by difference from an initial value comprising substrateplus nuclear-free homogenate. The nuclear-free homogen-ates contained approx. l,mole of volatile acid/ml., chieflyacetic acid, and this was not significantly depleted duringthe 55min. incubation period either in the presence orabsence of 30,umoles of propionate. Apart from the dis-appearance of a trace (less than 0 2,umole) of formic acid

present in the homogenate, consumption of propionic acidwas the only change detected.

In experiments that did not involve manometric measure-ments the consumption of volatile acid was measured bydifference from initial values determined at the time oftipping. In either case values quoted for rates of consump-tion of volatile acid represent the means of at least tworeplicate incubations.

Lactate was estimated by the colorimetric procedure ofBarker & Summerson (1941) after copper-lime treatmentof samples of the protein-free supernatant. Crystallinezinc lactate was employed as reference standard.

Total nitrogen in homogenate fractions and slices wasdetermined by the Kjeldahl procedure of McKenzie &Wallace (1954).

Protein was determined by the method of Lowry,Rosebrough, Farr & Randall (1951), with crystalline bovinealbumin as reference standard.Paper chromatography. Flask contents were treated with

Zn(OH)2 to remove protein and 0 75 samples evaporatedjust to dryness under reduced pressure. The residue wastaken up in 2ml. of water, acidified with H2SO4 (pH < 2)and mixed with 25g. of anhydrous NaSO4. The acids wereextracted in a Soxhlet apparatus for 6hr. with freshlydistilled peroxide-free diethyl ether and the ether wasevaporated. After redissolving in known volumes of watersamples were spotted on Whatman no. 1 paper with andwithout authentic acids and chromatographed. The foursystems employed were: A, ethanol-aq. NH3 (sp.gr. 0 88)-water (8:1:1, by vol.), as described by Jones, Dowling &Skraba (1953); B, diethyl ether-acetic acid-water (13:3: 1,by vol.) on Whatman no. 1 paper washed with 1% aq.NH3 and dried (Denison & Phares (1952); C, 3-methyl-butan-l-ol saturated with 4N-formic acid (Flavin &Ochoa, 1957); D, ethyl methyl ketone-water-acetone-formic acid (40:6:3:1, by vol.), as described by Hogstrom(1957).

Estimation of 14C. The 14C was estimated as 14CO2 bythe method of Brown & Miller (1947), by the vacuumtechniques described by Glascock (1954a). The dimensionsofthe combustion tube were increased to permit combustionof spots cut from paper chromatograms. The total lengthof the quartz combustion tube (internal diam. 10-12 mm.)was 125 cm. The length of the catalytic filling ('simpleband' filling of Niederl & Niederl, 1942) was 45-6cm.,maintained at 7000 with an electric furnace. Samples wereburnt at 800° with a second electric furnace preceding thecatalytic filling and combustion gases were trapped inliquid N2 in the multiple U trap described by Buchanan &Nakao (1952).

After metering (mercury manometer) 14CO2 was supple-mented with unlabelled CO2 to give a total pressure in thecounting tube of 200mm. Hg and with CS2 to give a partialpressure of 20mm. Hg and counted. Counting tubes of thetype described by Glascock (1954b) with a tungsten anodeand stainless-steel cathode were obtained from 20thCentury Electronics Ltd., New Addington, Surrey (typeGA 10/M). An external quenching circuit (Neher & Harper,1936) was employed and the operating potential was3760v. Commercial power supply and scaling equipmentwere used. Counting efficiency was 52-4% (counts aspercentage of disintegrations in the tube), and recovery ofcarbon was 100+ 0-5% from a range ofcompounds includingstearic acid and glutamic acid. Recovery of carbon from

Vtol. 9"i 415

R. M. SMITH AND W. S. OSBORNE-WHITEspots cut from paper chromatograms was quto 70mg./paper. Overall errors in the estirbased on replicate measurements over sever+2% when 104 counts were recorded.In experiments involving the recovery

compounds from paper chromatograms by cthe paper, papers were first sprayed lightly ato locate carrier acid spots, and location of 1mined with a strip-scanning device by empend-window Geiger-Muller tube (Generalmodel E.H.M.2). Radioactive areas werescissors, allowing about 0-25in. beyond theand burnt in quartz boats.

