Alcohol dehydrogenase activity in the uremic rat

Pergamon Press

Life Sciences Vol . 22, pp .1985-1992 Printed in the û.S .A .

AI

L

ALTIVI27t IN ~ URFNIIC RAT

Flsteb~an MezeY and James J . Potter Department of Medicine of The Baltimore City äospi tals and ~e Johns Hopacins Medical Institutions, Baltimore, Maryland 21224 (Received in final form April 17, 1978) SLhR4~Iat

An increase in liver alcohol dehydrngenase activity was äetected after 6 days Of tt,~n;a in the rat, with a ,~~,mm increase occurring after 10 days . ~e increase was docarented in the oxidative and reductive directions with ethanol and acetaldehyde as substrates, respectively : was not found in organs other than the liver and was not accompanied by ; m>~aa~ in other cytosoli.c enzymes . ~ enzyme in the anemic animal did mt differ in p~I optim~n for ethaml o~ddati .on, Rtn for ethanol and DF1û~ or in e].ects+ophoretic mobility fxcm the enzyme in the mnoal animal . .Ar7ra*ulpct~ny prevented the increase . It is suggested that the increase in liver alcohol dehydrogenase requires intact a~++ai glands and is most li]aely caused by the stress of surgery and the urenic state . In a previous study the hepatic activity of alcohol dehydrogenase (E .C . 1 .1 .1 .1) was elevated in rats maäe anemic bY ramval of approximately 85$ of the renal mass (1) . ~ increase in the enzyme activity could not be reprndu~oed by incubation of liver fran a no~xmal rat with urardc rat plasma, anemic human seam or urea, or by the daily oral admisListratian of urea for a period of 8 days . ~e aims of the present study were to define the time-course and organ specificity of the increase in alcohol dehydroc~enase activity after the production of unania, as well as the pmaperties and possible etiology of the increased enzyme activity . I~m~urnr c A~ ~~~ Mate Sprague-Dawley rats weighing, initially, betweai 100 and 120 g were used . i~nia was produced s tt+~+q+ cally as described by Hayslett et al (2) . Control animals had a sham surrRcal procedure consisting only of manipulation of each kidney with a clamp . L~emic and control animals were sacrificed at 2, 6, 10 and 14 days after surgery . Liver alcohol dehydnogenase was ~tPn;*,~ at all the time intervals, while the activity of alwhol dehydmgenase in other organs (gastric and int~t+ ;*,ai muoosa, brain and testis) and the hepatic activities of tyrosine a,n;mtra*,wf erase (E .C . 2 .6 .1 .5) arri tryptophan pyrmlase (E .C . 1 .13 .11 .11) were determined only in animals sacrificed 14 days after surgery. At the time of sacrifice the rats were anesthetized with ether, and blood was drawn fmn the aorta . ~e liver, brain and testis were reimved and rinsed in 1 .15$ IaCl . ~ intestine was ramved arri divided into pro~dnnal, middle and distal parts of equal length . ~e lamina of the stanach and of the sections of intestine were perfused with 1 .15$ KCl arri opened . ~e muoosa was scraped off in the wld . All tissues were hotrogetLi.zed with 4 whines of 0 .25 M sucrose in 0300-9653/78/0612-1985$02 .00/0

