Biochem. J. (1963) 88, 213

The Metabolism of Acetate and Glucose by the Isolated Perfused Udder2. THE CONTRIBUTION OF ACETATE AND GLUCOSE TO CARBON DIOXIDE

AND MILK CONSTITUENTS*

BY D. C. HARDWICK, J. L. LINZELL AND T. B. MEPHAMAgricultural Research Council Institute of Animal Physiology, Babraham, Cambridge

(Received 30 January 1963)

Recent work has emphasized that both glucoseand acetate are important metabolites in theruminant. Annison & Lindsay (1961) estimatethat 33 % of expired C02 is derived directly fromacetate in the fed sheep, and Annison & White(1961) find that, under these conditions, 23 %comes from glucose; both these values are sensitiveto the nutritional state of the animal. Considerablequantities of both glucose and acetate are taken upby the goat udder in vivo [Linzell (1960); some ofthe values for glucose uptake in this paper werewrong and an erratum has been published inJ. Physiol. (1962), 163, 1], but it is not easy todetermine in the whole animal the relative amountsof C02 coming from each substance in the meta-bolism of the udder itself (Wood, Gillespie, Hansen,Wood & Hardenbrooke, 1959). It is technicallyeasier in the isolated organ because (a) the additionof substrates can be controlled, (b) radioactivesubstances can readily be infused for long periods,so that steady-state levels are more likely to bereached, (c) the blood flow is known, and (d) themetabolism of only one organ is involved.Although glucose is not a significant precursor of

milk fatty acids in the ruminant (Balmain, Folley& Glascock, 1954; Kleiber et al. 1955), considerableamounts of milk citrate come from glucose in thecow (Kleiber et al. 1955; Tombropoulos & Kleiber,1961), and half the C02 from glucose may beproduced by routes other than the pentose phos-phate pathway (Black, Kleiber, Butterworth,Brubacher & Kaneko, 1957a). These discordantresults suggest that the relationship betweenglucose, acetyl-CoA and the tricarboxylic acidcycle in the ruminant udder is not clear.

Further work (D. C. Hardwick, J. L. Linzell &S. M. Price, unpublished work) to elucidate themetabolic routes of these substances by using in-hibitors and substituents for glucose gave resultsthat were difficult to interpret. In the presence ofacetate and amino acids, the following substanceswere tested as glucose substitutes: lactose, galac-tose, mannose, fructose, ribose, glycerol, pyruvate,,B-hydroxybutyrate, propionate, fumarate andmalate. None supported milk secretion, and only

* Part 1: Hardwick, Linzell & Price (1961).

mannose and propionate caused an oxygen uptakesimilar to that with glucose. Lactose, galactose,fructose, pyruvate and fl-hydroxybutyrate weretaken up by the gland, but at a rate lower thanthat for glucose. Transport into the cell or, in somecases, phosphorylation may have been limiting formany of these substances. With inhibitors,although milk secretion was decreased by potas-sium cyanide, 2,4-dinitrophenol and sodiumfluoride, other inhibitors (e.g. fluoroacetate andfluorocitrate) could not be assessed because theyhad non-specific effects on other tissues of the gland(e.g. they caused vasoconstriction and oedema).Hardwick, Linzell & Price (1961) have shown

that in the isolated udder maximal milk productionof normal composition depends on both glucose andacetate; it was not clearly shown, however, thatlactose and fat were synthesized from the addedglucose and acetate. We have therefore now studiedthe transfer of 14C from [14C]glucose and [14C]_acetate, not only to C02, but also to lactose, tri-glyceride fatty acids, triglyceride glycerol andcitrate in the milk formed in vitro by the perfusedudder.

METHODS

Treatment of mammary glands. Mammary glands weretaken from Welsh goats in early lactation (3-8 weeks)giving 22-34 ml./hr./gland. The perfusions and generalanalysis were carried out as described by Hardwick &Linzell (1960) and Hardwick et at. (1961), with modificationto enable the total respired 14CO2 to be collected. For thispurpose the Hooker-type oxygenators were replaced by asingle membrane-type 'lung' (Peirce, 1960) to provide anenclosed gas circuit and ensure that the venous bloodreached the lung without any prior gas exchange. Themembrane 'lung' was fed by a stromuhr-type pump thatalso measured the venous blood flow (Kisin & Tsaturov,1961). The oxygen, supplied from a spirometer, was circu-lated through the 'lung' and two C02 traps in a closedcircuit. (The first C02 trap, containing 200 ml. of O 1 N-NaOH, was changed hourly, and the second, containing200 ml. of N-NaOH, was checked at the end of the experi-ment.) The membrane 'lung' did not always supply suffi-cient oxygen and had to be supplemented from time to timeby the Hooker-type oxygenator which was not completelygas-tight. To ensure that exchange of C02 between theblood and artificial kidney fluid did not vitiate the measure-