EXPERIMENTAL AND RESI

Rate of metaboli8m of propionate b1slices and stability to preliminary incubabsence of substrate. The rate of mepropionate by sheep-liver slices was defive experiments with two different medand 2). When substrate was added wof placing in the bath the rate in K]bicarbonate medium was 0-44(±s.Emole/mg. of N/hr., and in the stande(with ATP and cytochrome c omittei(+s.E.M. 0-16) tmole/mg. of N/hr.The results in Table 1 were subjected

of variance. The analysis showed thaRinger bicarbonate medium there was nchange in activity (P > 0.05) on incul

Table 1. Rate of metabolism of propioruliver slices and stability to incubation in tsubstrate

Conditions of the experiments are descMaterials and Methods section. The standarcthat employed for homogenate metabolism I

and cytochrome c omitted. The gas phase(95:5), and propionate consumption was det36min. after incubation of the slices in tIsubstrate for the stated period. Initial voladetermined in control flasks to which acid wai

time of tipping. The results are the means +:

separate experiments. In each experimevessels were incubated and the mean value of tconsumption was used for the calculation.

Propionate co](,umoles/mg. o

Time of preliminaryincubation at 370

without substrate (min.)0

10204060

Krebs-Ringerbicarbonatemedium

0-60+ 0*150-63+ 0-170*65+ 0-130-62+0-060-72+ 0-08

antitative up for up to 60min. in the absence of substrate. In thenation of 14C standard medium the rate fell on incubation in theal years were absence of substrate, but the fall over the first

of labelled 20min. was not significant (P > 0-05), and in two

oombustion of further experiments (Table 2) no loss in activitywith indicator was observed on incubating slices in standardAC was deter- medium for l9min. in the absence of substrate.)loying a thin The rate in the standard medium was significantlyElectric Co. higher (P < 0.001) than in Krebs-Ringer bicar-cut out with bonate medium when substrate was present at thevisible spot, beginning of the incubation.

Sheep-liver slices thus exhibit considerablestability to preliminary incubation in their capacity

ULTS to metabolize propionate under the conditions

8heep-liver employed.r

sheep-lteer Location of activity in liver homogenates and)ation in the stability to incubation in the absence of substrate.ftabolism of Table 3 shows the mean activity of the various,termined in centrifugal fractions expressed as a percentage oflia (Tables 1 mean activity in the whole homogenates andrithin 2mmn. compared with the mean distribution of nitrogenrebs-Ringer in the fractions. The mitochondrial supernatant.M. 0- 13) P- itselfwas entirely inactive, the fraction metabolizingLrd medium propionate at the highest rate (per ml.) being thed) was 0-88 600g supernatant. The mitochondria had theIoanalysis highest specific activity in terms of nitrogen.

1 toinaKes- Study of the stability of the fractions to aEt in Krebs- preliminary incubation of 19min. in the absence ofLO significant substrate (Table 2) showed that very little loss of)ating slices activity occurred during this period either with

the whole homogenate (average loss 8%) or withthe 600g supernatant (average loss 5%; see alsoTable 4).

ate by sheep- Marked loss of activity (average 46%) occurredhe absence of on incubating the isolated mitochondria in the

absence of substrate, but this could be restored to aribed in the considerable extent by the addition of the mito-

I medium was chondrial supernatant after the preliminary incub-but with ATP ation of l9min. Under these conditions the meanwas 02+ CO2 loss of activity due to incubation of the mito-termined over chondria was decreased to 25%. This loss onhe absence of aging of the intrinsic capacity of mitochondria toutile acid was metabolize propionate was surprisingly small.s added at the From the results it appeared that the effect of theS.E.M. of three mitochondrial supernatant was twofold; it exertedthe propionate a marked stimulating effect on the rate of metabo-

lism of propionate by the mitochondria, and itprotected the mitochondria from inactivation

nrsumed during the 19min. period in the absence ofsubstrate.If N/hr.) The 600g sediment, consisting of unbroken cells,

I cell fragments, nuclei, erythrocytes and someStandard mitochondria, contained 16% of the activity of themedium homogenate but about 30% of the nitrogen. This1-08+ 0.19 fraction was discarded in the experiments described1-01+ 0-07 below, which were performed on the 600g super-0-89 + 0-02 natant (nuclear-free homogenate).0.74+0.05 Dependence of propionate metabolism on carbon0*64+0-15 dioxide and oxygen and stimulation by Mg2+ ions.

