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Pyridoxal-5-phosphate and Other Factors Affecting the Activity of Alanine Aminotransferase in the Blood of Sheep and Cattle
Res.
IIcf.
Sci. 1974. 16, 40- 46
Pyridoxal-s-phosphate and Other Factors Affecting the Activity of Alanine Alninotransferase in the Blood of Sheep and Cattle J.
W. B OYD AND G . W . R OBERTS
D epartlllent of Veterillary C lilliwl Stlldies, Ullil'ersity (~l Lil'erpoo l, Leallllrst, Nestoll, /IVirral SUMMA R.. Y. Th e alallille (//lIill o trall~Ierase (A LA T) actillities ill the blood ~r sOllie dOlllestic allilllais I/(/ I'e beelJ illlJestigated. H oloellz Ylll e lellels Ivere lIIeasllred ill the abscll ce of pyridoxal-5phosphate (PLP) alld apoC/lz yllle lel'els IIlere lII easllred as the stillllliatioll wllsed by addillg PLP. III th e g llillea-pig, rabbit alld dog, holoellz Ylll e ill blood cells is IOlver thall ill plaslll a. III rat, wt, pig, lIIall , horse, goat, sheep and Will the holoenz Ylll e is IIIl1 ch higher ill cells thall plasllla. T i,e .Yllil/ill ant allilllals, goat, sheep and CO IlJ, Illerej ollnd to be IIlIiqll e ill that their erythrocytes (olltaill relatil'ely large alllollllts (~f A LA T ill the apoC/lz ylll efo rlll . III sheep there was a positi lJc w rrelatioll betllJeell plasllla alld blood ce /l holoenz Ylll es and also betweell lilJer 1IOI0ellz Ylll e alld blood cell apoell z yllle. III experilllcllts with sheep PLP alld w rtisolle actillated IIlhereas DL-pellicillalllillc alld alllillo-ox yacetic acid illhibited A LA T actil'ity . It is postulated that the parallel jlllct//atioll obseY/!ed ill plasllla alld blood cell holoellz Ylll es is dll e to the eJJeets of' actilJators or illhibitors ill blood. THERE HA VE BEEN man y repo rts o f signi fica nt variati ons in the bl ood pl asm a activiti es o f alanine amin o transferase (L-alanine: 2 - 0XOglutarate amin o trans ferase, EC 3.6.12) formerl y ca ll ed glutami c pyru vic transa minase (Ro Llssel & Stallcup , J966; B oyd & Ford, 1967; B oots & Lud w ick, I970; B oots ct al., 1970; To ll crsrud & Gcdd e-Dahl , 1971 ). Attempts havc bcen m ade to rel atc vari ati ons in plasm a alanin c amino transfcrasc (ALA T ) to physiologica l and cn viro nmelltal facto rs, such as age, sex, bod y we ight, ph ys ica l activity, oestrus, pregnancy, lactati on, ll1ilk y ield , environmental temperature and season of the yea r. However, there is so me confli ct bet ween the findin gs o f differcnt invcsti ga to rs and evcn betwecn th e findings o f th e sa m e invcsti ga tors on diffcrent groups of animals. Pyridox al-s-ph os phate (PLP) is the cocnzyme w hi ch co mbin es w ith th c inactive apocnzym e of A LAT to convcrt it to the acti vc holoenzym e. It is known that m ost ALAT in animal tiss uc exists in the ho loenzyme fo rm. but th at som c m ay be prcsent as thc apoen zym e. In th eir assays of A LAT none of th e previo Lls invcsti ga to rs add ed PLP to their assa y
systcm s and so thcy w erc o nl y Illcasurin g end ogcno us holocnzy me. In the prescnt invcsti gati o n the ap ocnzy me was also meas ured by dc termining t1:c stimulating effcct o f PtP. B o th apoenzym c and holoenzyme were m casurcd in pl as n'l3 and bl oo d of vari o us spccics o f animals. Th c rel ati o nships bctwccn thc 2 fo rms o f AL AT wcre invcsti ga ted in shce p and ca ttl e. T he cffects o f pyrid oxine, PLP , pyrid ox ine antagoni sts and co rti sone o n ALA T activity in shce p wcrc investi ga ted .
M ETH ODS
Assay oj A lallill c A lllili otralisferase (ALAT) T hc 2 m cth o ds used wcrc m o difi ca ti o ns of th c m c th o d of w robl cwski & L a D uc (1 956). T hc first Ill cth o d was suitable fo r use w ith a sin g le bca m spcctroph oto m ctcr w hil c th e sccond mc th o d rcq uired a dou bl c bea m instrum cnt. T he 2 m cth o ds gavc co mpa ra bl c rcs ults.
Method
T
H e parini zed b lood was hacm o lyscd b y prepa rin g a I in 10 dilution ill wa ter and o· 5 ml of thi s hac lll olysate was pre-inc ubatcd eithe r w ith or w ith o ut 50 fLg P LP for IS min . T hcn 0'5 IllI so luti o n of J O mM
Alallille Alllillotrall.lj erase ill Blood N a- 2- oxoglutaratc, 400 mM DL alan in c and I 00 mM T ri s acetatc buffer pH 7 '4 was add cd . A ro utin c blank containin g no alanin e w as includcd with cach assay. T he assay w as also appli cd to scrum and ho m ogcnatcs preparcd from livcr bio psy sa mp les . Aftcr in cubati on for 30 min thc amin o transfcrase reacti o n was sto ppcd b y addin g o· 5 ml N-tri chl oroaccti c acid . Thc pro tein prccipitate was rcmoved by centrifu g in g and 1' 0 ml supcrn atant was transferred to a spec troph o to m ctcr cuvcttc and neut ra li zed b y a 1'0 ml N /3 NaOH soluti on . Then 1'0 1111 0' 5 mM rcdu ced NA D in 0'2 M T ris acc tate buffcr was addcd fo llo wed b y 101-'-1 lactatc dchyd roge nasc (L D H) (1 0 m g/ m l). T hc decrcasc in o ptical den sity at 340 nm indica ted th c am o unt of p yruvatc p rodu ccd b y th c amin o transfcrasc. With a li ght path o f J O mm a changc o f o ptical dcnsity o f 0'208 is cquivalent to 0" I-'- m o lc. Hencc th c units of cn zy m c acti vity ca n be calcu lated as I-'- m o les o f pyruvate fo rm cd per min pcr litrc o f blood fro m th e equati o n Activity (iu/l) = C han ge of O .D . per min x 963
TA BLE [ T il E M IC HA ELI S CONST ANT ( K M) fO R AL AN INE AMIN OTRA NSfE RA SE O F V ARI OUS A N IMALS
2-oxog lutarate L-alan inc Rat li ver supern ata nt (at 25 °C) Shecp blood , di alysed (a t 30°C) Shee p pla slll a, di alysed (a t 30°C)
O'l I
2 1'2
Distriblltion of ALA T ill SlIvcel/lllal' Fractiolls (~f LiIJer HOlll oge;l<7tes. Rat and ca ttl c liver samp les were hOI11.ogeni zed and fracti on ated by differential centrifuging and the vari ous cell fracti ons assayed for ALAT activity. The results (Tabl e ll) show th at m ost o f the activity is in the supernatant fracti on. TA BL E II
Method 2 H ae m o lysa tc or plasm a (0 " m l) was prc-incubatcd fo r 15 min with o r w itho ut 50 I-'- g PLP . Aftcr prcin cubati on thc fo ll owin g rcagcnts w erc add cd to givc fin al conccntrati ons o f 5 mM N a 2- o xoglutaratc, r 66 111M L-alanin e, 0'2 mM reduced NAD, IOI-'-g/ml LDB and 100 111M Tri s acctate, pH 7'4 in a fin al volu m c o f 3'0 ml. T hc o ptical dcnsity o f th c tcs ts Wcrc rcad again st blank cuvcttes co ntaining all rcagents except alanin e in a rcco rdin g do uble bca m spectro ph o to m ctcr w ith a cuvette ho ldcr maintain cd at constant temperature. At fir st both m eth ods wc rc ca rri ed o ut at 25 °C but later cxperimcnts we re perfor med at 30°C in acco rd ance w ith thc revi scd rcco mm end ati o ns of th c Intern ati o nal Uni on o f Bi ochcmi stry.