213

D. C. HARDWICK, J. L. LINZELL AND T. B. MEPHAMments, the 'kidney' was excluded from the circuit duringsome periods of the experiment. Neomycin (10 ,ug./ml.) andpenicillin (100 units/ml.) were used in the perfusion fluidand artificial kidney fluid because they gave a wider anti-bacterial cover than the streptomycin and penicillinpreviously used.The glands were starved for the first 2 hr. to remove as

much of the endogenous substrates and intermediarymetabolites as possible. After the second hour a substratemixture was infused into the perfusion fluid continuouslyat a rate appropriate for the amount of milk being formed.The mixture consisted of glucose, acetate and amino acidsapproximately in the proportions taken up by the glandin vivo (Hardwick & Linzell, 1960); to this mixture wasadded either uniformly labelled [14C]glucose ([U-14C]-glucose) (20,pc/100 ml.; 6-63,umc/mg. of glucose C) or[2-"4C]acetate (10p/c/100 ml.; 10,umc/mg. of acetate C).

Carbon dioxide. The CO2 output was determined everyhour by titrating the NaOH in the first trap betweenpH 9 0 (phenolphthalein) and pH 3.3 (bromophenol blue).The contents of the second trap were titrated at the end ofthe experiment; about 10% of the C02 was trapped here.A portion of the 0 1 N-NaOH from the first trap was

acidified with HCI and the C02 evolved was trapped asBaCO3 in 3 ml. of 0-IN-Ba(OH)2. The BaCO2 was centri-fuged, washed twice with water and suspended in ethanol.The CO2 from samples of perfusion fluid was similarlyevolved and collected with the use of cetyl alcohol toprevent frothing. Suspensions of BaCO3 were plated out on5 cm.2 aluminium planchets and dried, and the BaCO3 wasdetermined both by weighing and, after counting, by dis-solving in an excess of 0-05N-HCI and back-titrating with0-01N-NaOH with methyl red as indicator. The radio-activity of the respired gas was also monitored by passingthe gas from the 'lung', before absorption in caustic soda,through a 1 1. flask in which were mounted a matched pairof Geiger-Miiller tubes. The counting efficiency of thiswas 0.12%.

Acetate. This was isolated from plasma and kidney fluidsby steam-distillation at acid pH (bromophenol blue),determined by titration and counted as described byAnnison & Lindsay (1961).

Glucose. This was determined with glucose oxidase(Huggett & Nixon, 1957) and isolated as the phenyl-hydrazone (Baxter, Kleiber & Black, 1955), which wasdetermined by its extinction at 390 m,. in ethanol, and thissolution was dried on a planchet for counting.

Lactose. This was isolated, from 2 ml. of milk, as de-scribed by Lucas, Kaneko, Hirohara & Kleiber (1959). Thefirst precipitation of whey proteins was omitted and theheating at pH 6-8-7 was at 1000. The cation- and anion-exchange resin columns were each 8 cm. x 0-6 cm. Lactosewas further purified by chromatography on Whatman3MM paper by using propan-2-ol-water (4:1, v/v) andeluting with water; it was determined by copper reductionwith the reagents of Somogyi (1952) and Nelson (1944).