416 1965

OXIDATION OF PROPIONATE BY SHEEP LIVER

Table 2. Rate of metabolism of propionate by sheep-liver slices and centrifugal fractions of sucrosehomogenates measured before and after aging in the absence of substrate

Slices were incubated either in Krebs-Ringer bicarbonate medium or in the standard medium with ATP andcytochrome c omitted. Homogenates (1:6) were prepared in 0-25M-sucrose and centrifugally fractionated asdescribed in the Materials and Methods section. The 600g sediment was resuspended on 0-25M-sucrose andmade to the original whole-homogenate volume. The mitochondria were washed twice and resuspended in0-25M-sucrose to the volume of nuclear-free homogenate from which they were derived. The reconstitutednuclear-free homogenate was prepared by resuspending twice-washed mitochondria in the mitochondrial super-natant to the original volume of nuclear-free homogenate from which they were derived. All homogenateincubations were performed in the standard medium, and all incubations took place for 36min. at 370 after theaddition of substrate in a gas phase of 02+ CO2 (95:5). Initial volatile acid was determined at the time of theaddition of substrate. Each value quoted is the mean of duplicate incubations.

PreparationSlices in Krebs-Ringer bicarbonatemedium

Slices in standard medium (no ATPor cytochrome c)Whole sucrose homogenate

600g Sediment in original homogenatevolume600g Supernatant (nuclear-freehomogenate)Twice-washed mitochondria

Mitochondrial supernatant(microsomes+cell sap)

Reconstituted nuclear-freehomogenate

Mitochondria incubated for 19min. inabsence of substrate before addingmitochondrial supernatant

N content(mg./ml. ofhomogenatefraction or

Expt. mg./g. ofno. slices)121212121212121212

Time of preliminaryincubation without,substrate at 370... ...

30-229-430-22945-355-101-691-514-424-191-020-883-223-223-963.933-963.93

Propionate consumed(,umoles/mg. of N/hr.)22 min. 19 min.0-260-160-560-572-552-951-281-683-312-764-756-03NilNil2-953-04

0-360-270-590-612-272-791-081-002-942-822-942-79NilNil2-442-782-192-39

Table 3. Distribution of propionate-metabolizingactivity in centrifugalfractions of sucrose homogenatesof sheep liver

The results of Expts. 1 and 2 (Table 2) are expressed asmean distribution of total nitrogen and total activity.The results are derived from the values in Table 2 foractivity determined after 2min. incubation withoutsubstrate, and from measurements of the volumes recoveredin the various fractions.

Homogenate fraction

Whole homogenate600g Sediment600g Supernatant (nuclear-freehomogenate)

MitochondriaMitochondrial supematant(microsomes+ ccll sap)

14

Percentageof totalN

10030-669-4

15-351-9

Percentageof totalactivity

10016-475-0

Table 4 shows the complete dependence of propion-ate metabolism in the nuclear-free homogenate onthe presence of carbon dioxide (or bicarbonate)and on oxygen. Also shown is the marked stimula-tion in rate due to Mg2+ ions, determined bothafter 2 and 19min. incubation in the absence ofsubstrate.Dependence on ATP and the effect of dinitrophenol.