RES ULTS
Kinetic Properties and Distriblltion oj' ALA T ill Liver and Blood Kin etic Properties (!f A LA T ill Blood and Liller Salllpies. The Mi chaeli s constants (K111) for 2oxoglu tarate and L-alan ine were determined by varying the substrate concentrati ons. T he results (Tabl e I) show that the affinit y of ALA T for L-alanine is lo w co mpared w ith its affinit y for 2- oxog lu tarate.
DI STRIB UTI O N OF ALAN I NE AMI NOTRANS I' ERASE IN SU Il CELLU LA Il FIlA CTIONS OF LI VE R H O MOGENATES
Wh olc hOlllogenate Nuclca r fracti on Mitochondri al frac ti on Mi croso mal fracti on Su pern ata nt frac ti on Recovery in fracti ons (% ) Results are th e Ill ca n of
2
Rat
atti c
58 '25 0'67 0'85 0 ·62 55'25 102
0 ·67 0'07 0' 17 O 'l!
0'4 0 104
experim cnts: U ni ts arc iuff!; at
2s oC
Distrivlltioll (~f AL A T il1 Blood. Shee p bl ood was co llected into siliconized gla ss tubes containing disodiulTl EDTA anticoagulant. It was fractionated by centrifug ing into plasma, platel ets, leucocytes and erythrocy tes accordin g to the m ethod of Koj et al. (I 96o). T he results (T able III) show that the acti vity is mainl y in the ery throcytes. Dial ys is had no effect on the A LA T acti viti es o f vari ous fracti ons. Th e F!ffect (! f' PLP 0 1/. A LA T ActilJity ill the P/asllla arId Blood 4 Variolls Anilllal Species T he results (Table IV) are gro uped acco rding to the characteri sti cs o f the ALA T acti vity in bl ood cell s.
J.
42
w, Boyd alld C, W, Roberts
TAnLE III
Challges ill ALA T Actill ity ill PlaslI/a, w llO lc Blood, Blood Cells alld Liller ojElIJes, Bl ood and
D ISTIl IBUTl ON or ALAN INE AM INOTIlANSFEIlASE IN SHEEP nLOOD IN lulL OF THE OIl ICINAL nLOOD AT 30°C
J.
Wh o le blood Pbslll a Cell s an d pbtclcts Hecove r y
2, Wh o le bl ood Pb slll a Pia telets Le ucocy tes Erythrocy tcs Recove r y
- PLl)
+PLP
ro6 7 96 97%
227 10 193 89%
l SI 9 0 0 1 19
241 13 13 0 20 5 96
8S%
liver biopsy sa mples were obta ined from 4 ewes at m onthly interva ls for 3 to 4 m onths, The mean level s of activity are shown in Table V , T he activity in packed bl ood cell s (C) was calcul ated from the packed cell volume (PCV), th e plasma activity (P) and the w hole bl ood activity (B) using the formul a
C=
CD - P X
JOo - PCV) JOo 100 PCV
%
ALA T Actillity ill PlaslI/a, Blood al/d Liller oj Sheep Challges ill ALA T Actillity ill PlaslI/a alld W hole Blood oj Lall/bs, H eparinized bl ood was co ll ected from three 4-month-old lambs and again at about week ly interva ls for 2 month s, In each of the 3 lambs the holoenzym e activity of plasma flu ctuated in para ll el with w hole blood holoenzym e activit y, This is shown for one lamb in Fig, 1. In Fig , 2 th e variati ons in w hole bl oo d ALA T holoenzyme in all 3 lambs arc shown,
Statistical Relatiol/sllips betUJeell ALA T H oloell z yll/e alld ApoCllz yll/e il1 PlaslI/a, Blood alld Liller (!I' Sheep, T he data coll ected from the lambs and ewes were ana lysed to detect any signifi cant correl ati ons, The stati sti ca ll y signifi cant results arc g iven in Table vr. Doth regression equati ons were ca lcul ated for each pa ir of variables because it is uncertain whi ch equation g ives the better description of these relationships , T he signifi cant positi ve correlati ons between plasma holoenzyme and bloo d cell holoenzyme is also presented in th e form of a sca tter diagram (Fi g, 3),
TAnLE IV ALAN INE AMI NOTIlA NSFEIlASE A CTIVITY IN PLASMA AND BLOOD 0 1' DI FFEIlENT AN IMAL SPECIES
PIJsnu - PLP Low ccllu br acri vity G uin ca-p ig 16 '1 ±3'S rbbb it LS' 4± 2'3 Dog 1 1'2±3 '9 Hi g h cc ll ulJr act ivi t y Hat at Pi g Man H o rse
n lood +PLL'
- PLP
+PLP
16 '6±4' 1 17'S±3'3 12'8±4'O
10' 1 ± I '8 I [ '4± I' 2 11 '4± 3'l
15'3 ± 2'3 Il '4± 2' 4 1 L' S± 2'[
w ith li tt le st illlubti o n b y cocn zy nl e 19 '3± I' 4 2 1'6± r '6 1O'6±4'S 12' S ± S' 4 J7' 1 ±3'S 18 'o±2 'O 10'7 ± 7'I 13'S ± 7'2 8'S±2'3 8'8 ±2 '7
Hi g h ccllu br activity w ith consid erab le cocnzy lll e Goat 7'7± I ' 1 II 'O±O'7 hecp 6'r±I' 1 g'2± I '7 Ca rtle II'O±t '9 1]'6 ± 2'4
77'9 ± 19'4 17S'S ± 5 2 '7 96'o ± 7'S 59'6 ±3 0 '6 6S '9 ± 22'3 stilllubti o ll 90'3±37'o 111'O±1 7'8 42'7 ± 22 '4
Ea ch result is the m ean o f 4 samp les fro m d ifferent indi vid llals± SD Units are ill/ l at 2S oC
79 'O± 19'6 17 S'8 ± SS ' ! roS'o±7'3 70 '2±3 2 '7 74 ' S±28'3 166'S±4 2 '8 3TJ'8±4 L'O 128'S ± S3'4
A lanille A lllillotrallsferase ill Blood 500
43
500
400 Whole blood
400
300 200 f
3 00
100
--I
f
o
--I
200
20 Plasma
)0
100
o Time in days
Flc. I. Va riations in the activity o f ALAT holoenzyme (e ) and apoenzyme (4 ) in plasma and w ho le blood o f a normal la m b.
