Biochem. J. (1980) 188, 619-632 619Printed in Great Britain

Glutamate, Glutamine, Aspartate, Asparagine, Glucose and Ketone-BodyMetabolism in Chick Intestinal Brush-Border Cells

John W. PORTEOUSDepartment ofBiochemistry, University ofAberdeen, Marischal College, Aberdeen AB9 IAS, Scotland, U.K.

(Received 28 September 1979)

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cellsrespired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added D-glucose (5 or 10mM), L-glutamine (2.5 mM) and L-glutamate(2.5 mM) were the only individual substrates which stimulated respiration by chick cells;L-aspartate (2.5 or 6.7mM), glutamate (6.7mM), glutamine (6.7mM), L-alanine (1 or10mM), pyruvate (1 or 2mM), L-lactate (5 or 10mM), butyrate (10mM) and oleate(1 mM) did not stimulate chick cell respiration; L-asparagine (6.7mM) inhibited slightly;glucose (5mM) stimulated more than did 10mM-glucose. 3. Acetoacetate (10mM) andD-3-hydroxybutyrate (10mM) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mM) was consumed moreslowly than 5 mM-glucose; the dominant product of glucose metabolism duringvigorous respiration was lactate; the proportion of glucose converted to lactate wasgreater with 10mM- than with 5mM-glucose. 5. Glutamate and aspartate consumptionrates decreased, and alanine and glutamine consumption rates increased when theirinitial concentrations were raised from 2.5 to 6.7 or 10mM. 6. The metabolic fate ofglucose was little affected by concomitant metabolism of any one of aspartate,glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of theseindividual amino acids. 7. Chick cells produced very little alanine from aspartate and, incontrast to rat cells, likewise produced very little alanine from glutamate or glutamine;in chick cells alanine appeared to be predominantly a product of transamination ofpyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutaminewere formed from aspartate (2.5 or 6.7mM); aspartate and glutamine were formed fromglutamate (2.5 mM) but only aspartate from 6.7 mM-glutamate; glutamate was thedominant product formed from glutamine (6.7 mM) but aspartate only was formed from2.5 mM-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesizeglutamine; glutamine synthesis is always diminished by concomitant metabolism ofglucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10.Proline was formed from glutamine (2.5 mM) but not from glutamine (2.5 mM) + glucose(5 mM) and not from 2.5 mM-glutamate; ornithine was formed from glutamine(2.5mM)+glucose (5.0mM) but not from glutamine alone; serine was formed fromglutamine (2.5 mM) + glucose (5 mM) and from these two substrates plus aspartate(2.5mM). 11. Total intracellular adenine nucleotides (22,umol/g dry wt.) remainedunchanged during incubation of chick cells with glucose. 12. Intracellular glutathione(0.7-0.8mM) was depleted by 40% during incubation of respiring chick cells withoutadded substrates for 75min at 37°C; partial restoration of the lost glutathione wasachieved by incubating cells with L-glutamate + L-cysteine + glycine.

The work described here was carried out in The Recent observations on glucose, ketone body,Metabolic Research Laboratory, Radcliffe Infirmary, glutamine, glutamate and aspartate metabolism inOxford, England, U.K. during leave of absence from the the vascularly and luminally perfused small intestineUniversity of Aberdeen.

0306-3283/80/060619-14$01.50/1 (© 1980 The Biochemical SocietyVol. 188

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in vivo and in vitro have been reviewed (Porteous,1978, 1979). One of several advantages of the tissueperfusion technique is that it permits the study ofmetabolism of substrates supplied alternatively fromeach side of the intestine. A possible shortcoming ofthe technique is that the cellular site of metabolism isnot identified; it is generally assumed that theintestinal columnar absorptive epithelial cells (brush-border cells) are responsible for the observedmetabolism of substrates supplied by the vascularor luminal route. One purpose of the presentinvestigation was to ascertain the capacity ofisolated intact brush-border cells to metabolizealanine, pyruvate, lactate, oleate, butyrate, aceto-acetate, 3-hydroxybutyrate, glutamine, glutamate,asparagine and aspartate. Glucose metabolism hasalso been studied because this substrate is thedominant monomeric component of animal diets andthe dominant free monomer in the circulation;because glucose was apparently converted to lactateonly to a very small degree during its translocationfrom the lumen to the vascular circuit of perfusedrat small intestine (Windmueller & Spaeth, 1978)whereas a substantial conversion of glucose to lac-tate was observed in similar preparations (Hanson& Parsons, 1976, 1977, 1978), in everted sacs(Pritchard & Porteous, 1977) and in isolated ratjejunal brush-border cells (Towler et al., 1978); andbecause of the expected interplay between aminoacid and glucose metabolism. Observations onglutathione in isolated brush border cells areincluded because of the postulated role of thispeptide in amino acid translocation (Meister & Tate,1976). The prerequisite assurance that such isolatedbrush border cells are intact, representative of theabsorptive epithelium in vivo and essentially free ofother tissue components was provided by earlierstudies on cells isolated from rat jejunum (Towler etal., 1978). In the present study, analogous cells havebeen isolated from chick small intestine partly inorder to provide comparisons with parallel in-vestigations on cells isolated from rat small intestinein this study and by Watford et al. (1979) and partlybecause isolated chick intestinal brush-border cellssurvive longer under incubation conditions in vitrothan do the corresponding rat cells.

