539th MEETING, UXBRIDGE
933
maximum suppression of drug metabolism also failed to depress the bile flow rate,
as shown in Table 2. Bile salt and bilirubin excretions were not altered significantly.
The AT? content of the liver was assayed in frozen samples by the procedure of
Delaney & Slater (1970) after the administration of propyl gallate and compound
SKF 525-A, since a relationship between bile flow and liver AT? content has been
demonstrated (Eakins et al., 1969; Slater & Delaney, 1970). Neither agent caused any
significant change in the concentration of AT?.
The results obtained indicate that the cytochrome P-450 system is not directly
involved in the control of bile secretion, but, as shown by the low-protein-diet experiments, when the total amount of the endoplasmic reticulum is decreased there is an
inhibitory action on bile flow, possibly owing to a decline in bile salt production.
We thank the Medical Research Council for financial support. The low-protein diet was a
generous gift from Dr. A. C. M. McLean; compound SKF 525-A was kindly provided by
Smith, Kline and French, Welwyn Garden City, Herts., U.K.
Berthelot, P., Erlinger, S . , Dhumeaux, D. &Preaux, A.-M. (1970)Amrr.J. Physiol. 219,809-813
Conney, A . H., Davison, C., Gastel, R. &Burns, J. J. (1960)J.Pharniacol. Exp. Ther. 130,l-8
Delaney, V. B. & Slater, T. F. (1970)Biocliem. J . 116, 299-302
Eakins, M. N.,Slater, T. F. & Delaney, V. B. (1969) Biocheni. J . 115, 6 2 ~ - 6 3 ~
Klaassen, C. D. (1969)J . Phannacul. E q . Tlier. 168, 218-223
Klaassen, C. D. (1971)J . Phortnacol. E.uy. Tho. 176, 743-751
Lowry, 0. H.,Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)J . Biol. Chem. 193,
265-275
Malloy, H. T. & Evelyn, K. A. (1937)J. B i d . Chenr. 119, 481490
Marshall, W. J. & McLean, A. E. M . (1969)Biochem. Pharmacol. 18, 153-157
McLean, A. E. M. & McLean, E. K. (1966)Biochem. J. 100, 564-571
Omura, T. & Sato, R.(1962)J. Biol. Chem. 237, PC 1375-1376
Orrenius, S. (1965)J . Cell Biol. 26, 725-733
Rogers, L. A., Dixon, R. C . & Fouts, J. R. (1963)Biochem. Pharmacol. 12, 341-348
Slater, T. F. & Delaney, V. B. (1970)Biochem. J . 116, 303-308
Slater, T. F. & Delaney, V. B. (1971)Tox-icul. Appl. Pharmacol. 20, 157-174
Torrielli, M .V. & Slater, T. F. (1971)Biocheni. Pharmacol. 20, 2027-2032
Woodcock, B.G.& Wood, G. C.(1971)Biochem. Pharmacol. 20, 2703-2713
The Effects of Antioxidants on the Concentrations of Reduced and Oxidized
Nicotinamide-Adenine Dinucleotide and of Triglyceride in Rat Liver
after the Administration of Ethanol
PHILLIP ROSSITER and T. F. SLATER
Department of Biochemistry, Brunel University, Uxbridge, Middx. U B 8 3PH, U.K.
The acute effects of a single oral dose of ethanol on rat liver are well documented
(Mallov & Bloch, 1956; Di Luzio & Zilversmit, 1960; for review see Slater, 1972).
Major events after the ingestion of ethanol include a disturbance of the WAD+]/
[NADH] ratio and the development of a fatty liver; the latter aspect of liver damage is
not easily interpreted owing to its multifaceted origin. Di Luzio (1964)and Krebs (1968)
postulated that the redox shift from NAD+ to NADH was a main factor leading to fatty
deposition, and Di Luzio (1967) later implicated mitochondria1 membrane damage as
another important phenomenon in that respect. Further evidence of damage to intracellular membrane-bound organelles in ethanol poisoning came from studies by Lieber
et ul. (1971), determinations of lipid-peroxidation products by Comporti et a l . (1967)
and a decrease in the polyunsaturated moiety of liver fatty acids (Comporti et al., 1971),
interpreted as evidence of lipid peroxidation in the endoplasmic reticulum.
VOI. 1
934
BIOCHEMICAL SOCIETY TRANSACTIONS
Di Luzio (1963, 1964) has observed that the antioxidants NN'-diphenyl-p-phenylenediamine and a-tocopherol had beneficial effects on liver morphology after ethanol
administration and that they prevented the ethanol-induced triglyceride accumulation.
Comporti et al. (1967) demonstrated that NN'-diphenyl-p-phenylenediamine prevented
malonaldehyde production, an indication of lipid peroxidation, and maintained normal
liver lipid concentrations during ethanol poisoning, thereby indicating the possible
importance of peroxidative membrane damage in this type of injury. Other antioxidants, synthetic (Di Luzio, 1967) and naturally occurring (Kalish & Di Luzio,1966;
Comporti et al., 1967), have been shown to inhibit the ethanol-induced fatty liver.