Copyright © 1978 Pergamon Press

198 6

Alcohol Dehydrogenase in IIremia

Vol . 22, No . 22, 1978

0 .1 M Tris-i~Q buffer pH 7 .4 . Ztie inmoge~ates were oentrifl7cJeC1 at 106, 000 g far one hour, atrl alcohol dehydrogenase activity was ~~r>~ in the rwst l t ing ~~TT+A tants by the metYnd of Boruiici~ and Hrink (3) . ~e following ethanol corx~tratfons were used : 18 mM for liver and testis, 108 mM far intestinal mioosa and brain and 900 mM far gastric muoosa . Liver alcohol äehyrlrogenase activity was a]so determined in the rductive dixecttoau, with 12 mM acetaldehyde as a substrate (4) . Retinol oxidation by the liver ~~+~+~ tant was det~ernd~ed bY the rate of foanation of retinal at 410 au as described, previously (5) . Protein concentration was determined in the supernatants of the tissue lxnogenates by the method of Lowry et al (6) with bovine serum albu=nin used as a standard . Mid~ae] .is-Menten constants for ethanol or t~D~ were calculated from Lineweavex` Buck plots obtained from deteantnations of alcohol dehYdrogenase activity at mn-saturating ethanol concaitratians . Starch gel electropd~oresis of the liver supernatant was carried out as described by Pietrusziao and ~eo rell (7) . gjrrosine A,tri*,.,trAnaferase and trypbophan pyrroLsse were deteaninned in a 12,000 g supea~atant Of the liver inmogenate . Tyrosine ~ ; ~*ransferase was determined by the method of Diamondstone (8), while tryptophan pyrrolase was determined by the method of IQnx and Aa, "t+a~, (9) modified by the addition of hamatin to the reaction m xttu~e (10) . Plasna urea nitxoc,~ai was d~tpr++~i,~ as described bY Fearon (ll) . ~ a separate experiment liver alcohol deh ' tease activity was ~+pYt+~i t,~ 2 days after the ps+oducticn of  ~A bY total bilateral ~ and in shamr operated controls . In addition, the effect of Aa.,P*,Ai~-r~,y on the iT+~+~A in liver alcohol d~ydnoge~ase was ~+a. ,iT,~ . Bilateral adrenalectanies ar a sham surgical Pr+oc~edare consisting of manipulation of the adrenals with a fioroeps were perfiorm~ed 2 days after the nrr,A,rt ;rn of ttYR"t1;A , ~e group of ur~nic-ac~enalectanized animals was given oorticosterone acetate (Si .gma Q~anical Oo ., St . Louis, Nb .) in a dose of 1 .25 mg/100 g of body weight, infraperita~eally, twice a day, starting one day after the AArr~ ,A1 ~tCIRj, . uxemic ani sl~roperated mn-uranic control animals were sacrificed 10 days after the xienal surgery . ~e effect of oortioosterone acetate s~iminis,trA tjpn on liver alcohol dehydrogenase was also determined in non-uram_c animals . Qmtrol ari;~+~a were injected with isowlumetric amounts of the vehicle +~*, ; ,AiR were sacrificed 15 (ethanol 208) used far the Ynrrone injection . hours after the last injection .

~e

~e results are expresses as means ± S .E .M . ~+P.r,i *,~ by the Stur3ecut t s t test .

statistical significance was

~e final body and liver weights were s~+m i Ar in the urani.c and control animals . Plasma urea nitrogen was increased to s comparable level at all time periods after the producti oau of t, .,~, A bY surgery (Table 1) . A significant increase in the activity of liver alcohol ~ was obtained after 6 days pf nram; A , gnd 8 umSxiiRIm iSlCresse was Obt8illed after 10 days Of ttrr-wni A , ~e increase after 14 days of ureIId.a was of R; , ; 7 Ar negnit~e tp that found after 10 days of t ~ ;A . Similar changes were obtained when the enzyme activity was expressed per g of wet liver weight ar as total liver enzyme activity . ~e increase in the enzyme activity after 14 days of tmc+lniA was demonstrated also in the reductive direction with acetaldehyde as a substrate . ~e mean activity in the uraai.c animals was 13 .57 ± 0 .576 umoles/mg/hr as compared with 7 .71 _+ 0 .844 tmoles/mg/hr in the control a i,t,ai ~ (P < 0 .001) . 7n addition, retiml oxidation was increased after 14 days of nrPm;A fr~n a control value of 0 .076 ± 0 .0102 uroles/mg/hr to 0 .148 ± 0 .0161 umoles/mg/hr

Vol . 22, No . 22, 1978

Alcohol Dehydrogenase in IIremia

198 7

(P < 0 .01) . By contrast, *~,P+ ;a resulted in no significant changes in the hepatic activities of tyrosine ~; ~+a^~ferase and tryptoQhan pyrrolase . TABIE 1 Effect of Urania on Liver Alcohol Dehydrogenase Activity Urania

PL9stna Urea Nitrogen Uremic Oontml (mg 00 ml)