The Effects oj DL-pcllicillalllillc, A lllil'lo-oxyacetic Acid, Pyridoxine RCI, PLP and Cortisolle Acetate 0 /1 A LA T ill Shecp In these experiments control animals were given inj ecti ons o f sterile water. T h e results are shown in T able VlT. In the sheep treated with corti son e there w as an average increase o f 38% in pl asm a glucose.
Time in days
Ftc . 2. Vari atio ns in ALAT holoe nzyme acti vities in wh ole blood of 3 d iffere nt lambs kep t together as a group.
ti o ns used were sel ected aft er the studi es o n the substrate affiniti es o f va ri o us sa mp les (Ta ble I) . The Km. valu es re po rted by H op per & Sega l (1962) fo r rat liver ALAT were I ' I mM fo r 2oxoglutarate and 34 111M fo r L-a lanine. T he substrate concentrati ons used here were simi lar to those repo rted as optimal by H enry et al. (1960).
DISC USSIO N
T he simplest colo rimetri c method o f AL AT assay (Reitman & Frankel, 1956) was unsuitable for investigating the PLP stimul ation o f A LAT beca use PLP fo rm ed a colo ured compl ex w ith the colo ur reagent. With the spectropho tometric m ethod o f Wro bl ewski & La Due (1956) there were difficulti es of m eas urem ent due to the absorbance by blood haemoglo bin at 340 nm . In ea rl y experiments w ith a sing le beam sp ectrophoto m eter the m ethod was m odifr ed so that haemoglobin was precipitated befo re py ruvate determinati on. In later experim ents w ith a do uble beam spectro photo m eter assays co uld be perfo rmed w itho ut haem oglobin precipitatio n. The substra te concentra-
10
0
8
E N
c
6
0
'0
.c 0
E VI E
Q
000
4
0
0
2
0
7
Flc . 3. T he relationshi p between th e activ ities o f ALAT holocnzyme in plas ma and packed blood cell s in ewes. (Pla sma holoenzyme = Ulood cell holoe nzy me x 0'007 + 3'25 infl at 25 °C)
J.
44
W. Boyd alld Co W. Roberts TABLE V
ALANIN E AMIN OTIlANSFEIlA SE ApOENZY ME (A) AND H OLOENZYME (H) A CTIVITIES IN PLASMA, WH OLE BLOOD, BLOOD CELLS AND LIVER OF EWES Pbsm~
ALAT
Wh o le bl ood
Blood cell s
Liver
123 ±64 200 ±74 7 6 ±3 6 38
326± 167 S33± 184 207 ±95 63
37 2 ±84 381 ±92 9±27 2
H S0 3±1°4 H+A 6° 2 ±r oS A 0 09±roo A% (of H+A ) 14
H activ ity is mcasured without adding PLP A acti vity is th e activit y increa se stimulated by addin g PLP Units arc iu/ I o r kg at 2S oC A total of I S sa mples were collected fro m 5 ewes over a peri od of 3 months
TABLE VI SIGNIFICAN CE 0 1' RELATI ONSHIPS BETWEEN ALANINE AMINOTIlANSFEIlASE ApOENZYME (A) AND H OLOENZYME (H) IN LAMBS AND EWES Dependent variable y
Independent Reg ression va riable coeffi cient x X b
C o rrelation coeffi cient
Illtcrccpt +
Probabil ity P<
Lambs (a) Pla s m ~ H = Wh o le b lood H Wh o le bl ood H = PIa S I1l~ H
X Oo029 X 23 °77
+ +
0°98 32° 12
o oS3
0°00 1
Ewes (b) Pla s m ~ H = Whole bloo d H Wh o le bl ood H = Pb slll~ H (c) Pb s m~ H = Blood ccll H Blood cell H = Plasn13 H (d) Li ver H = Whole b loo d A Wh o le bl ood A = Liver H (e) Liver H = Di ood ce ll A Blood ce ll A = Li ver H
X OoO l9 x 24 °02 X Oo007 X 69°99 x 1°58 8 x 0 0287 X 0062 1 x 0°790
+
3° 2 4 9"4 0 3° 2 5 6So52 250 06 30 °34 243° 6 86 08
0 06R
ooor
+ + +
0°7 2 0°68 0°7 0
Both regression eq uati ons have bec n ca lculated for cach pair of va riables Activities wcre ex pressed in ill/I of pbsma, w hole blood or pa cked blood cells and in iu per kg of liver at 2S oC
TABLE VII EFFECT OF ACTIVATORS AN D INHIB ITo nS ON ALAT H OLOENZY ME (H) AND ApOENZYM E (A) IN PLASMA AND BLOOD CELLS 0 1' SHEEP
DL-pen ici ll amin e Amin o- oxyace ti c acid P yrid ox in e HCI P y rid oxa l-s -ph osph ate Cortisone acetate
D~ il y
Number o f shee p trea ted
dose (m g/ kg)
[ 2
10 2 °5
TO
S
5 5
2
2°2 1°0
Duration of treatillent (da ys)
7
ALAT ~s percenta ge of co ntrol v~llIes Ce ll H
Ce ll A
Plasm a H
PIasn13 A
37 a 26a 103 144 b 13 1C
77 76 99 T3 0 122b
43 a
43 c 33 a 100 120
22~
95 13 6a l OT
a Sta ti sti ca l signifi ca nce P < oooO! b P < 0°01 C .. .. P <0 002 The test anim als were dosed by intramusc ular inj ecti o no Co ntrol anim als wcre given inj ecti o ns o f sterile water
11 8
A lanille AIlI/'llotrall.s/erase ill Bloor! In an investiga tion of th e subcellular distributi on o f ALAT in li ver hom ogenates, Rowse ll et aI, (1963) found th at 90 to 95% of ALAT was in the so luble fraction of rat liver. In th e present work 94% of ALAT was in the cytoso l of rat liver. Shee p and cattl e have much lower Ii vcr ALA T than rats (Boyd, 1962) but aga in the ALAT in cattl e liver is mainl y in the cytosol (Table II) . T he di stribution of ALAT in the vario us cell fractions of human blood has been studied by Koj et al. (1960). They found that ALAT is present in plasma and erythrocytes but was n ot detectabl e in leucocytes and platel ets. The results in Table Il[ show a similar distributi on o f ALAT in sheep blood. However, whil e ALAT in human who le blood is only 3- 6 times hi gher than in plasma (Karmen et al., I955; Koj et al., 1960) the ALAT in sheep whole blood is about 16 times higher than in plasnu (Tabl e IIl). It has been reported that preincubati on of lysed human er ythrocytes w ith PLP stimul ates their activity by 27% (Cheney et al., 1965 b). Simi lar trea tment causes 72% stimul ati on o f sheep erythrocyte ALAT activity (Table TIl) . T he PLP stimul ation of ALAT in plasma and ha el1lolysed bl ood from 11 different animal species is sh own in Table IV. Plasma ALAT ranged fronl. 