The dependence of propionate metabolism in thenuclear-free homogenate on the presence of ATP isshown in Table 5. Substrate was added after 19min.incubation at 37°. The effect of ATP reached amaximum at about 1 mm.In a further experiment the effect on consumption

of propionate of 30,um-dinitrophenol was deter-mined. Duplicate pairs of flasks were incubatedcontaining in final volumes of 6ml. of standardmedium 30 ,umoles of propionate (added after19min.) and lml. of nuclear-free homogenate

Bioch. 1965, 95

Vol. 95 417

R. M. SMITH AND W. S. OSBORNE-WHITETable 4. Dependence of propionate netabonuclear-free honogenate on oxygen axdioxide and stimulation by Mg2+ ions

Homogenate (1:6) of sheep liver was preparesucrose and a nuclear-free homogenate prepar

of N/ml.). After the stated period at 370 in thsubstrate, propionate was added from the 5i5incubated for 36min. in standard medium wi(95:5) (complete), standard medium with N2+(no 02), standard medium with NaHCO3 omitphase 100% 02 (no C02), or standard mediumomitted and gas phase 02+CO2 (95:5) (noInitial volatile acid was determined at the timiValues for uptakes represent the means

incubations, except in the complete system forvalues are the means of six replicate incubation

Propionate cons

Time ofpreliminary (,umoles/mg. ofincubation without

substrate at 37° ... 2 min. 1

SystemCompleteNo 02

No C02No Mg2+ ions

2*79NilNil0-19

Table 5. Dependence of propionate methATP in the presence of oxygen + carbon dio.

The conditions of the experiment and the coi

the medium (excepting ATP) were as descrMaterials and Methods section. Concentraticfrom 0 to 1-5mM were present in the main chapresence of nuclear-free homogenate duringand equilibration period of 19 min. at 37° beforeofpropionate from the side arm to give a final cc

of 5mm. Consumption of propionate was me

36min., as described in the Materials and MethFinal volumes of 6ml. containing l Oml. ofhomogenate (3-11mg. of N) were employed, an

was 02+ CO2 (95:5).

Concn. of Propionateadded ATP consumed

(mM) (,umoles/mg. of I

0

0-51-01-5

0.01-22-42-6

containing 4-13mg. of N. After 36min. iwith substrate flasks without dinitropconsumed 5-0 and 5-l,moles of pwhereas no consumption was detectedcontaining dinitrophenol.From the above results and from the d

on oxygen it may be inferred that Imetabolism in these preparations was

'lismn in the on the process of oxidative phosphorylation. Thead carbon presence of ATP may also have served to protect

the mitochondria against the aging process.

e)d in 0-25M- by cytochrome cytochrome c

ed (4 215mg. stimulated the rate of consumption of propionateabsence of and oxygen by the nuclear-free homogenate. The

de arm and endogenous respiration was not increased byth 02+ Co2 additional cytochrome c. The maximum rate bothCO2 (95:5) of uptake of oxygen and consumption of propionateted and gas was reached with 5,uM-cytochrome c.

with MgC12 Rate of metabolism of propionate and acetate. In

Mg2+ ions)- a series of 18 experiments the rate of consumptionof tipping. of propionate by nuclear-free homogenates oftriplicate sheep liver was measured in the standard medium

.which bothafter 19min. incubation in the absence of substrate.

18.The mean rate of consumption of propionate was

Numed 2.39( + S.E.M. 0 15) ,uequiv./mg. ofN/hr. The values

N/hr.) obtained lay within the range 1.6-3.2/equiv./mg.19min. of N/hr.

In a series of three experiments under identical

2.62 conditions, no consumption of acetate was detected

either in the presence or absence of 0-67 mM-L-malate. In the same experiments propionate was

0-48 metabolized at rates within the normal range.Relationship of oxygen consumption to propionate

utilization. In 14 measurements with nuclear-freehomogenates a mean of 1.74(± s.E.M. 0-04),umoles

abolism on of oxygen were consumed for each ,umole of pro-

xide (95:5) pionate disappearing. The correlation between

mposition of total oxygen and propionate is approximatelyibed in the equivalent to the complete oxidation of half theDns of ATP propionate metabolized.Lmber in the Production of lactate. Leng & Annison (1963)the gassing with sheep-liver slices, and Pennington & Suther-the addition land (1956) with slices of sheep rumen epithelium,)ncentration found lactate to be a major end product of pro-

asured over pionate metabolism. In sheep-liver nuclear-freeiods section. homogenates, added propionate (final conen. 5mM)d gas pr-ase had no effect on the rate of lactate production

(eight observations), and thus the two processes ofmetabolism of propionate and production oflactate appeared to proceed independently.