Materials and Methods

ReagentsBovine plasma albumin (fraction V) and ovine

testicular hyaluronidase (type 36-231-2) were fromMiles Laboratories, Stoke Poges, Bucks. SL2 4LY,U.K.; the albumin was dialysed as described byKrebs et al. (1974). NAD+, NADH, NADP+,NADPH, the sodium salts of pyruvic acid, 2-oxoglutaric acid and oxaloacetic acid, and allenzyme preparations were from Boehringer Corp.,

Lewes, East Sussex BN7 1LG, U.K. L-Carnitinehydrochloride was from International Enzymes,Windsor, Berks. SL4 5NJ, U.K. Inorganic chemicalsglucose, ethanol and glycerol were analyticalreagent grade. Sodium L-glutamate, L-glutamine,L-aspartic acid, L-asparagine, L-alanine, acetoaceticacid, D-3-hydroxybutyric acid, sodium butyrate,sodium acetate, sodium citrate, sodium succinateand oleic acid were the best commerically availablematerials. L-Lactic acid was prepared by Mr. R.Hems. Decanoyl-L-carnitine, 4-pentenoic acid and4-bromo-octanoic acid were gifts from Dr. D. H.Williamson.Animals

Male Rhode Island Red x Light Sussex chicks(3-5 weeks old) were supplied by Orchard Farms,Great Missenden, Bucks., U.K., fed on Chick StarterCrumbs ACS (BOCM Silcock, Basingstoke, Hants.,U.K.) with free access to water, and were killed bydecapitation when 4-7 weeks old. Incubation ofisolated intestinal brush border cells was startedwithin the next 40min.Preparation ofchick intestinal brush-border cells

The whole of the small intestine below the gizzardand above the junction of the caeca with the largeintestine (Bolton, 1969; Hill, 1976) was excised andthe bilobal pancreas was removed. The lumen ofthe intestine was washed out at room temperature(about 200C) with 50ml of bicarbonate saline(Krebs & Henseleit, 1932) supplemented withdithiothreitol (1.0mM) and albumin (2.5 mg/ml), andequilibrated with 02/CO2 (95 :5), final pH 7.4. Thewashed intestine was slit longitudinally, cut intosegments (about 3 cm) and washed rapidly bysuspension in 2 x 50ml of the same medium at roomtemperature, followed by filtration on a domesticnylon sieve. Washed segments of intestine wereincubated at 37°C for 5min with gentle shaking in100ml of the same medium now also supplementedwith hyaluronidase (1.5 mg/ml) in a stopperedplastic vessel which was continuously gassed with02/CO2 (95 :5). These segments of intestine werefiltered as before, washed twice in 50ml of freshmedium without hyaluronidase, then agitatedvigorously with a plastic rod for 1 min at roomtemperature in a further 50ml of medium withouthyaluronidase to release brush-border cells from theunderlying tissue. The cell suspension was harvestedinto a polypropylene centrifuge tube by filtration ofthe segments on nylon mesh as before. The residualsegments were suspended and agitated again threetimes in the same way and filtered to fill a furtherthree centrifuge tubes with cell suspension. Cellswere sedimented by centrifuging (about 10OOgsustained for 15s at room temperature), and super-natant suspensions were discarded and the looselysedimented cells were gently resuspended in fresh

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AMINO ACID AND GLUCOSE METABOLISM IN CHICK 6

medium, then sedimented once more. The finalcombined cell sediments (3-5g wet wt. from eachintestine) were resuspended to a concentration of5-10mg of cell dry wt./ml in fresh medium of thesame composition as before except that the albuminconcentration was now 25 mg/ml.

Preparation ofratjejunal brush-border cellsThe cell isolation procedure was that described by

Towler et al. (1978) except that (i) the cell isola-tion, washing and incubation medium was thesingle medium described above, and (ii) apart fromthe incubation with hyaluronidase at 37°C, all otherpreparative stages were carried out at room tem-perature (about 20°C). This procedure differs in afew details from that described by Watford et al.(1979). The cells were finally suspended as describedfor chick cells.

Incubation conditionsFreshly isolated cells (10-20mg dry wt.) were

incubated in Krebs-Henseleit bicarbonate salinesupplemented with dithiothreitol (1.0mM), EGTA(0.1 mM) and albumin (25 mg/ml), with and withoutadded substrates as stipulated in the Results section;the total volume of the incubation system was 3 ml.The incubation system was contained in the mainwell of Gevers flasks (Krebs et al., 1974) shakencontinuously (100 oscillations/min) at 37°C; the gasphase was 02/CO2 (95:5). With very few excep-tions, respiration measurements and analyses of themetabolism of endogenous or added substrates were

thus performed on the same cell suspension; excep-tionally, cells and medium were incubated instoppered conical flasks with the same gas phase as

before. Reactions were terminated by acidificationwith ice-cold HCl04 (0.5 M); extraction of cells wascontinued with occasional vortex-mixing during 1 hat 0OC. Residual cell material was sedimented(3000g for 5min at 0OC), the supernatant extractwas retained and neutralized with KOH andKHCO3. In some experiments, cells were separatedrapidly from the incubation medium by sedi-mentation into 0.5 M-HClO4 using a modification ofthe special centrifuge tubes described by Hems et al.(1975); cell extract and medium were thenseparately processed and analysed.

A nalysesOxygen consumption was measured by Warburg

respirometry at constant CO2 concentration (Krebset al., 1974). Other substrates and products weredetermined spectrophotometrically (in neutralizedextracts of cells plus medium, of cells alone, or in themedium alone) as described in Bergmeyer (1974) bythe following authors: glucose (Bergmeyer, H. U.,Bernt, E., Schmidt, F. & Stork, H., pp. 1196-1201);pyruvate (Czok, R. & Lamprecht, W., pp.

Vol. 188

1446-145 1); lactate (Gutmann, I. & Wahlefeld,A. W., pp. 1464-1468); 2-oxoglutarate (Bergmeyer,H. U. & Bernt, E., pp. 1577-1580); oxaloacetate(Wahlefeld, A. W., pp. 1604-1608); alanine (Wil-liamson, D. H., pp. 1679-1685); aspartate andasparagine (Bergmeyer, H. U., Bernt, E., Mollering,H. & Pfleiderer, G., pp. 1696-1700); glutamate(Bernt, E. & Bergmeyer, H. U., pp. 1704-1708);glutamine (Lund, P., pp. 1719-1722); urea (Gut-mann, I. & Bergmeyer, H. U., pp. 1794-1798); NH3(Kun, E. & Kearney, E. B., pp. 1802-1806);D-3-hydroxybutyrate (Williamson, D. H. & Mel-lanby, J., pp. 1836-1839); acetoacetate (Mellanby,J. & Williamson, D. H., pp. 1836-1839); ATP(Lamprecht, W. & Trautschold, I., pp. 2101-2110);ADP and AMP (Jaworek, D., Gruber, W. &Bergmeyer, H. U., pp. 2127-2131). Amino acidsother than those listed above were determined byautomated column chromatography. Reduced andoxidized glutathione were determined as describedby Vifia et al. (1978).