6·J iu jl in sheep to 19'3 ittjl in rats. PLP stimulati on of plasma ALA T ranged from 3% in guin ea-pi gs to 34% in sheep. Haemolysed blood ALAT ranged from TO 'I iujl in g uin ea-pi gs to 175 iu jl in ca ts. PLP stimulati on of hac m olysed bl ood ALA T ranged from zero in rabbits to 200% in ca ttl e. It was possibl e to cl assify the various animals into 3 gro ups on th e basis of ha c moly sed blood ALAT (Tab le IV). In th e first gro up arc g uin ea-pi g, rabbit and dog w hi ch have whole bl ood ALAT simil ar to plasma ALAT indi ca ting rather low ALAT in bl oo d cell s. In the seco nd g roup arc the rat, ca t, pi g , horse and m an wh ich ev id entl y have mu ch hi gher ALA T activity in bl ood cell s than in plasma, but the ALA T in cell s is n ot greatl y stimulated by PLP. In the third gro up arc the ruminant animals, goa ts, shee p and cattl e w ith hi gh blood cellul ar ALAT whi ch is markedl y stimulated b y PLP. It can be ass ulll ed that th e ALA T activity m easured in th e absence
45
of added PLP is holoen zy m e and the activity in crease ca used by PLP is du e to apoenzyme. Thu s a uniqu e feature of these ruminant anim:tl s is the large amount of ALAT apoenzy me in their erythrocytes. In rats it has been found that plasma and erythrocyte ALAT apoenzymes increase in pyrid oxine defi ciency and arc redu ced to n ormal in response to vitamin thera py (Brin, 1964). However, a pyridoxin e deficiency in ruminants is unlikel y beca use of the end ogenous synthesis of B vitamins by ruminal mi croorga lli sm s. Moreover, treating normal sheep w ith large doses o f pyridoxine or PLP did not red uce th e ALA T a poenzymes in bl oo d (Table VII). As stated in the introducti on, several workers have reported rather large Au ctuations in plasm a ALA T in sheep and cattle. The present results show that in sheep the plasm a ho loenzym e Auctuates in parallel with bl ood cell holoenz yme. If the changes in pla sm a holoenzym e were caused by leakage of bl ood cell h o loenzym e there wo uld presumabl y be a simil ar relati onship between plasm a and cellular apoenzy mes. H owever, the apoenzymes remain relatively constant and arc not signifIcantl y related to each other. Since m ature erythrocytes Ca nJlot synthesize prote ins the actual amounts of .A LAT in ery throcytes Illust be fair! y constant, and so the Auctuati on in erythrocyte ho loenzyme activity m ust be due to the effects of acti vators or inhibitors. Tn rats, bl oo d h oloenzyme is activated by corti sone (Cheney et a/., 19653) and inhibited by DLpeni cill amine (Heddle ct al. , J963 ), w hile H o pper & Sega l (I962) have reported that amino-oxyaceti c acid is a powerful ill pitro inhibitor o f holoenzyme. In the present investigati on lIsing sheep it was fo und that corti sone and PLP were activators and DL-penicillamine :md amino-oxyacetic acid were inhibitors of bl oo d holoenzyme (Tabl e VII). It ca n be postulated that th e large Auctuati ons in plasm a and bl oo d cell holoenzym e acti viti es ill sheep and ca ttl e arc at least partly du e to certain products o f rumen fermentati on acting as activa tors or inhibitors. It is possible that the producti on o f PLP by rumen mi croorgani sms Auctuates and co uld be res ponsibl e for th e par.l l1e1 variati ons
J. w. Boyd al1d C . W. in blood cel l and plasma holoenzymes observed in sheep . No explanation can be offered for the significant relationship between ALA T holoenzyme in the liver and apoenzyme in blood cel ls of sheep (Tabl e VI) . It seems unlikely that variati on in one of these factors ca uses variation in the other. Presumably both of these vari ables are governed by a third factor which mi ght be genetical or nutritional. ACKNOWLEDGMENT T his wo rk was supported by a gra nt from the Agricu ltural Research Coun cil. ReceiIJedjof pllblicatit>1I Decelllber 4111, 1972 REFERENCES B OOTS , L. n . & LUDW ICK, T. M. (1970) ]. Dairy Sci. 53. 449 BOOTS, L. R. , L UDWI CK, T. M . & nADER, E. R. (1970) ].
Dairy Sci. 53. 1587
RO/Jerts
J.
B OYD, W. (1962) R es. vet. Sci. 3. 326 B OYD,]. W. & FOR D. E. H. ([ 967) ]. agric. Sci. 68. 385 BRIN, M . (1964) Tile R ed Blood Cell. E d. C. Bishop and D . M. S urgc n o r , p. 451. A cade mi c Prcss, N.Y. C HENEY, M . c., CUR RY , D. M . & BEATON, G. H. (19653)
J.
Call.]' Pllysiol. Pllarlllaw i. 43. 579 C HENEY, M .
c. , SABRY, Z.