Alcohol Dehydrogenase dontrol Ilremic uncles p

Days 2

13 .9 * 1 .96

60 .9 * 5 .34*

1 .19 * 0 .081

0 .98 * 0 .032

6

13 .0 * 1 .09

58 .3 * 9 .92*

1 .01 * 0 .042

1 .23 * 0 .016**

10

22 .3 * 0 .80

57 .0 * 4 .40*

1 .20 * 0 .070

2 .02 * 0 .100*

14

13 .6 * 0 .69

45 .5 * 1 .70*

1 .28 * 0 .137

1 .92 * 0 .213**

Uremia was prodined surgically as described in the methods . Alcohol dehydrogenase activity w®s determined bY the method of Hormichsen aryl Brink (3) . All values are means ± S .E .M . of 8 ari,~+ i~ . *Signif + icantly differently fran control value at P< 0 .001 . **Significantly different frown control value at P < 0 .05 . EUrther studies of liver alcohol dehydrogenase activity after 14 days of urenda revealed that the following ~,-r; es of the enzyme were similar in the uremic and control ani+~~~ : A pü ôptimm for ethanol oxidation of 10 .6, ICm constants of comparable magnitxde for ethanol (Table 2) and for NAD+ (B .5 X 10"6 M in the ur~nic as oa~ared with 3 .8 X 10 - 5 M in the control animals), and starch gel electrophoresis of the liver supernatants revealing a single band of activity migrating toward the cathode . Urpnda did not affect the activity of almhol dehydrogenase in organs other than the liver (Table 2) . Similar results were obtained when the enzyme activity in the various organs was assayed in the reductive direction with acetaldehyäe as a substrate . Zbtal nephrectcnry followed by sacrifice of the arL+~rA , 2 days later . resulted in an increase in plasma iu~ea nitrogen from a mean control level of 6 .7 ± 1 .41 to 62 .0 ± 1 .40 mg/100 ml (P <0 .001), but in no changes in liver alcohol dehydro9~re activity . ~e mean enzyme values in the control animals and in those gfi~ar total neptirec6any were 1 .24 ± 0 .311 and 1 .00 ± 0 .047 umoles/m3/hr, respectively . ~e an ;mala did not survive for more than 2 days after total nephreotarry to perniit the r~+orm; *~ tion of alcohol dehydrogenase activity at later time intervals . gil.ateral adP*,A1 ~~~ prevented the increases in hepatic alcohol dehydr+ogenase activity produced by  r,A. ;a (Figure 1) . AchnirListration of ocrtioosterone did not reverse the inhibitary effect of adrpsialectcniy, and it had no effect on liver alcohol d~lrdnogenase Lz non-urat~ic animals . DI9C[7SSION F~~+++AT+ tal uremia appears to be a unique irodel for the study of factors that nod.ify hepatic alcohol dehydxogenase activity . Previously, increases in hepatic

198 8

Alcohol Dehydrogenase in Uremia

Vol . 22, No . 22, 1978

TARTR 2

Effect of 14 Days of Uremia on Alcd~ol ~ Activity in Various Tisanes of the I~t

Tissue

Liver C~stric Muoosa

Alcohol ~ QJntrol Uremic Actl ty F~ for F`~haml ACtl ty i~n for F .`hhaml (M) lIIbleS/~ml`J*~r lIlbles~m[~Lr 1 .1 X 10-3

1 .28 ~ 0 .137

N .D . **

6 .37 * 0 .577

8 .0 X 10 -3

0 .282 *0 .069

1 .5 X 10-3 N .D .

1 .92 * 0 .214*** 5 .91 t 0 .619

Intestinal Muoosa Upper ~ird

9 .1 X 10 - 3

0 .252 *0 .054

Nhddle ~lit d

N.D.

0 .264 t 0 . 055

N. D .

0 . 225 * 0 .035

D~tstal ~1f,irr7

N .D .

0 .135 t 0 .017

N.D .