Citrate. This was eluted from the anion-exchange resinused, as described above, for de-ionizing the lactose. Theelution was performed with a formic acid gradient madeby running 6N-formic acid into 100 ml. of water in a100 ml. flask; the flow rate was about 0-5 ml./min. Citrateappeared between 60 and 90 ml. of effluent. The content ofthe fractions was determined by the method of McArdle(1955). The citrate was further purified by precipitation of

the sodium salt from ethanol, and, in some cases, by re-

chromatographing the acid on Dowex 2 resin as above.Fatty acid8. The milk fat was extracted in a Soxhlet

apparatus with ether-light petroleum (b.p. 40-60°) (1:1,v/v) either from 2 ml. of milk previously dried at 950 or

from the dried casein precipitate obtained during theseparation of lactose and citrate. This was hydrolysed with5 ml. of methanolic 12% (w/v) KOH for 3 hr. under refluxon a boiling-water bath, and the residue was evaporated todryness on a water bath. After dissolution in 5 ml. ofwater, the solution was acidified with about 1 ml. ofION-H2SO4, and the fatty acids and non-saponifiablematerial were extracted with ether which was washed wellwith water. The aqueous layer was preserved for glycerolextraction (see below). The fatty acids were extracted withN-NaOH, and the aqueous phase was washed with ether andacidified. The acid aqueous phase was extracted with etherwhich was washed well with water. This extraction methodwas used to ensure that no acetate was counted in the fattyacid fraction, so that any contamination of the milk byblood or tissue fluid would not interfere with the measure-

ments. The recovery of [14C]acetate added to milk fat wasless than 0-2%. Some propionate and butyrate may alsohave been lost. The fatty acids were determined byweighing and by titrating 50-500,ug. in 2 ml. of methanolagainst aq. 001N-NaOH in a Conway burette with phenolred as indicator. Normally these observations gave an

equiv. wt. of about 230, which agrees with published values(Macy, Kelly & Sloan, 1953). In the acetate experimentsthe acids were divided into volatile and non-volatilefractions by steam-distilling 20-50 mg. of material in a

10 ml. flask for 20-30 min.; about 30 ml. of distillate was

collected. The flask and residual fluid were extracted withether to recover the non-volatile acids, and the condenserand distillate similarly treated to obtain the volatile acids.Determinations were made as above. The non-volatileacids gave an equiv. wt. of about 260, but the volatile acidsoften had an equiv. wt. higher than that calculated frompublished data on goat-milk fatty acids, suggesting thepresence of a non-acidic impurity. Since the quantitieswere small it was decided to calculate the weight ofmaterial from the titration and an equiv. wt. of 135.

Glycerol. The specific radioactivity was determined afterseparation as the tribenzoate (Black et al. 1957a). This wasfiltered, washed with water and recrystallized from ethanolby adding 4 vol. of water. The ester was determined by itsextinction at 275 m,u in ethanol. This method of benzoyl-ating glycerol was not always successful.

Counting. All substances were counted on 5 cm.2aluminium planchets in a Nuclear-Chicago gas-flow counterwith a Micromil window. The counter efficiency was calcu-lated to be 30%. Counts were corrected to infinite thinnessby using curves for individual substances. Specific radio-activities are expressed as counts/min./mg. of C to ensure

that the values are comparable for the various substancesmeasured.

Calculations. The total counts in any one product(specific radioactivity x mg. of C in product) up to time tdivided by the total counts added as precursor (specificradioactivity x mg. of C in precursor) up to time t andmultiplied by 100 is referred to as the 'precursor quotient'.When the specific radioactivities of the precursor andproduct are constant this is a measure of the percentage ofthe precursor forming the product. In our experiments

214 1963

MAMMARY METABOLISM OF GLUCOSE AND ACETATEsuch steady states were not always reached or maintained.We have therefore used the whole period of infusion for thecalculation, and because of the slow appearance of radio-activity in the milk the estimates are minimal.

In contrast with this, the ratio of specific radioactivitiesof product to precursor (multiplied by 100) when pre-cursor and product have constant specific radioactivitiescan be used to measure the percentage of product comingfrom the precursor. This is referred to as the 'productquotient', and is similar to the 'transfer quotient' ofKleiber (1954).

RESULTS AND DISCUSSION

Two experiments with [U-14C]glucose and twowith [2-14C]acetate were done. Table 1 shows theprecursor and product quotients in all four experi-ments and illustrates the essential similarity of thecorresponding pairs of results. The detailed resultsof the two better experiments are given in Figs. 1([U-14C]glucose) and 2 ([2-_4C]acetate), and resultsquoted in the text are calculated from these. Thefindings will be discussed under four headings: theproduction of C02, the synthesis of lactose, thesynthesis of fatty acid and of glycerol, and therelation of glucose and acetate to the tricarboxylicacid cycle.

