Clin. Biochem. 11 (4) 156-158 (1978)

The Effects of Acute and Chronic Uremia in Rats on their Hepatic Microsomal Enzyme Activity U. K. TERNER, L. I. WIEBE*, A. A. NOUJAIM, J. B. DOSSETOR ' and E. J. SANDERS' Faculty of Pharmacy and Pharmaceutical Sciences University of Alberta, Edmonton, Alberta (Accepted January 20, 1978) CLBIA, 11, (4) 156-158 (1978) Clin. Bioehem. Terner, U.K., Wiebe, L.I., Noujaim, A.A., Dossetor, J.B., and Sanders, E.J.

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta. T H E E F F E C T S OF A C U T E AND CHRONIC U R E M I A IN RATS ON T H E I R H E P A T I C MICROSOMAL E N Z Y M E A C T I V I T Y The activity of the hepatic microsomal enzymes hexabarbital oxidase, aminopyrine demethylase, and p-nitrobenzoic acid reductase were determined in the chronically uremic rats, acutely uremic rats and controls. Plasma corticoid levels (corticostetone) were also measured in all groups. A significant reduction in the hepatic microsomal enzyme activity as compared to controls was observed in both groups, with the chronic group showing slightly g r eat er reduction. Furthermore, while plasma corticoid levels were significantly elevated in both groups, the acute uremic group had plasma corticoid levels which were almost twice the levels found in the chronically uremic animals. The significance of these observations are discussed.

PREVIOUS PUBLISHED W O R K F R O M OUR LABORATORIES ~'~)as well as work by others '47~ has shown that hepatic enzymes activity in vivo and in vitro is altered in chronic uremia, both in experimental animals and in man. Leber ~8,~ used a uremic rat model six days post-operatively to equate the effect of uremia on hepatic microsomal enzyme activity. However it has been shown that stress alone can also significantly a l t e r h e p a t i c m i c r o s o m a l e n z y m e a c t i v i t y c,o-1,~ I t was t h e r e f o r e o f i n t e r e s t to e l l u c i d a t e w h e t h e r t he m i c r o s o m a l m e t a b o l i s m in c h r o n i c u r e m i c a n i m a l s , d i f f e r s f r o m t h a t in a c u t e u r e m i c a n i m a l s s u f f e r i n g f r o m p o s t - o p e r a t i v e s t r e s s as a c o m p l i c a t i n g f a c t o r a nd f r o m t h a t in n o r m a l animals. T h e f i n d i n g s f r o m t he s e s t u d i e s a r e t h e s u b j e c t o f t h i s paper. EXPERIMENTAL METHODS Male Wistar rats (obtained from Woodlyn F a r m s Ltd., Guelph Ontario), in the weight range of 175-200 grams, at the onset of the experiment, were used. All were maintained as previously described ~ . Chronic uremia was induced by subtotal nephrectomy, removing 60 to 80% of the renal mass of the left kidney, followed by contra-lateral nephrectomy a week later. The method was similar to t h a t described by Kessner and Epstein ~3~. Control animals were obtained from the same group. 1. F acu l t y of Medicine 2. Department of Physiology *. Correspondence L.I. Wiebe, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta.