N/hr.) Production of ether-soluble non-volatile acids.Ether-soluble acids produced during the metabolismof propionate by nuclear-free homogenates were

detected by paper chromatography in four differentsolvent systems as described in the Materials andMethods section. Satisfactory resolution of a

mixture of malate, lactate, succinate, methyl-malonate and fumarate was achieved, and the

incubation presence of malate, succinate and fumaratehenol had demonstrated. No methylmalonate was formed.)ropionate, The acids were not detected unless propionate was

I in flasks added as substrate. From an assessment based on

spot sizes and limits of detection it was estimatedlependence that a total of 2 7,umoles of succinate, fumaratepropionate and malate had been produced during the loss ofdependent 4-7 ,umoles of propionate.

418 1965

OXIDATION OF PROPIONATE BY SHEEP LIVER

Evidence for the formation of propionyl-CoA.Metabolism of propionate to succinate and L-malate is consistent with the operation in sheepliver of the methylmalonate pathway of propionatemetabolism (Mazumder, Sasakawa, Kaziro &Ochoa, 1961). The first reaction in the sequence isthe formation of propionyl-CoA, presumably bymeans of the acetate-activating enzyme reportedby Hele (1954). Accordingly, attempts were madeto detect the formation of propionyl-CoA in thenuclear-free homogenate incubated with propionateand CoA. Formation ofCoA thio esters was detectedafter treatment with hydroxylamine, essentially bythe method of Stadtman & Barker (1950). Allflasks containing both propionate and CoA showeda well-defined spot chromatographically identicalwith propionylhydroxamic acid. Flasks containingpropionate only showed no spots.The formation of the material identified in this

way with propionylhydroxamic acid was thusdependent both on propionate and on CoA. Theamounts formed, however, were small and werenot visibly increased by the inclusion of hydroxyl-amine in flask contents during incubation at 37°.

Identification of methyl1malonate as an interme-diate. Flavin, Ortiz & Ochoa (1955) observed thatrat-liver homogenates oxidized methylmalonatein the presence of bicarbonate at about two-thirdsthe rate at which propionate was oxidized. In thepresent study it was found in two experiments thatmethylmalonate (5mM) was without effect on therespiration of sheep-liver nuclear-free homogenatesunder conditions where propionate was rapidlyoxidized. This finding, together with the failureto detect methylmalonate among the products ofpropionate metabolism, led to a closer examinationof the initial steps in the metabolism of propionateby sheep-liver homogenates.The conditions employed to allow the formation

of methylmalonate to be detected were similar tothose used by Flavin, Castro-Mendoza & Ochoa(1957) in studying carboxylating-enzyme activityin pig-heart preparations. Whole homogenates ofsheep liver were prepared in a high-speed blenderin 0 04M-tris-hydrochloric acid buffer, pH8, todisrupt mitochondrial structure, and fixation of14CO2 from NaH14C03 was measured in thepresence of propionyl-CoA. After alkaline hydroly-sis of CoA thio esters and extraction into ether,radioactive products were chromatographed withcarrier on paper, and the separated productsburnt to determine total activity as 14CO2 (Table 6).

Total radioactivity fixed into products wasdirectly related to the amount of homogenateprotein, although the rate declined with increasingprotein. By taking as the true rate 43-2m,uc/mg.of protein/hr. and applying the specific activity ofthe substrate 14CO2 (200mp,c/,umole), the rate offixation of 14CO2, and so of propionyl-CoA, was0-216,mole/mg. of protein/hr. Assuming that6 6mg. of protein was equivalent to 1mg. of N (avalue obtained by assay of whole liver), then thisrate becomes 1.4,umoles/mg. of N/hr. at 300.This compares satisfactorily with the mean rate of2.4,umoles/mg. of N/hr. obtained with nuclear-freehomogenates at 370, assuming that the rate offormation of propionyl-CoA was not limiting.The order in which the products began to accumu-

late with increasing protein is consistent with areaction sequence in which methylmalonate,succinate and malate were formed in that order.