(1) Carbon dioxide. The specific radioactivity ofCO2 reached a constant level in experiments withboth acetate and glucose, so that the ratios of

specific radioactivities of CO2 to those of precursors('product quotients') are valid measures of theproportion of C02 coming from each substance.Thus, whereas about 40% of expired C02 camefrom glucose, only 14% came from acetate. The'precursor quotient' suggests that 12% of addedglucose, but only 2-5% of acetate, appeared asC02. However, these values are unreliable becausetotal C02 output was not measured; during periodswhen the 'kidney' was excluded, accumulation ofNa+ ions from sodium acetate probably boundsignificant quantities of C02 in the plasma, a factoverlooked at the time. Table 1 also gives valuescalculated from the average C02 production in iso-lated perfused udders (36 observations) and maybe more correct. In any case these are, of necessity,minimum figures, because of the time lag of 2-3 hr.between the entry of glucose or acetate into theudder and the reaching of steady-state levels ofC02 specific radioactivity. The size of the initialpool of intermediates will affect the time requiredto reach the steady state, but not the value of thesteady-state level, which is controlled by therelative entry of labelled and unlabelled precursors.We tried to reduce the initial pool size by starvingthe gland for 2 hr. Clearly at steady state 40-50%of the C02 produced was from neither the glucosenor the acetate added. Possible sources of un-labelled carbon are the added amino acids (by

Table 1. Transfer of 14C from uniformly labelled [14C]glUC08e and [2_14C]acetate to carbon dioxideand milk constituents

Four perfusion experiments were carried out. Experimental details are given in the text. In Expt. 128 someunlabelled glucose (29% of the total) was infused before the [U-14C]glucose. In Expt. 130 the gland was anoxicbetween 1-5 and 55 hr. owing to partial 'lung' failure; the labelling of citrate was higher during this period. (Thevalue in parenthesis is calculated from the specific radioactivity of the milk citrate obtained during the period ofanoxia.)

Labelled substrate ... ... ... ... ...

Expt. no. ... ... ... ... ... ... ...

Period of infusion (hr.) ... ... ... ...

C02LactoseGlycerolTotal fatty acidsVolatile fatty acidsNon-volatile fatty acidsCitrate

C02 (measured)C02 (estimated from average C02 output)LactoseGlycerolTotal fatty acidsCitrate

[U-L4C]Glucose

128 134

[2-14C]Acetater1130 136

4th-lOth 2nd-8th 2nd-9th 2nd-11Product quotients

(percentage of product formed from precursor)

47499.70-15

3977

> 230-47

140-10-57

> 34> 42

n

Ith

140-27

> 2> 48

90

30 41 44 (72) 33Precursor quotients

(percentage of precursor transferred to product)

512-55-80-080-020-1

11-71225-30-270-10-38

4-510-50-0350-02

10-70-54

2-550-170-0613-10-5

Vol. 88 215

D. C. HARDWICK, J. L. LINZELL AND T. B. MEPHAMtransamination) and tissue stores of fat and carbo-hydrates. It was calculated that such a CO2production in a 10 hr. experiment represents lessthan 1 % of the weight of the gland tissue. Theutilization of unlabelled material can be variable, asillustrated in Fig. 2; between 5 and 9 hr. there wasa fall in the specific radioactivities not only ofproducts such as C02 and citrate but also of that ofplasma acetate.The greater amount of C02 coming from glucose

than from acetate is consistent with the highuptake of glucose by lactating glands of consciousgoats. From the finding of Black et al. (1957a)(and by assuming the validity of their calculations)and the present results, it may be calculated that13 % of the total C02 may come from addedglucose via the tricarboxylic acid cycle; thusBlack et al. (1957 a) calculated that at least 50% ofglucose-generated C02 comes from the pentosephosphate pathway in the cow, and, if the restcomes from the glycolysis route, one-third isproduced before the tricarboxylic acid cycle andtwo-thirds within it; thus from our results two-thirds of one-half of 40 %, i.e. 13 %, of all the C02produced comes from glucose via the tricarboxylicacid cycle. It seems likely therefore (on theassumption made by Black et al. 1957 a) that

glucose and the methyl carbon of acetate supplyapproximately equal amounts of the total C02produced by the tricarboxylic acid cycle. It ispossible that the carboxyl carbon of acetate maycontribute more carbon to C02 than does themethyl carbon, because of possible exchange andsynthetic reactions at the a-oxoglutarate level,which would dilute the methyl carbon after thecarboxyl carbon had been released as C02 (Wein-man, Strisower & Chaikoff, 1957). The results ofBlack, Kleiber, Smith & Stewart (1957b) suggestthat such dilution is of minor importance.