]. elill. N lltf. 16. 337
I. & BEATON, G. H. (1965b) A lii.
J. G., M CH EN IlY, E. W. & B EATON , G. H. (1963) Carl. I Biocll elll . Pllysiol. 41.121 5 H ENRY, R. J., C1 Il AM OIU, N., GOLUB, O. J. & BEIlKMAN, S . (1960) A lii . I elill. Palh. 34. 38 [ H OPPER, S. & SECAL, H . L. (1962)]. Bioi. C/WII. 237. 3[ 89 KARMEN, A ., WROBLEWSKI, F. & LA DUE, J. S. (1955) ]. elill. IIl11est. 34. 126 Ko], A ., ZC Ll CZYNSK I, j. M . & FREN DO, ]' (1960) c/ill. C hilli . Acta 5.339 R EITMA N, S. & FIlANKEL. S. (1957) A lii . ]. elill. Patlr. 28. 56 R OUSSEL, ]. D. & STALLCUP, O. T. (1966) A lii .]. Ilet. R es. 27. 1527 R OWSELL, E. V ., TURNEIl, K. V . & C ARNIE, J. A. (1963) Biochelll . J. 89. 65P TOLLEIlSRUD, S. & GEDDE-DAHL, T. W. (1 971) Acta Vet. Scalld. 12. 393 WIlOBlEWSKI, F. & LA DUE, J. S. (1956) Proc. Soc. expo Bioi. M ed. 91. 569 H EDDLE,
IIcf.
Sci. 1974. 16, 40- 46
Pyridoxal-s-phosphate and Other Factors Affecting the Activity of Alanine Alninotransferase in the Blood of Sheep and Cattle J.
W. B OYD AND G . W . R OBERTS
D epartlllent of Veterillary C lilliwl Stlldies, Ullil'ersity (~l Lil'erpoo l, Leallllrst, Nestoll, /IVirral SUMMA R.. Y. Th e alallille (//lIill o trall~Ierase (A LA T) actillities ill the blood ~r sOllie dOlllestic allilllais I/(/ I'e beelJ illlJestigated. H oloellz Ylll e lellels Ivere lIIeasllred ill the abscll ce of pyridoxal-5phosphate (PLP) alld apoC/lz yllle lel'els IIlere lII easllred as the stillllliatioll wllsed by addillg PLP. III th e g llillea-pig, rabbit alld dog, holoellz Ylll e ill blood cells is IOlver thall ill plaslll a. III rat, wt, pig, lIIall , horse, goat, sheep and Will the holoenz Ylll e is IIIl1 ch higher ill cells thall plasllla. T i,e .Yllil/ill ant allilllals, goat, sheep and CO IlJ, Illerej ollnd to be IIlIiqll e ill that their erythrocytes (olltaill relatil'ely large alllollllts (~f A LA T ill the apoC/lz ylll efo rlll . III sheep there was a positi lJc w rrelatioll betllJeell plasllla alld blood ce /l holoenz Ylll es and also betweell lilJer 1IOI0ellz Ylll e alld blood cell apoell z yllle. III experilllcllts with sheep PLP alld w rtisolle actillated IIlhereas DL-pellicillalllillc alld alllillo-ox yacetic acid illhibited A LA T actil'ity . It is postulated that the parallel jlllct//atioll obseY/!ed ill plasllla alld blood cell holoellz Ylll es is dll e to the eJJeets of' actilJators or illhibitors ill blood. THERE HA VE BEEN man y repo rts o f signi fica nt variati ons in the bl ood pl asm a activiti es o f alanine amin o transferase (L-alanine: 2 - 0XOglutarate amin o trans ferase, EC 3.6.12) formerl y ca ll ed glutami c pyru vic transa minase (Ro Llssel & Stallcup , J966; B oyd & Ford, 1967; B oots & Lud w ick, I970; B oots ct al., 1970; To ll crsrud & Gcdd e-Dahl , 1971 ). Attempts havc bcen m ade to rel atc vari ati ons in plasm a alanin c amino transfcrasc (ALA T ) to physiologica l and cn viro nmelltal facto rs, such as age, sex, bod y we ight, ph ys ica l activity, oestrus, pregnancy, lactati on, ll1ilk y ield , environmental temperature and season of the yea r. However, there is so me confli ct bet ween the findin gs o f differcnt invcsti ga to rs and evcn betwecn th e findings o f th e sa m e invcsti ga tors on diffcrent groups of animals. Pyridox al-s-ph os phate (PLP) is the cocnzyme w hi ch co mbin es w ith th c inactive apocnzym e of A LAT to convcrt it to the acti vc holoenzym e. It is known that m ost ALAT in animal tiss uc exists in the ho loenzyme fo rm. but th at som c m ay be prcsent as thc apoen zym e. In th eir assays of A LAT none of th e previo Lls invcsti ga to rs add ed PLP to their assa y
systcm s and so thcy w erc o nl y Illcasurin g end ogcno us holocnzy me. In the prescnt invcsti gati o n the ap ocnzy me was also meas ured by dc termining t1:c stimulating effcct o f PtP. B o th apoenzym c and holoenzyme were m casurcd in pl as n'l3 and bl oo d of vari o us spccics o f animals. Th c rel ati o nships bctwccn thc 2 fo rms o f AL AT wcre invcsti ga ted in shce p and ca ttl e. T he cffects o f pyrid oxine, PLP , pyrid ox ine antagoni sts and co rti sone o n ALA T activity in shce p wcrc investi ga ted .
M ETH ODS
Assay oj A lallill c A lllili otralisferase (ALAT) T hc 2 m cth o ds used wcrc m o difi ca ti o ns of th c m c th o d of w robl cwski & L a D uc (1 956). T hc first Ill cth o d was suitable fo r use w ith a sin g le bca m spcctroph oto m ctcr w hil c th e sccond mc th o d rcq uired a dou bl c bea m instrum cnt. T he 2 m cth o ds gavc co mpa ra bl c rcs ults.