Conduct ofexperiments andpresentation ofresultsTo avoid circadian variations in metabolic activi-

ties, all animals were killed between 10.00h and11.00 h. The amounts of each substrate and productpresent in (a) the incubation medium alone and (b)the medium plus cells, was always determined atzero time and again (c) in the medium plus cells atthe end of each incubation period. Zero-timedeterminations on the medium alone served to checkthe initial substrate concentrations, those on mediumplus cells indicated any initial contribution ofsubstrate or product by the cells to the incubationsystem. The difference between determinations (b)and (c) gave the gross consumption or production ofa given solute (i) in the absence and (ii) in thepresence of any added substrate; results wereexpressed as umol/min per g dry wt. of cells. Grossrates of consumption of 02 by rat brush-border cellsare shown in Table 1. Gross rates of consumption orproduction of a number of solutes by chick cellsincubated in the absence of added substrates areshown in Table 2. As all experiments were con-ducted in the paired manner described above, thechange in consumption or production of each solutecould be calculated in each experiment as thealgebraic difference between results (i) and (ii)described above; in Tables 3 and 4, the means of anumber of such determinations and calculations aregiven, together with standard errors and the sig-nificance of the differences between values of (i) and(ii) as determined by the paired t test.

All experiments on rat brush-border cells werecarried out during January and February 1977.Seasonal variations in metabolic activities of wholeileal tissue have been reported (Neptune, 1965).Most of the results reported here are concerned with

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J. W. PORTEOUS

the metabolism, by suspensions of chick brush-border cells, of glucose and amino acids at relativelylow (L) initial concentrations (Table 3) or atrelatively high (H) initial concentrations (Table 4);these experiments were conducted in the sequence H,L, H, L over the 8-month period February-September1977. Four sets of results were thus available fromcells incubated in the absence of added substrates,and two pairs of sets of results were likewiseavailable from cells incubated with added substratesat different times during the 8 month period. Nostatistically significant differences could be dis-cerned amongst any of the four sets of resultsobtained in the absence of added substrates; theseresults have therefore been collated as a single set(Table 2). Rates of consumption or production ofsolutes incubated in the presence of low initialconcentrations of substrates have likewise beentreated as a single set since no statistically sig-nificant seasonal variations could be detected (Table3). Results obtained after incubating cells withrelatively high concentrations of substrates havebeen treated as a single set (Table 4); in thisparticular instance, the numbers of experiments inthe two subsets were too small to permit testing forseasonal variations.

Results

General characteristics of the isolated cellsThe microscopic appearance of both rat and chick

cells matched that described by Towler et al. (1978)but the ratio wet wt./dry wt. of the cells was lowerthan that previously observed (about 9 for rat cellsand about 10 for chick cells); the decrease in thisratio [from a value of 13 reported by Towler et al.(1978)] was probably caused by the inclusion ofdithiothreitol in the isolation and incubation medium(Watford et al., 1979). Respiration by the rat cellson endogenous substrates or on added glucose(10mM) declined gradually with time so that the rateat 30min was about 80% of that observed during thefirst 10min. Under the same conditions, chick cellsrespired at a steady rate for 60min at 37°C;experiments with rat and chick cells were conductedunder conditions in which oxygen, glucose andindividual amino acids were consumed linearly withtime of incubation (Tables 1, 3 and 4).

Rat and chick brush-border cells compared; rates ofrespiration and substrate consumptionThe rates of respiration by rat brush-border cells

in the absence of added substrates and in thepresence of added glucose (10mM) were indis-tinguishable (Table 1) from those reported byWatford et al. (1979); glutamate (10mM) failed tostimulate rat cell respiration in contrast to themarked stimulation observed with 5mM-glutamate

by Watford et al. (1979), while glutamine (10mM)stimulated respiration (Table 1) more markedly thandid 5mM-glutamine (Watford et al., 1979). Thesevariations in respiration rate in response to differentconcentrations of these two amino acids do not seemto have been noted before; similar concentration-dependent variations will be noted below in the ratesof respiration on added substrates and in the rates ofconsumption of four amino acids by chick brush-border cells. The remaining observations on res-piration by rat brush-border cells (Table 1) areconsistent with those reported by Watford et al.(1979) except that a marked stimulation of rat cellrespiration by added succinate was observed; thislatter observation was also made and commentedupon by Towler et al. (1978).The rate of respiration on endogenous substrates

in chick cells (Table 2) was about 40% greater thanin rat cells (Table 1). Of the substrates tested withchick cells, only D-glucose (5 mM), L-glutamate(2.5 mM) and L-glutamine (2.5 mM) raised respirationrates (Table 3) above the already high rate ofrespiration on endogenous substrates (Table 2).Higher initial concentrations of glucose stimulatedrespiration less than did 5.0mM-glucose, and higherinitial concentrations of glutamate or glutaminefailed to stimulate chick cell respiration (Tables 3and 4). L-Asparagine depressed the respiration rateobserved on endogenous substrates (Table 4);

Table 1. Ratjejunal brush-border-cell respiration ratesBrush-border cells were isolated from rat jejunumas described by Towler et al. (1978) except that theisolation and incubation media were those describedin this paper. Results are shown for the rate ofrespiration in the absence of added substrates andfor the gross rates of respiration in the presenceof named substrates. Values shown are means+ S.E.M. for the number of experiments indicated inbrackets. Values for respiration rates on addedsubstrates were tested by the unpaired t test againstvalues for respiration in the absence of addedsubstrates: ***P<0.001; *P<0.05. All substrateswere added at IOmM except for citrate (5 mM).

Substrate addedNoneD-GlucoseL-GlutamateL-GlutamineL-AspartateAcetateSuccinateCitrateEthanolGlycerol

Respiration rate(,umol 02/min per g dry wt.)

10.2 + 0.9 (22)20.6 + 1.3*** (25)10.3 (2)19.5 + 1.1*** (14)11.8+0.9 (9)10.6 + 1.8 (7)17.4± 1.0*** (21)13.4 + 1.6* (6)9.2 (2)

12.4 (2)

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AMINO ACID AND GLUCOSE METABOLISM IN CHICK

Table 2. Consumption and production (or release) ofsolutes by chick brush-border cells incubated in the

absence ofany added substratesCells were isolated from chick jejunum, thenincubated, in the absence of any added substrates,as indicated in the Materials and Methods section.Values shown are means+ S.E.M. for the numberof experiments shown in brackets. The significancesof the differences between mean values and zeroare given as *P < 0.05; **P < 0.01; ***P < 0.001 .