(2) Synthesis of lactose. The importance ofglucose as a precursor of lactose was first suggestedby Foa's (1912) work with perfused sheep udders,and demonstrated by Folley, Popjak & Malpress(1952) in the rabbit and by Barry (1952) in thegoat. The present results showed that this is alsotrue for the isolated perfused udder. The 'productquotient' shows that 77 % of the lactose producedcame from added glucose, whereas only 0 3% camefrom added acetate. The specific radioactivity ofthe plasma glucose fell during the experiment with[U-14C]glucose, indicating a dilution by unlabelledglucose. Part at least ofthis dilution is accounted forby the influx of unlabelled glucose from the kidney,which was in circuit for 30 min. during the sixth

(counts/min./mg. of C)

(counts/min./mg. of C)

Citrate (mg./100 g.)

Lactose (g/100 g.)

_ Fat (g./100 g.)

Hourly yield (% of previous)

10

200 0

100

10-6-2-10050a-

v4t: @--*--It _ _ Lactose

0

_ = _ ____ Fat

I -I I

1 2 3 4 5 6 7 8Time of perfusion (hr.)

Fig. 1. Perfusion of one goat mammary gland with [U-_4C]glucose (Expt. 134). The gland, weighing 360 g.,

yielded 22 ml. of milk/hr. before the experiment (average of 7 days, shown as 100% on the Figure). The infusionof substrates with [U-14C]glucose was started at 2 hr. In the next 6 hr. 9-75 g. of glucose and 3-4 g. of acetatewere taken up. Some non-labelled glucose entered the circuit in the sixth hour (black bar). The mean bloodflow during perfusion was 39 ml./100 g./min.; the 02 consumption was about 2-2 ml./100 g./min., at maximum.The horizontal broken lines for milk composition represent the average contents for 3 days before the ex-

periment, and those for specific radioactivity the level of radioactivity of the infused glucose.

.

0C.

0ICto

C)._

MI

216 1963

1.

MAMMARY METABOLISM OF GLUCOSE AND ACETATE

hour of perfusion. If the 'product quotient' iscalculated from the plasma specific radioactivity,rather than from that of the infused substrate,100% of lactose came from plasma glucose (Fig. 1);25 % of the added glucose was converted intolactose, the highest 'precursor quotient' observedin these experiments. The specific radioactivity wasconstant after the third hour, suggesting smallpools of intermediates relative to the rate of lactosesecretion.

(3) Synthes8i offat. The importance of acetate asa precursor offatty acids in the ruminant udder wasfirst demonstrated in the whole animal by PopjAk,Folley & French (1951), and the same group showedthat glucose was not an important precursor offatty acids (Bahmain et al. 1954).

(a) Fatty acids. The present results also showthat acetate was an important precursor of fattyacids, whereas negligible quantities of glucose wereincorporated into these compounds, the reverse ofthe findings with lactose. The 'product quotients'indicate that at least 50 % of the total fatty acidswere derived from acetate, whereas 0-5 % camefrom glucose. Constant specific radioactivity wasnot reached so that the values are minimal. It is

unlikely that the results with [1-14C]acetate wouldbe different, because the C2 moiety is not believedto be broken during fatty acid synthesis. Thevolatile fatty acids, unlike the non-volatile fraction,did reach a constant specific radioactivity whichwas equal to that of the acetate being given; thus90-100% of the volatile fatty acids came fromacetate in our perfusion (Fig. 2).

It should be emphasized that these experimentswere done in the absence of added triglycerides andhigher fatty acids. When whole blood is used,relatively small quantities of these substances areavailable from this source, and variable amountswill be present in the udder, though it is not certainhow much of this is available for milk fat. The'product quotient' indicates that at least 30 % ofthe non-volatile fatty acids can be synthesizedfrom added acetate in vitro, but we do not yetknow whether all the fatty acids present in thisfraction are labelled.The cow udder takes up neutral fat from the

blood in vivo (Lintzel, 1934), and the findings ofRiis, Luick & Kleiber (1960) suggest that 50 % ofthe fatty acids of the milk come from colloidalfatty material in the plasma. On the other hand, in

(counts/min./mg. of C)

(counts/min./mg. of C)

Citrate (mg./100 g.)