0 .149 t 0 .025

Brain

1 .3 X 10-2

0 .275 t 0 .065

7 .4 X 10 - 3

0 .224 * 0 .037

Zèstis

8 .3 X 10~

0 .068t 0 .013

1 .7 X 10- 3

0 .103t 0 .018

Alcd~wl dehydxogenase activity was determined by the method of eonnichaen and Brink (3) . Ethanol coa~centratioa~s in the reaction ndxtures to obtain maximan activities were : 18 mM for liver and testis ; 108 mM for intest;nal muoosa and brain ; and 900 mM for gastric muoosa . ~e enz~m~e activities are expressed as means S .E .M. of 8 ari++~la . * mg of protein ** N .D . = not measured *** Significantly different frvn control value at P <0 .05 alcohol dehydrngenase activity were reported to oa .-ur after ethanol administration (12,13) ; other investigators, however, have beg unable to confi .rn these f; ~ ; ~ (14,15) . ~e increase in hepatic alcad~ol dehydrogenase activity in uremia was dencnstrated in the oxidative directicai with ethanol and retiml as substrates and in the reductive direction with acetaldehyde as a substrate . It was mt absexved in organs other than the liver, and was not associated with increases in the activities of either tyrosine ~+mrr~fe=-~ ~ ~yp~ phan pyrrolase which, like alcohol dehydmgenase, are enzyaes present in the soluble fraction of liver knmogenates . Howevier, both tyrosine amimtransferase and trypbophan pyrrolase have short half-lives and changes in their activities may not coincide with changes in other labile liver proteins . Previous studies of hepatic enzyme activities in ramia indnoed by bilateral n~ahrectomy revealed iSlCreâSeB Zn a8partdte 8IId a1 ani r,a ami m+rgngferàse8 WiLich are located in the soluble and mitochondrial fractions of liver hamgenates, but in m changes in lactate dehydrogenase which is present in the soluble fraction (16) . ~e increase in hepatic alcohol d~hydrogenase activity did not occur acutely after the experimental pmoduction of uremia, but r+?~+,; ~ the pmesenoe of uremia for a few days, suggesting that the enzyme has a slow turnover . ale nedlanism far the increase of the enzyme in ~ a appears to be hnrmcx~ since

Alcohol Dehydrogenase in IIremia

Vol . 22, No . 22, 1978

198 9

2 .0

t w .c r ô a P Ô Ô

i 0.5

o_

c~~ Go

,~oF cov Peu

~o

~F

Jv

eFmo J~oo~

avo P

~yo bvo Jôô~ a~aw

Pece

FIG . 1 Effect of adrenalectomy on increases in hepatic alcohol dehych~ogenase activity prodaoed by uremia in rats . Bilateral adx+enaleotcmies were perfonoed 2 days after and the Fm ;male sacrificed 10 days after the p~mduction of urania . Fach bar r~apr~ te the mean enzyme activity ± S .E .M. of 4 animals . * Qortioostei~cne acetate was administered in a dose of 1 .25 mg/ 100 g body wei.c~t, intraperitoneally, tsuioe a day starting one c3ay after the aa*,P*,Al e~,;~., ** Significantly different from the value of the other groups at P < 0 .05 . ac3r,as,alpc~r prevented the increase . Sze (17) observed that increases in liver alcohol dehydrogenase psoduoed by dzmnic administration of ethanol in adore, were abolished by aA~lsac~~r and that the administration of eortioostexna~e restored the ~+~+~++~~,7 effect of ethanol on the enzyme . In this study, by contrast, the administration of oorticosteLnne did not reverse the effect of adre~alectany in preventing the increase in the enzyme produced bY uxsoia . In both this study and in that of Sze the ~++;++ ; Atration of oortieosteinne dial mt result in ;*,~"+-PaaP s in liver alcohol äehydrogenase in intact an ;mala , sin~a+ ;*' that aortiwsteroids could not be the cause of the increase in the enzyme but are permissive at best . In a recent study free this labaratoxy, increases in hepatic alcohol dehydrogenase, of the seine a~agnituc3e as those fend in uremia were demonstrated after 7 days of stress induced by ; ~+ii izatian of rats far 2 .5 hass a day (E . Mezey, J .J . Potter and R. . KV~eti~ansky, submitted far publication) . ~e incxeases in the enzyme coincided with peak increases in plas
1990