SoluteOxygenGlucosePyruvateLactateAlanineSerineProlineOrnithineAmmoniaGlutamate2-OxoglutarateGlutamineAspartateAsparagineUrea

Rates of consumption (-) andproduction or release (+)(pmol/min per g dry wt.)-14.1 +0.69*** (14)+0.03 + 0.03 (8)+0.05 +0.03 (13)-0.19 +0.14 (12)+0.21 + 0.05** (14)+0.46 + 0.04 (3)+0.57 + 0.08 (3)+0.05 + 0.03 (3)+0.35 + 0.31 (12)+0.18 +0.06** (16)+0.02 + 0.006* (13)+0.80 + 0.08*** (10)+0.11+0.03** (13)+0.09 + 0.02 (4)-0.03 + 0.01 (3)

L-aspartate failed to stimulate chick cell respiration(Tables 3 and 4) and it was notable that aspartatealso failed to stimulate rat brush-border cell res-piration (Table 1). Pyruvate (1 or 2mM), L-lactate (5or 10mM), L-alanine (1 or 10mM), butyrate(10mM) and oleate (1 mM) also failed to stimulatechick brush-border cell respiration. The scope forquantitative comparison of respiration rates by chickand rat cells is limited (Tables 1, 3 and 4; Watford etal., 1979); both species of intestinal brush-bordercell appear to use glucose and to a lesser extent,glutamine and glutamate, as respiratory substrates.

Glucose was more rapidly consumed by chickbrush-border cells than was any other substratetested but there was a significant decrease in the rateof glucose consumption when the initial concen-tration was raised from 5 to 10mM; decreases inrates of consumption were also observed whenthe initial concentrations of glutamate and aspartatewere raised from 2.5 to 6.7mM (Tables 3 and 4).In contrast, rates of consumption of glutamine bychick cells increased as the initial concentration wasincreased (Tables 3 and 4); similarly, the rate of con-sumption of L-alanine increased from 1.7 to6.4,umol/min per g dry wt. when the initial aminoacid concentration was raised from 1 to 10mM(results not tabulated). The rates of consumptionof acetoacetate (10mM) and D-3-hydroxybutyrate

(10mM) were 4.2 and 3.5,umol/min g dry wt.of cells (results not tabulated) and these rates ofsubstrate consumption were exceeded only by therates of glucose and alanine consumption; 20% ofthe acetoacetate consumed appeared as D-3-hydroxybutyrate, whereas 40% of the hydroxy-butyrate consumed appeared as acetoacetate.Neither of these ketone bodies stimulated chick-cell respiration, in contrast to the stimulation of rat-cell respiration noted by Watford et al. (1979). Therates of glucose consumption by chick cells (Tables3 and 4) were comparable with that observed byWatford et al. (1979) for rat brush-border cells. Therate of consumption of 2.5 mM-glutamate by chickcells (Table 3) was the same as that reported for5.0mM-glutamate by rat cells (Watford et al., 1979)but this rate was markedly diminished in chick cellswhen the initial glutamate concentration was raised(Table 4). Glutamine consumption by chick cells(Tables 3 and 4) proceeded at only 20% of the ratereported for rat cells (Watford et al., 1979).

The metabolic fate of glucose and of selected aminoacids in respiring chick brush-border cells

Table 2 collates results obtained in the absence ofdeliberately added substrates. The high rate of 02consumption by chick cells has already been noted.The only other measurements that could be shownto be significantly different from zero were the meanrates of production or release of 2-oxoglutarate,alanine, glutamate, glutamine and aspartate. Withthe exception of glutamine and ornithine, the meanrate of release of amino acids lay between 0.2 and0.6,umol/min per g dry wt. of cells (of the aminoacids determined solely by automated columnchromatography, only serine, proline and ornithineare shown as representative examples). This slowrelease of amino acids could signify metabolicproduction within the cells or extracellular proteo-lysis by the cells of the albumin in the incubationmedium; the present experiments do not distinguishbetween these possibilities but the rate of release orproduction of any given amino acid was quiteconstant. Ammonia production and lactate con-sumption were more variable from day to day thanthe production or consumption of other solutes inthe absence of added substrates.

Glucose metabolism. The rapid consumption ofglucose (Table 3) was accompanied by an incre-ment in 02 consumption, by increased rates ofpyruvate, lactate and alanine production, and bysmall increases in glutamate and oxoglutarateproduction. On the ammonia consumed, 80% wasaccounted for by the increased alanine and gluta-mate formation though the extreme variability of thevalues for ammonia consumption leave the precisionof this last conclusion in some doubt.

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J. W. PORTEOUS

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Glutamate metabolism. Glutamate was con-sumed at only 20% of the rate of glucose con-sumption (Table 3) and gave only 45% of thestimulation of 02 consumption; the only notableproducts of glutamate metabolism observed wereaspartate and glutamine, the two together accountingfor 74% of the glutamate consumed, but it willbe noted that ammonia consumption was insuf-ficient to account for the glutamine produced. Therate of consumption of glutamate was not sig-nificantly altered by the concomitant presence ofglucose (Table 3) and the rate of production ofaspartate was not significantly altered under thesecircumstances but there was a significant rise in therate of production of oxoglutarate; the respirationrate, and the rates of glucose consumption and ofpyruvate and lactate production were not distin-guishable from the rates observed when glucose wasadded alone but the rate of alanine production wasgreater than the sum of the rates in the presence ofthe individual substrates; the mean rate of glutamineproduction decreased when glucose was also beingmetabolized and the individual values obtained weremore variable. This last point will be taken up againlater.