Acute uremia was induced in rats by subtotal nephrectomy, followed immediately by contra-lateral nephrectomy using the procedures described above for chronic uremia. Tissue preparation - - animals under mild other anesthesia were exsanguinated and the entire liver was removed and placed in ice cold 0.25M sucrose solution. Livers were blotted dry and weighed, then minced and homogenized in 3 volumes of 0.25M sucrose, using a Potter-Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 15,000 x g, for 20 minutes, at 2°-4°C., in a Sorvall refrigerated centrifuge, using a fixed angle rotor. The su p er n at an t was then collected, care being taken so as not to include the lipid layer on the surface of the contents of the centrifuge tubes. Determination of Hexobarbital Oxidase Activity The assay procedure of Cooper and Brodie ~m for measurement of hexobarbital metabolism was used. To the reaction mixture of 1.6 ml, containing 2~ mole of sodium hexobarbital, 20 bt mole nicatinamide, 1 ~ mole NADP, 25~ mole glucose-6-phosphate, 25 ~ mole Mg C12, was added 1.9 ml of 0.1M Phosphate buffer pH 7.4 and 1 ml 15,000 x g supernatant. A f t e r incubating for 1 hour, in a Dubnoff Metabolic Incubator, a t 37°C, the reaction was stopped by the addition of 0.1 M phosphate b u f f e r pH 5.5 and l g NaC1 and the unmetabolized hexobarbital was extracted. Assays were carried out in duplicate. Determination of Aminopyrine Demethylase The reaction mixture for this assay contained 10 ~ mole aminopyrine, 1~ mole NA~DP, 12 ~ mole glucose-6-phosphate, 25 ~ mole MgCI~, 100 I~ mole nicatinamide, 50 ~ mole semicarbazide in 2.5 ml. To this was added 3.0 ml of 0.1M phosphate buffer pH 7.4 and finally, 0.5 ml of 15,000 x g supernatant, to make up a total volume of 6 ml, in a 25 ml Erlenmeyer flask. The sample flasks were incubated, aerobically, for 30 minutes, at 37"C, in a Dubnoff Metabolic Incubator. The quantity of formaldehyde formed from the substrate was determined by the method of Nash c15~, as modified by Cochin and Axelrod c~e~. Determinations of enzyme activity were carried out in triplicate. Determination of Para-nitrobenzoic Acid Reductase The incubation mixture for the determination of r a t liver microsomal para-nitrobenzoic acid reductase activity was as follows: To 20 ml beakers were added 3.5 ml of reaction mixture containing 61~ mole para-nitrobenzoic acid, 2 ~ mole N A D P , 25 ~ mole nicotinamide. The reaction mixture was increased to 5.0 ml with the addition of 0.1M phosphate buffer. The beakers containing the substrate and the cofactors were placed in a Dubnoff Metabolic Incubator and a lucite chamber was placed over the beakers. While the mixture was being incubated, the lucite chamber was flushed with nitrogen. One ml of the 15,000 x g supernatant was then added to each beaker and the reaction mixture was then incubated, at 37"C, for 30 minutes and then arrested by the addition of 4.0 ml of 20% TCA. The quantities of free and conjugated metabolites were determined according to the method of Bratton and Marshall c1~. Spectrometric determinations were performed, using a Unicam P1800 spectrophotometer. Microsomal Cytochrome P-4500 was determined essentially as described by Omura and Sato ~s~. A Unicam PI800 was

E F F E C T OF CHRONIC AND A C U T E UREMIA ON MICROSOMAL ENZYME ACTIVITY

157

TABLE 1 EFFECT OF CHRONIC AND ACUTE UREMIA ON MICROSOMAL ENZYME ACTIVITY

TREATMENT

Control

HEXOBARBITAL OXIDASE

A M I N O P Y R I N E P-NITROBENZOIC N-DEMETHYLASE FREE

ACIDREDUCTASE TOTAL

CYTOCHROME P-450"

MICROSOMAL

PROTEIN,~.

5.36 ~ 0.19

0.92 ± 0.06

0.31 ± 0.04

0.53 ± 0.04

30.45 4- 1.45

21.36 ± 0.56

Chronic Uremia

2.29 ± 0.15 P _< 0.001

0.43 =t= 0.02 P _< 0.001

0.25 ± 0.02 P ffi 0.025

0.41 ± 0.02 P _< 0.001

17.60 ± 1.62 P _< 0.001

19.48 -~ 0.78 P _< 0.1

Control

5.55 ~ 0.15

0.91 ± 0.03

0.32 ± 0.02

0.54 ± 0.03

31.70± 1.12

21.76 ~ 0.56

Acute Uremia

3.39 ~ 0.18 P _< 0.001

0.45 < 0.06 P _< 0.001

0.24 ~ 0.02 P _< 0.025

0.42 ± 0.01 P _< 0.005

24.20 =k 1.90 P _< 0.01

19.17 ± 0.99 P _< 0.1

Chronic vs Acute

2.29 ± 0.15

0.43 ± 0.02

0.25 m 0.02

0.41 ± 0.02

17.60 ± 1.62

19.48 ~ 0.78

3.39 ± 0.18 P ffi 0.005

0.45 =t= 0.06 P > 0.5

0.24 ~ 0.01 P > 0.5

0.42 ~ 0.01 P > 0.5

24.20 ± 1.90 P _< 0.005

19.17 ~ 0.99 P > 0.5

V$

vs

Activity of Hepatic Microsomal Enzymes expressed as ;anoles metabolite formed per gram of liver (25% homogenate) ~ s.e. of mean per 60 min for hexobarbital oxidase and aminopyrine N-demethylase, while p-nitrobenzoic acid reductase is expressed per 30 rain. All values represent the mean values of 6 rats with the exception of chronic uremia where 10 rats were used. *m~ moles/g of liver mg/g of liver used to determine the difference between sample and reference cuvettes. The difference in optical density was determined as a change between 450-490 m}z and the quantity of cytochrome P-450 was determined using the molar extinction coefficient of 91raM -1 cm "ms~. All determinations were performed in duplicate.