Alcohol Dehydrogenase in Uremia

Vol . 22, No . 22, 1978

p~resenoe of intact t+~~~ glands, and may be caused by the stress of surgery and the iuemic state . The e~ct haama~sal ioechanian for the increase in the enzyme activity, however, r+enains to be dero~i *,~ , In humans, higher hepatic alcohol dehyrlrogenase activity has been demonstrated in patients dying suddenly frcm trauma or acui-.e illness, than in patients dying frun diranic illness (18,19) . This may be due to decreased enzyme activity in the latter group . However starch gel electrop~tnresis of specimens with hick alod~ol ~~+~~~ activity reviealed, in additi.cai to the usual enzymes, an amdic form. This form w®s subsequently isolated and found to have many of the ~aracteristic;s of other forms of alcohol dehydrogenase, but a much higher Km for ethanol (20) . In the rat, in contrast to the human, starch gel electxoptnresis yields only one band migrating toward the cathode (4,21) . The increase in hepatic alcarol dehydrogienase in ur~aai.a dial not entail a dzange in the n*,~,P*+ ;Pa of ~ ~~ suc3~ as pH optimm for ethaml oxidation, Km for ethanol and NAD+, or a change in electnoproretic mobility, sVia+ ; 7 that the increase in activity is due to an increase in the quantity of the enzyme rather than the appearance of a new form . The physiological mle of alcohol dehydrogames i8 iaila~own. ~si ~a ethanol, it has been shown to oaddize a nutrber of potential physiologic substrates (22) . Hence, dzanges in the activity of the enzyme could result in alteration of no~anal aietabolisn . This woxjc was supported by Grant AA 00 626 from the U.S . Public Health Service . REFEI~.~ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .

E. ME2EY, R. E. V~L, J. J. PO'iTIIt and J. W. 1~1E, J. Lab . ßin. Med. _86 931-937 (1975) . J. P . HAYSIEIT, M. IQ~SFIGARiAN airl F. H. E~x~rN , J. Qin . Invest . _48 10021006 (1969) . R. R. HCtaJICHSIId and N. G. BRINIC, Methods of ho (S . P. Cblowick ard N. O. Raplan, HBs .) 1 pp . 495-50~, Aaaä~nic Press, New York (1955) . A. I . C3~ZII~i, R. PIET4iL15ZI~, J . HE[~EL, F. F. BECKER and E. RUBIIJ, Arch . Biorhem. Bicphys . 171 348-360 (1975) . E. MEZEY and P. R. BOLT, bcp. Irbl . Pathol . 15 148-156 (1971) . O. H. Il.~1fbC, N. J. F~LX~i, A. L. FARR an~ R. J. RAP~r3., J. Biol . Chen . 193 265-275 (1951) . R. PIEIItiB~ and H . , Axrh . Biocraa. B.icphys . _131 288-298 (1969) . T. I . DIAbDt~6'DOt~, Anal . Bioehen. 16 395-401 . W. E. KNDX and V. H . Ai]ElißA(.~i, J. B~1 . Chen. 214 307-313 (1955) . R. L. BIAI~ and E. RUN, in Methods of ].ô(D . B. McOormick and L. D. Wright, FY3s .) 18B pp .~ß, Press, New York (1971) . .J. 33 902-907 (1939) . W. R. FFAtYJNT, Biod~en R. D. HAWRaTS, H . KAIANP and J. M. KHANNA, C8I1 . J. fhysiol. Phareaool . _44 241-257 (1966) . S . P . MLSTrr"rç ard A. GAR.SI~, Aust . Ann. Med . .18 227-231 (1969) . C. S . r.rRa~ ard L. M. r~r~AUr.T , Sciesioe 162 91~918 (1968) . F. 7.C1BCN ard E. MEZEY, J. Lab. ßin. Med. 7`7 110-121 (1971) . K. P. MAIER, G. HOPPE-SEYC~EEt, H. TALRE, J.Ff~FR.ICH, P . ~and W. GEI~C, F~ . J. Clin . invest . 3 201-207 (1973) . P. Y. SZE, Bioehen. Med. 14 156-~61 (1975) . E. A7Ei7F~0, M. SM11H, D . n. HC]PKII~ECXQ and H. HARRIS, Ann. Hum. Genet. _38 31-37 (1974) . T. R. LI and L. J . MAGNES, Biodran. Bicphys . Iàes . Oomm
Vol . 22, No . 22, 1978 21 . 22 .

Alcohol Dehydrogenase in Uremia

H . JORNVAi~. and O. N~[ü7VIC, EUr . J . Biod~em . _29 167-174 (1972) . H . ~g70i2E[~, Haxvey Lect . 61 17-41 (1965) .

1991