Lactose (g./100 g.)

Fat (g./100 g.)

Hourly yield (% ofprevious)

7000 -

6000 -

5000 -

4000 -

3000 -

2000-1000 -

0-200-100 -

0-200100

6i148_

50_l

Plasma volatile, fatty acids

,_ -~',, -Milk volatile fatty acids- Substrate volatile

I>/ -,/ fatty acidsX' ",' - / ~Milk total fatty acids

',I~ _ ~.. Milk citrateI RkMilk non-volatileI ~~~-*-~~~~-~~ co2 ~~~fatty acids

Milk-fat glycerol

| | =*.-*-t-* ._ . ._ Lactose

a Citrate

- - .--- - - -- Lactose

. ______ ==;===; ~~ Fat

I_I I0 1 2 3 4 5 6 7 8 9 10 11

Time of perfusion (hr.)

Fig. 2. Perfusion of one goat mammary gland with [2-14C]acetate (Expt. 136). The gland, weighing 413 g.,

yielded 34-4 ml. of milk/hr. before the experiment (average of 7 days, shown as 100% on the Figure). Theinfusion of substrates with [2-14C]acetate was started at 2 hr. In the next 9 hr. 28-2 g. of glucose and 9-6 g.

of acetate were taken up. The mean blood flow during perfusion was 65 ml./100 g./min.; the 02 consumption wasabout 4 ml./100 g./min. at maximum. The horizontal broken lines for milk composition represent the average

contents for 3 days before the experiment, and those for the specific radioactivity the level of radioactivity ofthe infused acetate.

Vol. 88 217

.

0Ca0

I

0C).

l-

U_

D. C. HARDWICK, J. L. LINZELL AND T. B. MEPHAM

the isolated perfused udder, without added tri-glyceride, D. C. Hardwick, J. L. Linzell & P. V. F.Ward (unpublished work) have shown that milktriglyceride secreted in the presence, or even thesmall quantity secreted in the absence, of acetatedoes not differ markedly in fatty acid compositionfrom that of normal milk. Ganguly (1960) suggeststhat the cow udder is capable of synthesizing allthe milk fatty acids from acetate, but he did notdistinguish between C16 and C18 acids. Such abilitywould be surprising, in view of the findings byPopjak, French, Hunter & Martin (1951) that[14C]acetate contributed largely to acids up to C16but little to C,8 acid synthesis for milk fat in thewhole goat.

(b) Glycerol synthesis. Glucose is generallyaccepted as the likely precursor of milk-fat gly-cerol, but Glascock (1958) concluded that this doesnot take place in the mammary gland whereasLuick (1960) considered that it does, althoughLuick & Kleiber (1961) indicated that this isdifficult to prove in the whole animal. We foundthat the perfused udder can indeed synthesizeglyceride glycerol from glucose; at least 23% camefrom glucose but only 2 % from acetate. The rate ofrise in glycerol specific radioactivity resembled thatof fatty acids: in both cases very little radioactivityappeared in the first 3-4 hr., after which the specificradioactivity rose throughout the experiment anddid not reach steady state in 8 hr., in contrast withthe curves for lactose, C02 and citrate. The muchslower rise compared with that of lactose indicatesthat the gland has a larger store of milk triglycerideor precursors than of lactose or precursors either inthe cells or in the alveoli. The similarity betweenthe glycerol and fatty acid curves suggests eitherthat the pools are of triglyceride or that the glyceroland fatty acid-precursor pools are of similar size.In addition the initial period of 3-4 hr. when littleor no radioactivity appeared in the fat may alsomean that there is a pool of preformed triglyceride,possibly in the alveoli, with which newly synthe-sized material does not readily mix.

(4) Formation of citrate. Kleiber et al. (1955)were the first to demonstrate that glucose contri-butes to milk citrate in the whole cow. In ourexperiments both glucose and acetate contributedto the synthesis of milk citrate, and the 'productquotient' shows that approx. 30% of this substancecame from each precursor (Figs. 1, 2). This is con-sistent with the relative quantity of carbon fromglucose and acetate estimated to be entering thetricarboxylic acid cycle on the basis ofC02 labelling.This similarity in citrate labelling was in markedcontrast with the different extents of fatty acidsynthesis from acetate and glucose. Our results inthe ruminant may be compared with those ofSpencer & Lowenstein (1962) in a non-ruminant.