Intracellular location of the reaction sequence ofthe, methylmalonate pathway. An experiment wasperformed to detect the distribution betweenmitochondria and cytoplasm of the overall enzymicactivity responsible for the conversion of pro-pionyl-CoA into methylmalonate, succinate andmalate.

Table 6. Radioactive products formed on incubating NaH14CO3 and propionyl-CoA withsheep-liver homogenate

A homogenate of sheep liver was prepared in 0-04M-tris-HCI buffer, pH8, in a high-speed blender to disruptmitochondrial structure. Samples of the homogenate were incubated with propionyl-CoA (1mM), GSH (5mM),MgCl2 (3mM), ATP (3mM) and NaH14CO3 (10mM; 200m,uc/,umole) in tris-HCl buffer, pH7.4, for 20min. at 30°.After hydrolysis of CoA thio esters, the ether-soluble products were separated by paper chromatography andburnt before measurement of radioactivity as 14CO2 in the gas phase. Experimental details are given in the textand in the Materials and Methods section.

Sum of separated products

Separated products (m,uc/hr.)

Protein (mg.) Methylmalonate Succinate0-425 15-5 1-80-85 18-6 13-71-58 26-3 29-13-16 25-5 51-0

.

TotalFumarate Malate (m1tc/hr.)

0-8 0-3 18-31-5 1-3 35-02-6 5-4 63-45.4 19-1 101-0

Rate(m,uc/mg. ofprotein/hr.)

43-241-440-232-1

Vol. 95 419

420 R. M. SMITH AND W. S. OSBORNE-WHITEMitochondria were isolated, washed twice with

0-25M-sucrose and disrupted in 0-04M-tris-hydro-chloric acid buffer, pH8, by high-speed blendingboth in the presence and absence of the mitochon-drial supernatant (microsomes plus cell sap).Samples of these homogenates and of the mito-chondrial supernatant were then incubated with[2-14C]propionyl-CoA in the presence and absenceof bicarbonate and ATP.The three homogenates were referred to as:

disrupted mitochondria (4.12mg. of protein/ml.),mitochondria disrupted in mitochondrial super-natant (10 15mg. of protein/ml.) and mitochondrialsupernatant (7.20mg. of protein/ml.). The twomitochondrial preparations contained equivalentconcentrations of mitochondria, which were en-riched approximately twofold with respect to theconcentration of the mitochondrial supernatant.

After paper chromatography in the presence ofcarrier the radioactive products detected by thestrip scanner were: methylmalonate (R.0-76),succinate (RFO.66), fumarate (RFO-81), malate(RFO35), traces of citrate (RFO.22) and malonate(RFO-54) and considerable activity (up to 10% ofthe total 14C recovered) in an unidentified product(RFO086) that travelled ahead of fumarate.

This material (X2) was partially resolved from asecond unidentified component (XI, RFO82) thatwas itself poorly resolved from fumarate. Com-ponent XI was present in all flasks after incubationbut may have been a contaminant of the [2-14C]-propionic acid. The unknown component X2 wasformed in quantity from propionyl-CoA onlywhen ATP was present. The three substances,fumarate, XI and X2, were not sufficiently wellresolved for separate determination of 14C and sohave been estimated together. In flasks where theradioactivity in the unknown substances wasgreater than 3m,uc (9m,tc/hr.), however, morethan half of this was attributable to componentX2, as judged by the strip-scanner records.The results ofthe experiment are given in Table 7.