Method
T
H e parini zed b lood was hacm o lyscd b y prepa rin g a I in 10 dilution ill wa ter and o· 5 ml of thi s hac lll olysate was pre-inc ubatcd eithe r w ith or w ith o ut 50 fLg P LP for IS min . T hcn 0'5 IllI so luti o n of J O mM
Alallille Alllillotrall.lj erase ill Blood N a- 2- oxoglutaratc, 400 mM DL alan in c and I 00 mM T ri s acetatc buffer pH 7 '4 was add cd . A ro utin c blank containin g no alanin e w as includcd with cach assay. T he assay w as also appli cd to scrum and ho m ogcnatcs preparcd from livcr bio psy sa mp les . Aftcr in cubati on for 30 min thc amin o transfcrase reacti o n was sto ppcd b y addin g o· 5 ml N-tri chl oroaccti c acid . Thc pro tein prccipitate was rcmoved by centrifu g in g and 1' 0 ml supcrn atant was transferred to a spec troph o to m ctcr cuvcttc and neut ra li zed b y a 1'0 ml N /3 NaOH soluti on . Then 1'0 1111 0' 5 mM rcdu ced NA D in 0'2 M T ris acc tate buffcr was addcd fo llo wed b y 101-'-1 lactatc dchyd roge nasc (L D H) (1 0 m g/ m l). T hc decrcasc in o ptical den sity at 340 nm indica ted th c am o unt of p yruvatc p rodu ccd b y th c amin o transfcrasc. With a li ght path o f J O mm a changc o f o ptical dcnsity o f 0'208 is cquivalent to 0" I-'- m o lc. Hencc th c units of cn zy m c acti vity ca n be calcu lated as I-'- m o les o f pyruvate fo rm cd per min pcr litrc o f blood fro m th e equati o n Activity (iu/l) = C han ge of O .D . per min x 963
TA BLE [ T il E M IC HA ELI S CONST ANT ( K M) fO R AL AN INE AMIN OTRA NSfE RA SE O F V ARI OUS A N IMALS
2-oxog lutarate L-alan inc Rat li ver supern ata nt (at 25 °C) Shecp blood , di alysed (a t 30°C) Shee p pla slll a, di alysed (a t 30°C)
O'l I
2 1'2
Distriblltion of ALA T ill SlIvcel/lllal' Fractiolls (~f LiIJer HOlll oge;l<7tes. Rat and ca ttl c liver samp les were hOI11.ogeni zed and fracti on ated by differential centrifuging and the vari ous cell fracti ons assayed for ALAT activity. The results (Tabl e ll) show th at m ost o f the activity is in the supernatant fracti on. TA BL E II
Method 2 H ae m o lysa tc or plasm a (0 " m l) was prc-incubatcd fo r 15 min with o r w itho ut 50 I-'- g PLP . Aftcr prcin cubati on thc fo ll owin g rcagcnts w erc add cd to givc fin al conccntrati ons o f 5 mM N a 2- o xoglutaratc, r 66 111M L-alanin e, 0'2 mM reduced NAD, IOI-'-g/ml LDB and 100 111M Tri s acctate, pH 7'4 in a fin al volu m c o f 3'0 ml. T hc o ptical dcnsity o f th c tcs ts Wcrc rcad again st blank cuvcttes co ntaining all rcagents except alanin e in a rcco rdin g do uble bca m spectro ph o to m ctcr w ith a cuvette ho ldcr maintain cd at constant temperature. At fir st both m eth ods wc rc ca rri ed o ut at 25 °C but later cxperimcnts we re perfor med at 30°C in acco rd ance w ith thc revi scd rcco mm end ati o ns of th c Intern ati o nal Uni on o f Bi ochcmi stry.
RES ULTS
Kinetic Properties and Distriblltion oj' ALA T ill Liver and Blood Kin etic Properties (!f A LA T ill Blood and Liller Salllpies. The Mi chaeli s constants (K111) for 2oxoglu tarate and L-alan ine were determined by varying the substrate concentrati ons. T he results (Tabl e I) show that the affinit y of ALA T for L-alanine is lo w co mpared w ith its affinit y for 2- oxog lu tarate.
DI STRIB UTI O N OF ALAN I NE AMI NOTRANS I' ERASE IN SU Il CELLU LA Il FIlA CTIONS OF LI VE R H O MOGENATES
Wh olc hOlllogenate Nuclca r fracti on Mitochondri al frac ti on Mi croso mal fracti on Su pern ata nt frac ti on Recovery in fracti ons (% ) Results are th e Ill ca n of
2
Rat
atti c
58 '25 0'67 0'85 0 ·62 55'25 102
0 ·67 0'07 0' 17 O 'l!
0'4 0 104
experim cnts: U ni ts arc iuff!; at
2s oC
Distrivlltioll (~f AL A T il1 Blood. Shee p bl ood was co llected into siliconized gla ss tubes containing disodiulTl EDTA anticoagulant. It was fractionated by centrifug ing into plasma, platel ets, leucocytes and erythrocy tes accordin g to the m ethod of Koj et al. (I 96o). T he results (T able III) show that the acti vity is mainl y in the ery throcytes. Dial ys is had no effect on the A LA T acti viti es o f vari ous fracti ons. Th e F!ffect (! f' PLP 0 1/. A LA T ActilJity ill the P/asllla arId Blood 4 Variolls Anilllal Species T he results (Table IV) are gro uped acco rding to the characteri sti cs o f the ALA T acti vity in bl ood cell s.
J.
42
w, Boyd alld C, W, Roberts
TAnLE III
Challges ill ALA T Actill ity ill PlaslI/a, w llO lc Blood, Blood Cells alld Liller ojElIJes, Bl ood and
D ISTIl IBUTl ON or ALAN INE AM INOTIlANSFEIlASE IN SHEEP nLOOD IN lulL OF THE OIl ICINAL nLOOD AT 30°C
J.