They observed incorporation of [14C]citrate intofatty acids in the supernatant fraction of rat-mammary-gland homogenates.Both fatty acids and citrate are generally

believed to be synthesized from acetyl-CoA and,if both glucose and acetate contribute to thesecompounds solely via acetyl-CoA in the mammarygland, then the ratios of the specific radio-activities of milk fatty acid and citrate should bethe same, whether the glucose or the acetate of thesubstrate mixture is labelled. That this is not sosuggests that the assumptions made, in particularthat acetyl-CoA is the sole precursor of these sub-stances and that the cell contains an homogeneouspool of acetyl-CoA, should be examined.

Since citrate is synthesized from oxaloacetateand acetyl-CoA, glucose may contribute to citratevia either or both of these substances. The rate atwhich new oxaloacetate (or other C4 acid) isrequired is determined by losses of cycle acids fromthe system either by synthetic reactions or into themilk. According to Deuel (1957) and Lindsay(1959) glucose is the most likely source of such C4acids. Utter & Keech (1960) have shown thatmarmmalian liver will carboxylate pyruvate tooxaloacetate (cf. Utter, 1959), and Freedman &Nemeth (1961) have obtained evidence for asimilar reaction in young guinea-pig liver. Thereaction has not been demonstrated in mammarytissue but is included in Scheme 1 to indicate apossible route from glucose to citrate.The rate of loss of citrate to the milk can be

calculated for the normal lactating udder, byassuming that the calculations of Black et al.(1957 a) are approximately correct and that glucoseand acetate are the major C02 precursors. Theresult shows that, for every 15 carbon atoms con-verted into C02 by the tricarboxylic acid cycle,only one is lost as milk citrate. However, the C02labelling suggests that glucose and acetate con-tribute approximately equal amounts of carbon tothe cycle, so that the loss of citrate to the milkrepresents only about two-fifteenths of the carboncontributed by glucose to the tricarboxylic acidcycle. The loss of citrate is not enough therefore toaccount for the entry of all the glucose, and, if allthis glucose enters the cycle via oxaloacetate, thereshould be other, much greater, losses of cycle acids.Such losses are rendered less likely by the conclu-sion of Black & Kleiber (1957) that dilution ofcycle acids by the influx of C4 components is slightcompared with the turnover in the cycle.The results presented indicate that glucose and

acetate do not both contribute to the same pool ofacetyl-CoA. It is possible that pyruvate derivedfrom glucose is decarboxylated to acetyl-CoA ata site where it is not available for the synthesis ofmilk fatty acids. This possibility is indicated in

218 1963

Vol. 88 MAMMARY METABOLISM OF GLUCOSE AND ACETATE 219

glucose | |AcetateFat

Lactose Glycerol

Phosphoenol- Fatty acidspyruvateX"\F a

+ PyruvateCO2\ Acetyl-CoA

7F xaloacetate Acetyl CoA/ 1V CitrateJ

Mitochondrion Milk citrate

Scheme 1. Possible relationships of glucose and acetatemetabolism in the lactating goat mammary gland.

Scheme 1 by an intramitochondrial pool of acetyl-CoA synthesized from both pyruvate and acetateand an extramitochondrial pool synthesized onlyfrom acetate. The Scheme assumes that milkcitrate is made in the mitochondria. This has notbeen investigated but seems likely, although Srere& Lipmann (1953) have shown the possibility ofcitrate synthesis in cell-supernatant fractions ofpigeon liver.The two possibilities, synthesis of oxaloacetate

from pyruvate and the existence of two intra-cellular pools of acetyl-CoA, are not mutuallyexclusive, and Scheme 1 suggests possible meta-bolic pathways and sites for these. Whether eitheror both of these suggestions is true, it seems thatthere is a block between pyruvate and the acetyl-CoA that can be used for fatty acid synthesis. Thisis supported by two experiments where pyruvatewas substituted for acetate in the full substratemixture in one of two mammary glands. The fatconcentration of the milk and the total milksecreted were lower on the side receiving pyruvatethan on the side receiving acetate. The resultsuggests that pyruvate cannot be used for fatsynthesis, but this will have to be confirmed by,for example, experiments with [14C]pyruvate. Itwould also seem to be worth while to investigatewhether an enzyme is absent or an inhibitor present.