Glutamine metabolism. Glutamine was con-sumed at 65% of the rate of glutamate con-sumption, an observation that is quantitativelyconsistent with observations by Windmueller &Spaeth (1975, 1976) on the different fates of thesetwo amino acids when absorbed from the intestinallumen; the only notable products detected wereaspartate and proline and these together accountedfor less than half of the glutamine consumed (Table3). Respiration was stimulated to an extent that wasindistinguishable from that observed with addedglutamate. When glucose was added along withglutamine, the rate of glutamine consumption be-came more variable; about 10% of the consumptionwas accounted for by a rise in glutamate pro-duction. The rates of respiration, glucose con-sumption, pyruvate and lactate production wererestored to values closely similar to those seen inother columns (Table 3) whenever glucose wasadded to the cells. There was a notable increase inalanine production to balance the change fromproduction of NH3 when glutamine alone wasmetabolized to a net consumption of ammonia whenglutamine and glucose were metabolized simul-taneously; a small production of ornithine (0.2,mol/min per g dry wt.); and a small serine formation(which was noted in this and only one othercircumstance). When aspartate, glutamine andglucose were added together to the chick cells (Table3), aspartate was consumed at approximately therate previously observed for glutamine consumptionfrom glutamine and glucose, but consumption ofglutamine was now obliterated, glutamate was

formed (presumably as a product of aspartate andglucose metabolism) and there was an enhancedproduction of alanine; otherwise the results ofglucose metabolism were quantitatively closelysimilar to those seen before. A small production ofserine was again evident.

Aspartate metabolism. Aspartate when addedalone to the chick cells failed to stimulate respiration(Table 3). The only notable products detected wereglutamate and a much smaller but significantamount of alanine and some glutamine; togetherthese products accounted for about 55% of theaspartate consumed. Concomitant addition ofglucose produced the familiar pattern of increasedrespiration rate and of glucose metabolism; the rateof consumption of aspartate was not affected butglutamine was no longer formed while the rates ofalanine and glutamate formation increased, and wereaccompanied by a consumption of ammonia.

Serine, proline, ornithine and urea formation. Ofthe amino acids determined solely by automatedcolumn chromatography, only serine, proline andornithine showed any increased rates of synthesis;the increases were small and occurred only in thecircumstances noted (Table 3 and text above).Direct spectrophotometric determinations of gluta-mate, aspartate and alanine gave results in quanti-tative accord with those obtained for these sameamino acids by column chromatography of the samesamples. Thus, though results from only threecolumn chromatographic analyses are reported,results on serine, proline and ornithine productionare probably reliable (Tables 2 and 3). The for-mation of small quantities of ornithine from glu-tamine and glucose noted in the text above is ofinterest in the light of the observation by Wind-mueller & Spaeth (1974) of incorporation ofradioactivity from [14C]glutamine into citrulline byperfused rat intestine. This matter is discussedfurther by Watford et al. (1979).

Results shown in Table 3 for urea consumption orformation are too small in number to draw finalconclusions but it is perhaps significant that onlywhen glucose or aspartate were added to chickcells was any measurable production of urea noted;analogous observations were made whenglutamate + glucose or aspartate + glucose were sub-strates added at relatively high concentration (Table4). Presumably only under these four circum-stances is the intracellular accumulation of argininesufficient to elicit the activity of an endogenousarginase.Summary. The broad picture that emerges from

Table 3 is that the rate of respiration on glucose andthe rate of consumption of glucose is little affectedby the concomitant presence of any of the aminoacids listed; production of pyruvate and lactate isalso little affected by the presence of these amino

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AMINO ACID AND GLUCOSE METABOLISM IN CHICK

acids but alanine production invariably increasesunder these circumstances. This last observation willbe discussed later. On the other hand, the metabolicfate of the individual amino acids is affectedquantitatively as detailed above, by the concomi-tant metabolism of glucose.

The effects of increasing the initial concentrations ofsubstrates

This broad picture of glucose, glutamate,glutamine and aspartate metabolism in chick in-testinal brush-border cells was generally confirmedwhen cells were incubated with higher initial con-centrations of substrates. But, as already noted, themean rates of consumption of glutamate andaspartate were markedly decreased and the meanrate of glutamine consumption was increased athigher initial substrate concentrations. The rates ofalanine formation were not significantly changed byaltering the substrate concentrations (Tables 3 and4); the most notable quantitative differences inmetabolic patterns observed with different initialsubstrate concentrations are described below.

Changes in the metabolism ofamino acids. No netformation of glutamine from glutamate (6.7 mM) wasobserved (Table 4; compare Table 3); the addition ofboth glucose and glutamate at higher initial con-centrations (Table 4) stimulated glutamate con-sumption (which it did not at lower substrateconcentrations; Table 3) and increased the pro-duction of oxoglutarate even more markedly (com-pare Tables 3 and 4).The net conversion of glutamine to glutamate and

NH3 was absent at low substrate concentrations butprominent at higher initial glutamine concentrations(Table 4; compare Table 3). The concomitantaddition of glucose and glutamine, at the higherconcentrations, diminished glutamine consumptionwithout affecting significantly the rates of ammoniaand glutamate production observed from glutaminealone; these observations contrast with those madewhen the same substrates were added singly andtogether at lower concentrations (Table 3).

Whereas aspartate was consumed from a mixtureof glutamine, aspartate and glucose at low initialconcentrations (Table 3) with no significant netglutamine consumption or production, it wasglutamine that was consumed when the initialconcentrations of these three mixed substrates wererelatively high (Table 4); glutamate was formedrapidly and aspartate was formed slowly under theseconditions.

At the higher initial aspartate concentration(Table 4) production of glutamate plus glutamineappeared to account for aspartate consumptionbut there was an inexplicable net production ofNH3;these events contrasted with those observed when alower concentration of aspartate was supplied to the

cells (Table 3). Aspartate consumption was mark-edly stimulated by the concomitant addition ofglucose at the higher substrate concentrations (Table4) but not at the lower concentrations (Table 3).Whether glucose was present or absent at the higherinitial aspartate concentration (Table 4), a sub-stantial fraction of the aspartate consumed could beaccounted for as glutamate and a lesser part asglutamine. This was true only in the absence ofglucose at lower aspartate concentrations; no glu-tamine was formed if glucose was present (Table 3).It was notable that the only circumstance in whichglucose failed to stimulate respiration was when itwas added together with glutamine and aspartate atthe higher initial concentrations (Table 4).