Microsomal Protein Determination The microsomal protein content was determined by a method based on the colorimetric determination of Lowry et al ~m. Determinations were performed in triplicate. Determinations were performed, using a standard curve, produced by using known concentrations of bovine serum albumin.

Determination of Plasma Corticoids A competitive protein binding technique was used to measure the total plasma corticoids (corticosterone, hydrocortisone). The procedure used was similar to that described by Jeffrey and Noujaim t2°~. Plasma corticoid determinations were performed in duplicate.

Determination of Blood Urea Nitrogen BUN levels were measured in control and uremic animals using Eskalab@ BUN reagent tablets (Smith Kline Laboratories, Palo Alta, California). Blood samples for plasma corticoid and serum BUN analyses were taken when the animals were sacrificed. All analyses were carried out using a minimum of 6-10 animals per test group. Data are expressed as mean -- s.e.m. The level of significance between test groups was calculated using the two-tailed Students t test. Data from groups were considered to differ significantly when P ~ 0.05. TABLE 2 THE EFFECT OF CHRONIC AND ACUTE UREMIA ON PLASMA CORTICOID AND BUN LEVELS TREATMENT

Control . . . . . . Chronic Uremic . . . . . . Acute Uremic . . . . . .

P L A S M ACORTICOSTORONEa

11.4 ± 0.8 36.4 -4- 3.9 P < 0.001" 513 ± 3.0 P _< 0.001' (P _< 0.025) +

BUN b

33.0 202.8 P < 18~ P _< (P >

± 1.8 ± 15.7 0.001" ± 12.8 0.001 0.5) +

*P values compared to controls -{-P values Chronic versus Acute a. Values expressed as ~g/100 ml plasma b. Values expressed as mg/100 ml plasma Values in the table represent the mean ± standard error of mean of 10 control; 10 chronic uremic and 6 acute uremic rats.

Preparation of Hepatic Tissue For Electron-Microscopic Examination Samples of liver tissue, obtained from chronic uremic and control r a t liver, were fixed with 3.5% glutaraldehyde in 0.1.M phosphate buffer, followed by 1.0% osmium tetroxide in phosphate buffer. The tissue was then imbedded in Araldite 502 and sections were stained with 5% aqueous uranyl actate and Reynold's lead citrate ~s~. RESULTS The a c t i v i t i e s of t h e v a r i o u s hepatic microsomal enzymes, ( h e x o b a r b i t a l oxidase, a m i n o p y r i n e demethylase, p a r a - n i t r o b e n z o i c acid r e d u c t a s e ) , the q u a n t i t y of cytochrome P-450, a n d the microsomal p r o t e i n were d e t e r m i n e d in normal, chronic uremic a n d acute u r e m i c r a t s (Table 1). The a c t i v i t y of all enzyme p a r a m e t e r s m e a s u r e d was s i g n i f i c a n t l y reduced in both chronic and acute u r e m i c rats, when compared to controls. F u r t h e r m o r e , the t r e n d of the p a r a m e t e r s m e a s u r e d was to lower a c t i v i t i e s in the chronic uremic a n i m a l s , compared to a c u t e l y u r e m i c a n i m a l s . The a c t i v i t y of the hexob a r b i t a l oxidase enzymes was s i g n i f i c a n t l y reduced in the chronic u r e m i c s when compared to acute u r e m i c r a t s (P--< 0.005). The a m i n o p y r i n e demethylase activi t y a n d the p a r a - n i t r o b e n z o i c acid r e d u c t a s e activity, while s o m e w h a t lower in the chronic u r e m i c rats, were n o t s i g n i f i c a n t l y d i f f e r e n t in the two u r e m i c g r o u p s (Table 1). The q u a n t i t y of cytochrome P-450 was, however, s i g n i f i c a n t l y reduced i n the c h r o n i c u r e m i c a n i m a l s when compared to acute u r e m i c s (P--