SUMMARY1. A substrate mixture of glucose, acetate and

amino acids containing either uniformly labelled['4C]glucose ([U-14C]glucose) or [2-14C]acetate has

been infused for 6-9 hr. into the perfusion fluid offour isolated perfused mammary glands of lactatinggoats.

2. In an experiment with [U-14C]glucose, 77%of the milk lactose, 23% of milk triglycerideglycerol, 41 % of milk citrate, 0-5% of milk tri-glyceride fatty acids and 39% of the expired C02came from added glucose.

3. In another experiment with [2-14C]acetate,only 0- 3 % of the milk lactose, 2 % of milk tri-glyceride glycerol and 14% of expired C02 camefrom added acetate. However, 48% of the milktriglyceride fatty acids, 90% of the volatile fattyacids and about 33% of the milk citrate came fromthis precursor.

4. The labelling of milk fatty acids by [2_14C]-acetate but not by [U-14C]glucose is contrastedwith the equal labelling of milk citrate by both.The possibilities of two acetyl-CoA pools and of thecarboxylation of pyruvate are discussed.

5. The slow initial rise in the specific radio-activities of both milk glycerol and fatty acids, ascompared with that of lactose, suggests theexistence of larger stores of triglyceride and pre-cursors than of lactose and precursors.

We are grateful to Mr I. R. Fleet, Mr R. D. Burton andMiss G. Chapman for able technical assistance, and to MrR. R. R. Richbell, Mr J. W. Cornwell and Mr D. C. Hallfor the care of the animals. Dr E. C. Peirce, II, andSpinlab, Knoxville, Tenn., U.S.A., kindly lent us themembrane lung. Dr J. M. Boissard gave us generousassistance with the problem of avoiding bacterial con-tamination during perfusion, and neomycin was given byE. R. Squibb and Co., Liverpool.

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The Arrangement of the Peptide Chains in y-Globulin

BY J. B. FLEISCHMAN, R. R. PORTER AND E. M. PRESSDepartment of Immunology, St Mary's Hospital Medical School, London, W. 2

(Received 29 January 1963)

Although N-terminal amino acid analysis of y-globulin suggested that the number of peptidechains/molecule depended on the species of origin(Porter, 1960), other workers (Edelman, 1959;Edelnan & Poulik, 1961; Fran6k, 1961; Ramel,Stellwagen & Schachman, 1961) showed that, withall the y-globulins examined, reduction in 6M-urealed to a uniform fall in molecular weight from about150 000 to about 50 000. This evidence indicatedthat all y-globulins have the same number ofchains/molecule and that therefore some of the N-terminal amino acids must be unreactive. Theproducts obtained after reduction in urea areinsoluble except in urea solutions, and hence aredifficult to fractionate and have lost all biologicalactivity. It was observed, however (Porter, 1962;Fleischman, Pain & Porter, 1962), that, when y-globulin was reduced with mercaptoethanol in theabsence of urea, five out of twenty disulphidebonds were broken, and that, though the molecularweight at neutral pH was unchanged, acidificationcaused the dissociation into two products whichcould be separated on Sephadex G-75 columnswith 100% recovery and which were soluble andretained biological activity. The molecular weightsof the two components (Pain, 1963) indicated that

there were two larger chains A and two smallerchains B in a molecule of y-globulin. As chains Aand B retained their antigenic activity theirrelation to the three pieces formed by papaindigestion of y-globulin (Porter, 1959) could beestablished and led to the postulation of a diagram-matic structure for y-globulin (Porter, 1962),shown in Scheme 1. The papain-digestion pieces Iand II both contain an antibody-combining siteand are considered to be identical in their generalstructure (Palmer, Mandy & Nisonoff, 1962;Stelos, Radzimski & Pressman, 1962).We now describe a more detailed character-

ization of the products of reduction of wholey-globulin and of the papain-digestion pieces. Theresults substantiate most of the features of thefour-chain structure, the location of papainhydrolysis and the position of the antibody-combining site.

EXPERIMENTAL

Materiat8y-Globulin from horse, human and rabbit serum was

prepared by chromatography on diethylaminoethylcellulose(Sober & Peterson, 1958). Some preparations of rabbity-globulin were also made by precipitation with Na2SO4