Asparagine was tested only at an initial con-centration of 6.7mm (Table 4). Although it was quiterapidly consumed (1.l,umol/min per g dry wt. ofcells) with formation of an equimolar amount ofNH3, net aspartate and glutamate formation to-gether accounted for only half of the asparagineconsumed. Like aspartate, asparagine depressed therate of respiration observed on endogenous sub-strates (Table 4).

Changes in the metabolism of glucose. It hasalready been remarked that the presence or absenceof selected amino acids had little effect on themetabolic fate of glucose other than to increasealanine formation (Tables 3 and 4). On the otherhand, changes in the initial concentration of glucosedid affect markedly some aspects of glucose meta-bolism. Thus, the glucose-stimulated respiration ratein the presence of 10mM-glucose was only 30% ofthe glucose-stimulated rate when glucose was addedat an initial concentration of 5mm (Tables 3 and4). The mean gross respiration rate in the pres-ence of 10mM-glucose (15.3 ±0.96,umol 02/minper g dry wt. of cells in seven determinations) was82% of that in the presence of 5mM-glucose(18.7 + 0.68,umol 02/min per g dry wt. of cells innine determinations) the two results being sig-nificantly different by the unpaired t test (P< 0.05).The reason for this diminution in respiration rate inthe presence of a higher concentration of glucose arenot clear but the phenomenon could be of consider-able significance for whole-body metabolism; chickplasma contains about 15 mM-glucose. The ratiolactic acid produced/pyruvic acid produced in thepresence of added glucose in all experiments shownin Tables 3 and 4 was 12.2 + 0.69 (mean + S.E.M forten sets of results); assuming that the lactatedehydrogenase-catalysed reaction was close toequilibrium in each instance, the small variationabout the mean value of this ratio suggests that thecytosolic NAD+/NADH ratio was held constantwhether the initial glucose concentration was 5 or10mM. On the other hand, the proportion ofglucose metabolized to lactic acid depended

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markedly on the initial glucose concentration.When glucose (5mM) was added to respiring cells,62.7 + 1.3% of the glucose consumed was convertedto lactic acid (Table 3); when glucose (10mM)was added, 86.8 + 4.2% of the diminished amountof glucose metabolized appeared as lactic acid(Table 4). Wide variations have been reportedin the proportion of glucose metabolized to lacticacid by different and by analogous preparationsof intestine (see Porteous, 1978, 1979). Postulateshave been made concerning the regulation ofthe conversion of glucose to lactic acid (Porteous,1977) and it may be deduced (Porteous, 1978,1979) that the proportion of glucose converted tolactic acid could vary with the direction of entryof glucose into the highly polarized brush-bordercell. The primary mechanisms determining thepronounced increase in the proportion of glucosemetabolized to lactic acid when the initial glucoseconcentration is increased are not clear from thepresent investigations, but an increased rate ofglucose consumption is not associated with theincreased conversion of glucose to lactic acid(Tables 3 and 4).

Adenine nucleotide concentrations in chick brush-border cells

Chick brush-border cells were incubated (a)without added substrates, (b) in the presence of5 mM-glucose, (c) in the presence of glucose(5 mM) + glutamate (2.5 mM) + glycine (2.5 mM) +cysteine (0.2 mM) for 0, 15, 30 and 45min underthe conditions detailed in the Materials andMethods section. Cells were then separated fromthe incubation medium and centrifuged into 0.5M-

HCl04 using slight modifications of the proceduredescribed by Hems et al. (1975). The results from atypical experiment are shown in Table 5. A decreasein the ATP content of each of three sets of cells wasnoted between 30 and 45min of incubation, butthere was a slight overall rise in ATP content of cellsincubated with rather than without substrates. TheADP content of cells rose during the first 15 min or30min of incubation but the AMP content remainedalmost constant with time in the presence or absenceof added substrates. Setting aside these relativelysmall variations, the total mean adenine nucleotidecontent of the cells was 22,mol/g dry wt. and theproportions ATP:ADP:AMP were 0.7:1.0:0.2.The incubation medium was devoid of ATP andAMP but contained ADP equal to about half theADP content of cells incubated without substrates,and equal to about 30% of the ADP content of cellsincubated with the named substrates; the ADP in themedium did not increase during a 45-min incu-bation.

Glutathione in chick intestinal brush-border cellsTypical results from one of three experiments are

shown in Fig. 1. Oxidized glutathione was not foundin either the cells or the incubation medium at anytime interval. Of the total reduced glutathione presentin the incubation system at any time interval, 90%was associated with the cells. Assuming even distri-bution of the glutathione through cell water, theinitial intracellular glutathione concentration wastypically 0.7-0.8 mm. Rapid depletion of cell gluta-thione occurred during incubation without addedsubstrates (Fig. 1); similar losses occurred inisolated hepatocytes (Vifia et al., 1978) but could be

Table 5. The adenine nucleotide content ofbrush-border cellsChick jejunal brush-border cells were isolated and incubated in stoppered flasks, then separated from the incubationmedium as described in the Materials and Methods section. The results shown apply to the cells only; further detailsare given in the text. The results shown are from a single experiment with cells isolated from one intestine; two otherbatches ofisolated cells gave similar results.

Added substratesNone

Glucose (10mM)

Glucose (10mM) + glutamate (1 mM)+ glycine (1 mM) + cysteine (0.2 mM)

Incubationperiod (min)

015304501530450153045

Intracellular [adenine nucleotidesl(,umol/g dry wt.)

r '-ATP7.37.87.36.28.08.48.07.38.79.08.86.8

ADP8.0

12.213.113.68.3

11.612.411.710.610.514.913.3

AMP2.92.42.62.82.92.42.43.92.32.12.32.3

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AMINO ACID AND GLUCOSE METABOLISM IN CHICK

Time (min)

Fig. 1. Reduced-glutathione content of chick brush-border cells

Chick jejunal brush-border cells were isolated andincubated in stoppered flasks, then separated fromthe incubation medium as described in the Materialsand Methods section. The results shown apply to thecells only; further details are given in the text.*, Cells incubated without added substrates; *,cells incubated with L-glutamate (1 mM) + glycine(1 mM) + L-cysteine (0.2 mM). Two further experi-ments gave essentially similar results.

prevented by including 0.2 mM-methionine in thehepatocyte incubation medium. This manoeuvre didnot prevent depletion of the glutathione content ofisolated chick brush-border cells (results not shown).Attempts to sustain the initial intracellular gluta-thione by incubating chick cells with the directprecursors of glutathione synthesis (Fig. 1) exacer-bated the initial depletion of glutathione but laterrestored the intracellular glutathione concentrationto about the same (depleted) level found in cellsincubated without added substrates. This pheno-menon has not been investigated further. No obviouschanges in the adenine nucleotide content of the cellsoccurred in these experiments (Table 5).