It is clear that the mitochondria were responsiblefor most of the formation of dicarboxylic acidsobserved, although some conversion occurred withthe mitochondrial supernatant, and a smallstimulation was apparent when the two componentswere incubated together. The unknown productX2, which in flasks containing ATP may be assessedas half the total of Xl + X2+ fumarate, was alsodependent for its formation on the mitochondrialfraction. From the strip-scanner records theformation of component X2 was clearly dependenton ATP.By applying the specific activity of the substrate

propionyl-CoA (330m,uc/,umole) to the rate offormation of radioactive succinate, malate andmethylmalonate (80m,uc/hr. by mitochondria with

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Vol. 95 OXIDATION OF PROPIONATE BY SHEEP LIVER 4211-65mg. of protein), and converting mg. of mito-chondrial protein into mg. of N (6.6mg. of mito-chondrial protein _ 1mg. of N), a rate of formationof dicarboxylic acids of about 1 ,umole/mg. of N/hr.at 300 is derived. This value is well below the rateof metabolism of propionate by mitochondria ofabout 5 4,umoles/mg. ofN/hr. at 370 in the standardmedium when substrate was added at 2min.(Table 2). The difference between this rate and therate obtained from the results in Table 6 is un-explained but may represent one or more ofseveral differences in the medium; in particular, theconcentration of bicarbonate was lower in theexperiment referred to in Table 7.The experiment serves to demonstrate the

existence of an ATP-dependent formation ofmethylmalonate, succinate and malate from pro-pionyl-CoA in mitochondria.

DISCUSSION

Leng & Annison (1963), working with sheep-liverslices, found that incubation with labelled pro-pionate caused introduction of label into glucose.The pattern of labelling was consistent with theformation of glucose from propionate via a sym-metrical intermediate such as succinate. No netsynthesis of glucose occurred, however, andlactate was the major product. Lactate wasalso the sole product detected by Pennington &Sutherland (1956) after metabolism of propionateby slices of sheep-rumen epithelium. In thepresence of malonate, however, radioactive pro-pionate gave rise to succinate of high specificactivity, and Pennington & Sutherland (1956)concluded that metabolism of propionate occurredvia fixation of carbon dioxide to form succinate.With the exception of a doubt as to the rate of

formation of propionyl-CoA, the results of thepresent paper are entirely consistent with themetabolism of propionate proceeding in sheep-liverhomogenates via a mitochondrial methylmalonatepathway (Mazumder et al. 1961; Mazumder,Sasakawa & Ochoa, 1963). The failure to detectmethylmalonate among the ether-soluble reactionproducts may be ascribed to the extraordinarystability of the thio ester bond of methylmalonyl-CoA (Stadtman, Overath, Eggerer & Lynen, 1960)leading to a failure to extract the acid into ether.The rate of fixation of 14CO2 by high-speed-blenderhomogenates in the presence of propionyl-CoA isadequate to account for the rate of metabolism ofpropionate by sucrose homogenates, which inturn is more than adequate to account for the ratein liver slices.

Nuclear-free homogenates, however, appear todiffer from slices in the nature of the products.No lactate whatever was produced by homogenates

from propionate, whereas it appeared to be thesole product in slices of liver or rumen epithelium.The dicarboxylic acids that accumulate in nuclear-free homogenates may represent precursors of thelactate produced by slices.The correlation between the rate of consumption

of oxygen and that of propionate was unexpected.It is consistent with the accumulation of dicar-boxylic acids, which was approx. 57% of thepropionate consumed on a molar basis. Pennington& Sutherland (1956) found that up to 50% (38-49%in four observations) of metabolized propionateappeared as lactate in slices ofrumen epithelium.The extent to which endogenous substrates

contribute to the consumption of oxygen whenpropionate is present cannot be gauged, and so thefraction of metabolized propionate that is com-pletely oxidized is not known. The correlationbetween total oxygen and propionate removed,however, suggests that a connexion exists betweenthe rate at which tricarboxylic acid-cycle inter-mediates are oxidized and the rate of consumptionof propionate.

We thank Dr H. R. Marston, F.R.S., for suggesting thisproblem, which was undertaken as part of a general investi-gation of cobalt deficiency in sheep. Thanks are due toDr E. A. Cornish, Mr A. G. Constantine and Miss M. J. Evansof the C.S.I.R.O. Division of Mathematical Statistics forstatistical analysis of results.

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