Wh o le blood Pbslll a Cell s an d pbtclcts Hecove r y
2, Wh o le bl ood Pb slll a Pia telets Le ucocy tes Erythrocy tcs Recove r y
- PLl)
+PLP
ro6 7 96 97%
227 10 193 89%
l SI 9 0 0 1 19
241 13 13 0 20 5 96
8S%
liver biopsy sa mples were obta ined from 4 ewes at m onthly interva ls for 3 to 4 m onths, The mean level s of activity are shown in Table V , T he activity in packed bl ood cell s (C) was calcul ated from the packed cell volume (PCV), th e plasma activity (P) and the w hole bl ood activity (B) using the formul a
C=
CD - P X
JOo - PCV) JOo 100 PCV
%
ALA T Actillity ill PlaslI/a, Blood al/d Liller oj Sheep Challges ill ALA T Actillity ill PlaslI/a alld W hole Blood oj Lall/bs, H eparinized bl ood was co ll ected from three 4-month-old lambs and again at about week ly interva ls for 2 month s, In each of the 3 lambs the holoenzym e activity of plasma flu ctuated in para ll el with w hole blood holoenzym e activit y, This is shown for one lamb in Fig, 1. In Fig , 2 th e variati ons in w hole bl oo d ALA T holoenzyme in all 3 lambs arc shown,
Statistical Relatiol/sllips betUJeell ALA T H oloell z yll/e alld ApoCllz yll/e il1 PlaslI/a, Blood alld Liller (!I' Sheep, T he data coll ected from the lambs and ewes were ana lysed to detect any signifi cant correl ati ons, The stati sti ca ll y signifi cant results arc g iven in Table vr. Doth regression equati ons were ca lcul ated for each pa ir of variables because it is uncertain whi ch equation g ives the better description of these relationships , T he signifi cant positi ve correlati ons between plasma holoenzyme and bloo d cell holoenzyme is also presented in th e form of a sca tter diagram (Fi g, 3),
TAnLE IV ALAN INE AMI NOTIlA NSFEIlASE A CTIVITY IN PLASMA AND BLOOD 0 1' DI FFEIlENT AN IMAL SPECIES
PIJsnu - PLP Low ccllu br acri vity G uin ca-p ig 16 '1 ±3'S rbbb it LS' 4± 2'3 Dog 1 1'2±3 '9 Hi g h cc ll ulJr act ivi t y Hat at Pi g Man H o rse
n lood +PLL'
- PLP
+PLP
16 '6±4' 1 17'S±3'3 12'8±4'O
10' 1 ± I '8 I [ '4± I' 2 11 '4± 3'l
15'3 ± 2'3 Il '4± 2' 4 1 L' S± 2'[
w ith li tt le st illlubti o n b y cocn zy nl e 19 '3± I' 4 2 1'6± r '6 1O'6±4'S 12' S ± S' 4 J7' 1 ±3'S 18 'o±2 'O 10'7 ± 7'I 13'S ± 7'2 8'S±2'3 8'8 ±2 '7
Hi g h ccllu br activity w ith consid erab le cocnzy lll e Goat 7'7± I ' 1 II 'O±O'7 hecp 6'r±I' 1 g'2± I '7 Ca rtle II'O±t '9 1]'6 ± 2'4
77'9 ± 19'4 17S'S ± 5 2 '7 96'o ± 7'S 59'6 ±3 0 '6 6S '9 ± 22'3 stilllubti o ll 90'3±37'o 111'O±1 7'8 42'7 ± 22 '4
Ea ch result is the m ean o f 4 samp les fro m d ifferent indi vid llals± SD Units are ill/ l at 2S oC
79 'O± 19'6 17 S'8 ± SS ' ! roS'o±7'3 70 '2±3 2 '7 74 ' S±28'3 166'S±4 2 '8 3TJ'8±4 L'O 128'S ± S3'4
A lanille A lllillotrallsferase ill Blood 500
43
500
400 Whole blood
400
300 200 f
3 00
100
--I
f
o
--I
200
20 Plasma
)0
100
o Time in days
Flc. I. Va riations in the activity o f ALAT holoenzyme (e ) and apoenzyme (4 ) in plasma and w ho le blood o f a normal la m b.
The Effects oj DL-pcllicillalllillc, A lllil'lo-oxyacetic Acid, Pyridoxine RCI, PLP and Cortisolle Acetate 0 /1 A LA T ill Shecp In these experiments control animals were given inj ecti ons o f sterile water. T h e results are shown in T able VlT. In the sheep treated with corti son e there w as an average increase o f 38% in pl asm a glucose.
Time in days
Ftc . 2. Vari atio ns in ALAT holoe nzyme acti vities in wh ole blood of 3 d iffere nt lambs kep t together as a group.
ti o ns used were sel ected aft er the studi es o n the substrate affiniti es o f va ri o us sa mp les (Ta ble I) . The Km. valu es re po rted by H op per & Sega l (1962) fo r rat liver ALAT were I ' I mM fo r 2oxoglutarate and 34 111M fo r L-a lanine. T he substrate concentrati ons used here were simi lar to those repo rted as optimal by H enry et al. (1960).
DISC USSIO N
T he simplest colo rimetri c method o f AL AT assay (Reitman & Frankel, 1956) was unsuitable for investigating the PLP stimul ation o f A LAT beca use PLP fo rm ed a colo ured compl ex w ith the colo ur reagent. With the spectropho tometric m ethod o f Wro bl ewski & La Due (1956) there were difficulti es of m eas urem ent due to the absorbance by blood haemoglo bin at 340 nm . In ea rl y experiments w ith a sing le beam sp ectrophoto m eter the m ethod was m odifr ed so that haemoglobin was precipitated befo re py ruvate determinati on. In later experim ents w ith a do uble beam spectro photo m eter assays co uld be perfo rmed w itho ut haem oglobin precipitatio n. The substra te concentra-
10
0
8
E N
c
6
0
'0
.c 0
E VI E
Q
000
4
0
0
2
0
7
Flc . 3. T he relationshi p between th e activ ities o f ALAT holocnzyme in plas ma and packed blood cell s in ewes. (Pla sma holoenzyme = Ulood cell holoe nzy me x 0'007 + 3'25 infl at 25 °C)
J.
44
W. Boyd alld Co W. Roberts TABLE V
ALANIN E AMIN OTIlANSFEIlA SE ApOENZY ME (A) AND H OLOENZYME (H) A CTIVITIES IN PLASMA, WH OLE BLOOD, BLOOD CELLS AND LIVER OF EWES Pbsm~
ALAT
Wh o le bl ood
Blood cell s
Liver
123 ±64 200 ±74 7 6 ±3 6 38
326± 167 S33± 184 207 ±95 63
37 2 ±84 381 ±92 9±27 2
H S0 3±1°4 H+A 6° 2 ±r oS A 0 09±roo A% (of H+A ) 14
H activ ity is mcasured without adding PLP A acti vity is th e activit y increa se stimulated by addin g PLP Units arc iu/ I o r kg at 2S oC A total of I S sa mples were collected fro m 5 ewes over a peri od of 3 months
TABLE VI SIGNIFICAN CE 0 1' RELATI ONSHIPS BETWEEN ALANINE AMINOTIlANSFEIlASE ApOENZYME (A) AND H OLOENZYME (H) IN LAMBS AND EWES Dependent variable y
Independent Reg ression va riable coeffi cient x X b
C o rrelation coeffi cient
Illtcrccpt +
Probabil ity P<
Lambs (a) Pla s m ~ H = Wh o le b lood H Wh o le bl ood H = PIa S I1l~ H
X Oo029 X 23 °77
+ +
0°98 32° 12
o oS3
0°00 1
Ewes (b) Pla s m ~ H = Whole bloo d H Wh o le bl ood H = Pb slll~ H (c) Pb s m~ H = Blood ccll H Blood cell H = Plasn13 H (d) Li ver H = Whole b loo d A Wh o le bl ood A = Liver H (e) Liver H = Di ood ce ll A Blood ce ll A = Li ver H
X OoO l9 x 24 °02 X Oo007 X 69°99 x 1°58 8 x 0 0287 X 0062 1 x 0°790
+
3° 2 4 9"4 0 3° 2 5 6So52 250 06 30 °34 243° 6 86 08
0 06R
ooor
+ + +
0°7 2 0°68 0°7 0
Both regression eq uati ons have bec n ca lculated for cach pair of va riables Activities wcre ex pressed in ill/I of pbsma, w hole blood or pa cked blood cells and in iu per kg of liver at 2S oC