Possible endogenous substratesChick intestinal brush-border cells exhibited a

high rate of respiration on endogenous substrates;this respiration was not readily enhanced by additionof any one of several substrates other than lowconcentrations of glucose, glutamate, glutamine(Tables 2, 3, 4 and accompanying text). Theendogenous substrates supporting this high rate ofrespiration have not been identified with certainty;they could include fatty acids. Known inhibitorsof fatty acid catabolism gave equivocal results.

Decanoyl-L-carnitine (1.0mM) did not inhibit endo-genous respiration, 4-pentenoic acid (0.8mM) gavevariable results and 4-bromo-octanoic acid (0.8mM)inhibited endogenous respiration by 20%.

Discussion

For reasons that have been reviewed elsewhere(Porteous, 1978, 1979),preparations of intestine thatare perfused in vivo or in vitro by both the luminaland vascular routes are best suited to studies oftranslocation of solutes in either direction throughwhole intestinal tissue and to elucidation of theconcomitant overall metabolic fate of such solutes.Suspensions of isolated cells, as employed here,cannot distinguish between the possible differentmetabolic fate of substrates entering the cells acrosstheir functionally distinct brush-border and baso-lateral membranes. The use of such cell suspensionsdoes, however, identify positively the particular intes-tinal cell component responsible for the observedmetabolic events, permits direct and continuousmeasurements of oxygen consumption under physio-logically appropriate conditions and allows moredetailed studies of intermediary metabolism than ispossible with the perfusion technique. The isolatedcell suspensions are thus ideal for rapid quantitativesurveys of the metabolic potential of the intestinalepithelium as illustrated by results reported here andelsewhere (Towler et al., 1978; Watford et al., 1979;Porteous et al., 1979; Porteous, 1978, 1979;Morrison & Porteous, 1980); indications of theoperation of putative regulatory mechanisms arealso quickly revealed.

Glutamine synthesis in chick brush-border cellsGlutamine catabolism in small intestine is well

established (Hanson & Parsons, 1977, 1978; Wind-mueller & Spaeth, 1974, 1975, 1976, 1978; Watfordet al., 1979; this paper, Tables 1 and 4). Wind-mueller & Spaeth (1974) suggested that mammalianintestine may also synthesize glutamine, and Pinkus& Windmueller (1978) reported a positive arterio-venous difference in the glutamine concentration ofblood perfusing chick and guinea-pig small intestine.Results shown in Table 3 appear to be the first todemonstrate the capacity of intestinal brush-bordercells to synthesize glutamine and to support thesuggestions and observations from Windmueller'slaboratory. The apparent deficiency in NH3 con-sumption during glutamine synthesis cannot betaken as evidence against glutamine synthesis; it ispossible that other reactions not measured in thisstudy were producing more NH3 when addedglutamate was the sole known substrate (Table 3).The positive evidence for a net synthesis ofglutamine when glutamate (2.5mM) was the soleadded substrate seems secure (Table 3); the mean

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glutamine formed accounted for 60% of the meanglutamate consumed. The variability about the meanvalue for the rate of glutamine formation was suchthat, for the number of experiments performed,P<0.1. Statistically this is insufficient to provideclear-cut proof of conversion of glutamate toglutamine, but marked variability in the rate ofsynthesis of glutamine from glutamate is notunexpected. It is consistent with the susceptibilityof mammalian glutamine synthetase from severalsources to allosteric inhibition of its activity byglycine, serine or alanine in the presence of Mn2+, orof Mg2+ and trace concentrations of Mn2+ or ofMg2+ and orthophosphate (Meister, 1974); seeTables 2 and 3. The slower mean rate of synthesis ofglutamine from glutamate in the presence of glucose(Table 3) could then be caused by partial allostericinhibition of glutamine synthetase by the increasedintracellular concentration of alanine that might beexpected to arise from the 10-fold increase in the rateof formation of alanine under these circumstances(Table 3). The increased variability in the rate ofsynthesis of glutamine in the presence of glucose(Table 3) could be a reflection of quite smallvariations from one experiment to another in theexact intracellular concentrations of allosteric effec-tors, including alanine, during incubation of cellswith both glutamate and glucose. The partiallyinhibited enzyme would presumably exhibit a sig-moid response of reaction velocity to changes insubstrate concentration and would thus also respondmost markedly to any small variations in theintracellular concentrations of substrates close to theKm values; Meister (1974) reports Km=0.18, 2.3and 2.5 mM for NH3, ATP and L-glutamate respec-tively during assay of the uninhibited enzyme invitro. This last value is identical with the extra-cellular glutamate concentration used in the presentexperiments (Table 3). It is notable that a slow rateof glutamine synthesis was also observed fromaspartate, presumably via the glutamate that wasformed slowly from low or high initial concen-trations of aspartate (Tables 3 and 4); again,concomitant metabolism of glucose diminished oreliminated glutamine synthesis, presumably becauseof inhibition of glutamine synthetase by the alaninewhich was then formed.

Net glutamine formation was not observed wheneither glutamate, or glutamate + glucose, were pre-sented at the higher initial concentrations to chickbrush-border cells (Table 4). The precise mechan-isms that prevent glutamine synthesis from higherconcentrations of glutamate alone are not obviousbut could be important in defining the role ofintestine in whole-body metabolism of glutamate andglutamine. It may be significant that y-glutamyl-phosphate is a known intermediate in glutaminesynthesis, and that glutamate is known to inhibit

(Ki= 4.5 mm) other transphosphorylations cata-lysed by highly purified glutamine synthetases(Meister, 1974).

Diferences between rat and chick intestinal brush-border cells

The observation that 10mM-glutamate did notstimulate rat-cell respiration (Table 1) whereas5mM-glutamate did (Watford et al., 1979); and that10 mM-glutamine stimulated rat-cell respiration(Table 1) more than did 5 mM-glutamine (Watford etal., 1979) requires further investigation. The slowproduction of alanine from 6.7 mM-glutamate orglutamine by chick cells (Table 4) contrasts with therapid production of alanine from 5 mM-glutamate orglutamine by rat cells (Watford et al., 1979). Chickcells consumed glutamine (Tables 3 and 4) muchmore slowly than did rat cells (Watford et al., 1979);this alone could, but does not necessarily, accountfor much of the diminished rate of alanine formationfrom glutamine by chick cells as compared with ratcells. The rates of consumption of 5.0mM-glutamateby rat cells (Watford et al., 1979) and of 2.5 mM-glutamate by chick cells (Table 3) were comparable,but raising the initial glutamate concentration to6.7mm diminished markedly the rate of glutamateconsumption by chick cells (Table 4). At com-parable rates of glutamate consumption, chick cellsproduced alanine (Table 3) at only 10% of the rateof production by rat cells (Watford et al., 1979).Since aspartate alone gave rise to a slow butsignificant rate of alanine formation at rates ofaspartate consumption comparable with glutamateconsumption (Table 3), the enzymes thought tocatalyse the conversion of glutamine or glutamate toalanine via malate or oxaloacetate and pyruvate inrat cells (Watford et al., 1979) are presumablypresent in chick cells; but the enzyme (or enzymes)must then be present at much lower activity in chickcells, or be strongly inhibited under the conditions ofincubation used (Table 3). It has then yet to bedecided by experiment whether the marked elevationof the rate of alanine production that wasobserved whenever glucose was a substrate inaddition to glutamate, glutamine, aspartate orglutamine and aspartate (Tables 3 and 4) was due toalleviation of the possible inhibition of the enzymescatalysing the conversion of malate or oxaloacetateto pyruvate, or whether the carbon skeleton ofalanine arose predominantly from glucose cata-bolism in the chick cells. Watford et al. (1979)observed a 1.5-2.5-fold increase in the ratios alanineproduced/glutamate consumed and alanine pro-duced/glutamine consumed when glucose was alsopresent in the incubation medium. In the presentexperiments the ratio alanine produced/amino acidconsumed rose 5- to 15-fold (Table 3) or 2- to20-fold (Table 4) when glucose was added to an

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AMINO ACID AND GLUCOSE METABOLISM IN CHICK 631

incubation medium containing one of glutamate,glutamine or aspartate. Such increases were notapparent in experiments on glutamine and glucosemetabolism by vascularly perfused rat intestine(Tables 1 and 5 of Hanson & Parsons, 1977). Thecircumstantial evidence is thus that most of thealanine produced by chick cells (Tables 3 and 4)arose from transamination of pyruvate produced byglucose metabolism. If rat intestinal brush-bordercells do produce substantial concentrations ofalanine from glutamate (Watford et al., 1979) thenglutamine synthesis from glutamate via any gluta-mine synthetase that was present could well beinhibited by the alanine produced. In contrast, chickbrush-border cells produce alanine only very slowlyfrom glutamate or aspartate (Tables 3 and 4);glutamine synthesis from glutamate or aspartate isthen possible, as already indicated (Table 3).

Values for changes in NH3 production or con-sumption (Tables 2, 3 and 4) were (with threeexceptions) statistically insignificant. But it is notice-able that the enhanced production of alanine thatalways resulted from the incubation of chick cellswith glucose or with glucose + glutamate, glutamine,aspartate or glutamine and aspartate (Tables 3 and4) was also always associated with a small meanconsumption of ammonia (Table 3) in contrast tothe small mean production of NH3 when cells wereincubated without substrates (Table 2) or withglutamine alone (Table 3). Exceptions to this generalrule were seen only when cells were incubated withhigher concentrations of glutamine + glucose orglutamine + aspartate + glucose (Table 4) because,as already pointed out, glutamine at the higherconcentration was predominantly catabolized toglutamate and NH3. Rat brush-border cells alwaysshowed a small mean production of NH3 whenincubated without substrates or with glucose, and asubstantial production when incubated with gluta-mate or glutamine, or with these amino acids plusglucose (Watford et al., 1979).

Other observations on chick brush-border-cellmetabolismAmongst the other observations made in the

present study, the most interesting concern the highrate of chick cell respiration on endogenous sub-strates; the outstanding role of glucose in stimulatingrespiration (except when both glutamine and aspar-tate were also present); the predominant conversionof glucose to lactic acid (even when gross respirationrates were high) and the partial dependence of theextent of this metabolic conversion upon the initialglucose concentration; the variation in the meta-bolic fates of individual amino acids when theirinitial concentrations were varied or when glucosewas also being metabolized. Each of these obser-

vations, like those on glutamine synthesis anddegradation, could be of considerable significancefor an understanding of the role of small intestine inwhole body metabolism. The observed quantitativevariations in metabolism just referred to mightreflect no more than the response of integratedintracellular metabolic fluxes to the imposed changesin extracellular substrate concentrations; equally,true regulatory (allosteric) mechanisms controllingboth vectorial and scalar catalytic events in cellmetabolism may be involved and require elucidation.Care has been taken to establish the functionalintegrity of isolated rat brush-border cells (Towler eta!., 1978) and results on the adenine nucleotidecontents of chick brush-border cells (Table 5)provide further evidence of functional integrity. Butthe results on the persistent diminution of theintracellular glutathione content of the chick cells(Fig. 1) suggest that further work on stabilizationof the cells and their components is required.

I thank the Medical Research Council for a grant todefray laboratory expenses involved in the work reportedhere. I thank Professor Sir Hans Krebs for the generoushospitality and support that he provided in The Meta-bolic Research Laboratory, Radcliffe Infirmary, Oxford.My warm thanks go also to Pat Lund, Dermot William-son, Marion Stubbs, Reg Hems and David Wiggins formaking me welcome; and to Madge Barber, Morag Stuartand Rose Farrell for their willing help on manyoccasions. I am indebted to Professor D. Kerridge,University of Aberdeen, for the statistical analysis ofresults presented here.

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