TABLE VII EFFECT OF ACTIVATORS AN D INHIB ITo nS ON ALAT H OLOENZY ME (H) AND ApOENZYM E (A) IN PLASMA AND BLOOD CELLS 0 1' SHEEP
DL-pen ici ll amin e Amin o- oxyace ti c acid P yrid ox in e HCI P y rid oxa l-s -ph osph ate Cortisone acetate
D~ il y
Number o f shee p trea ted
dose (m g/ kg)
[ 2
10 2 °5
TO
S
5 5
2
2°2 1°0
Duration of treatillent (da ys)
7
ALAT ~s percenta ge of co ntrol v~llIes Ce ll H
Ce ll A
Plasm a H
PIasn13 A
37 a 26a 103 144 b 13 1C
77 76 99 T3 0 122b
43 a
43 c 33 a 100 120
22~
95 13 6a l OT
a Sta ti sti ca l signifi ca nce P < oooO! b P < 0°01 C .. .. P <0 002 The test anim als were dosed by intramusc ular inj ecti o no Co ntrol anim als wcre given inj ecti o ns o f sterile water
11 8
A lanille AIlI/'llotrall.s/erase ill Bloor! In an investiga tion of th e subcellular distributi on o f ALAT in li ver hom ogenates, Rowse ll et aI, (1963) found th at 90 to 95% of ALAT was in the so luble fraction of rat liver. In th e present work 94% of ALAT was in the cytoso l of rat liver. Shee p and cattl e have much lower Ii vcr ALA T than rats (Boyd, 1962) but aga in the ALAT in cattl e liver is mainl y in the cytosol (Table II) . T he di stribution of ALAT in the vario us cell fractions of human blood has been studied by Koj et al. (1960). They found that ALAT is present in plasma and erythrocytes but was n ot detectabl e in leucocytes and platel ets. The results in Table Il[ show a similar distributi on o f ALAT in sheep blood. However, whil e ALAT in human who le blood is only 3- 6 times hi gher than in plasma (Karmen et al., I955; Koj et al., 1960) the ALAT in sheep whole blood is about 16 times higher than in plasnu (Tabl e IIl). It has been reported that preincubati on of lysed human er ythrocytes w ith PLP stimul ates their activity by 27% (Cheney et al., 1965 b). Simi lar trea tment causes 72% stimul ati on o f sheep erythrocyte ALAT activity (Table TIl) . T he PLP stimul ation of ALAT in plasma and ha el1lolysed bl ood from 11 different animal species is sh own in Table IV. Plasma ALAT ranged fronl. 6·J iu jl in sheep to 19'3 ittjl in rats. PLP stimulati on of plasma ALA T ranged from 3% in guin ea-pi gs to 34% in sheep. Haemolysed blood ALAT ranged from TO 'I iujl in g uin ea-pi gs to 175 iu jl in ca ts. PLP stimulati on of hac m olysed bl ood ALA T ranged from zero in rabbits to 200% in ca ttl e. It was possibl e to cl assify the various animals into 3 gro ups on th e basis of ha c moly sed blood ALAT (Tab le IV). In th e first gro up arc g uin ea-pi g, rabbit and dog w hi ch have whole bl ood ALAT simil ar to plasma ALAT indi ca ting rather low ALAT in bl oo d cell s. In the seco nd g roup arc the rat, ca t, pi g , horse and m an wh ich ev id entl y have mu ch hi gher ALA T activity in bl ood cell s than in plasma, but the ALA T in cell s is n ot greatl y stimulated by PLP. In the third gro up arc the ruminant animals, goa ts, shee p and cattl e w ith hi gh blood cellul ar ALAT whi ch is markedl y stimulated b y PLP. It can be ass ulll ed that th e ALA T activity m easured in th e absence
45
of added PLP is holoen zy m e and the activity in crease ca used by PLP is du e to apoenzyme. Thu s a uniqu e feature of these ruminant anim:tl s is the large amount of ALAT apoenzy me in their erythrocytes. In rats it has been found that plasma and erythrocyte ALAT apoenzymes increase in pyrid oxine defi ciency and arc redu ced to n ormal in response to vitamin thera py (Brin, 1964). However, a pyridoxin e deficiency in ruminants is unlikel y beca use of the end ogenous synthesis of B vitamins by ruminal mi croorga lli sm s. Moreover, treating normal sheep w ith large doses o f pyridoxine or PLP did not red uce th e ALA T a poenzymes in bl oo d (Table VII). As stated in the introducti on, several workers have reported rather large Au ctuations in plasm a ALA T in sheep and cattle. The present results show that in sheep the plasm a ho loenzym e Auctuates in parallel with bl ood cell holoenz yme. If the changes in pla sm a holoenzym e were caused by leakage of bl ood cell h o loenzym e there wo uld presumabl y be a simil ar relati onship between plasm a and cellular apoenzy mes. H owever, the apoenzymes remain relatively constant and arc not signifIcantl y related to each other. Since m ature erythrocytes Ca nJlot synthesize prote ins the actual amounts of .A LAT in ery throcytes Illust be fair! y constant, and so the Auctuati on in erythrocyte ho loenzyme activity m ust be due to the effects of acti vators or inhibitors. Tn rats, bl oo d h oloenzyme is activated by corti sone (Cheney et a/., 19653) and inhibited by DLpeni cill amine (Heddle ct al. , J963 ), w hile H o pper & Sega l (I962) have reported that amino-oxyaceti c acid is a powerful ill pitro inhibitor o f holoenzyme. In the present investigati on lIsing sheep it was fo und that corti sone and PLP were activators and DL-penicillamine :md amino-oxyacetic acid were inhibitors of bl oo d holoenzyme (Tabl e VII). It ca n be postulated that th e large Auctuati ons in plasm a and bl oo d cell holoenzym e acti viti es ill sheep and ca ttl e arc at least partly du e to certain products o f rumen fermentati on acting as activa tors or inhibitors. It is possible that the producti on o f PLP by rumen mi croorgani sms Auctuates and co uld be res ponsibl e for th e par.l l1e1 variati ons
J. w. Boyd al1d C . W. in blood cel l and plasma holoenzymes observed in sheep . No explanation can be offered for the significant relationship between ALA T holoenzyme in the liver and apoenzyme in blood cel ls of sheep (Tabl e VI) . It seems unlikely that variati on in one of these factors ca uses variation in the other. Presumably both of these vari ables are governed by a third factor which mi ght be genetical or nutritional. ACKNOWLEDGMENT T his wo rk was supported by a gra nt from the Agricu ltural Research Coun cil. ReceiIJedjof pllblicatit>1I Decelllber 4111, 1972 REFERENCES B OOTS , L. n . & LUDW ICK, T. M. (1970) ]. Dairy Sci. 53. 449 BOOTS, L. R. , L UDWI CK, T. M . & nADER, E. R. (1970) ].
Dairy Sci. 53. 1587
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