Slater et al. (1964~)and several other groups have studied the time-course of the
changes in liver [NAD+]/[NADH] ratio after dosing with ethanol and observed a
maximal change at about 1h after dosing. The rate of development of the fatty liver after
ethanol administration is linear up to about 24h after dosing (Wooles, 1966), and
similar results were obtained in the present study, which was directed to evaluating the
importance of the changes in the [NAD+]/[NADH] ratio to the development of the
fatty liver. For this purpose the effects of three antioxidants that attenuate the triglyceride accumulation were studied for their actions on the [NAD+]/[NADH]ratio in
liver after ethanol administration.
Female albino Wistar rats weighing 100-15Og were dosed intraperitoneally with antioxidant at 48, 24 and 2h before being dosed with ethanol. At zero time ethanol was
administered orally (1 :1, v/v, in water; 6g of ethanol/kg body wt.); control rats received
an isoenergetic (isocaloric) glucose solution. Rats were killed 1h later and the liver was
assayed for total NAD+ and NADH by the method of Slater et al. (1964b).
The accumulation of triglyceride in the liver was studied in rats given ethanol as
detailed above; triglyceride determinations were carried out on livers of rats killed at
6, 12 and 18h after the dosing. Liver extracts were treated according to the method of
Folch et al. (1957) for removal of non-triglyceride material and then the triglyceride
was determined by the method of Van Handel & Zilversmit (1957).
The effects of antioxidants on triglyceride increase 18h after dosing with ethanol
Table 1. Efects of antioxidants on the accumulation of triglyceride and on the
[NAD+]/[NADH] ratio in rat liver after the administration of ethanol
For experimental details see the text. Values are expressed as means*s.E.M., with the
numbers of rats used in each group in parentheses. Antioxidant dosages were:
NN'-diphenyl-p-phenylenediamine and butylated hydroxytoluene, 600mg/kg body wt. ;
propyl gallate, 150mg/kg body wt.
Agent
Corn oil
NN'-Dipheny l-pphenylenediamine
Corn oil
Butylated hydroxytoluene
0.9 % NaCl
Propyl gallate
Ethanol
Triglyceride
(mg/g of liver)
11.56f 1.00 (4)
20.58 f 1.90 (5)
14.07 f2.91 (3)
13.25f1.55 (4)
11.56f 1.00 (4)
20.58 f 1.90 (5)
15.34f2.51 (3)
17.93 f 1.68 (3)
14.10 f2.67 (6)
28.10f3.98 (6)
13.95f2.13 (6)
19.49f2.76 (6)
P
[NAD+]/mADH]
ratio
P
5.2 f0.27 (6)
1.68 f0.48 (6)
4.60 f0.39 (6)
<5%
1.38 f0.49 (6)
=.5 %
3.95 f0.25 (4)
1.30+0.10 (4)
5.17f0.56 (4)
1.2250.17 (4)
>5%
4%
3.00f.0.24 (4)
1.68 f.0.42 (4)
4.50f 0.50 (4)
1.25f.0.13 (4)
(1 P:
>5%
(t test)
1973
539th MEETING, UXBRIDGE
935
Time (h)
Fig. 1. Time-course of the ethanol-induced shift in the [NAD+]/[NADH] ratio and
triglyceride deposition in rat liver
For experimental details see the text. 0,Triglyceride concentration in liver of control
and ethanol-treated (----) rats; m, [NAD+]/[NADH] ratio in liver of ethanoltreated rats (from Slater et al., 1964a).
(-)
were studied in rats treated as detailed above. The antioxidants studied were NNdiphenyl-p-phenylenediamine,butylated hydroxytoluene (BHT) and propyl gallate. The
last two antioxidants were used by Di Luzio (1964) as components of an antioxidant
mixture known as G-50.
The results obtained (Table 1) for the effects of antioxidants on ethanol-stimulated
changes in the [NAD+]/[NADH]ratio show no significant difference between the means
of antioxidant +isoenergetic-glucose-treated groups and antioxidant +ethanol-treated
groups. From this it was concluded that the antioxidants have no significant effect on
the absorption or oxidation of ethanol in viuo.
The accumulation of triglyceride in the liver was studied up to 18h after dosing
with ethanol (Fig. 1) and found to be significantly elevated at 18h. These findings are
shown in relation to the time-course of ethanol-stimulated changes in the “AD+]/
[NADH] ratio (Slater et al., 1964a). In view of these data NAD+ and NADH determinations were carried out at 1h after dosing with ethanol and triglyceride determinations
at 18h after dosing with ethanol.
Observations on the ethanol-induced fatty liver reveal that the antioxidants tested
caused significant inhibition of the rise in triglyceride concentration despite their lack
of effect on cellular redox levels (Table 1). We infer from these findings that the
antioxidant-mediated protection against the ethanol-induced fatty liver is largely
independent of changes in the [NAD+]/[NADH] ratio.
Comporti, M., Hartman, A. & Di Luzio, N. R. (1967) Lab. Invesf. 16, 616-624
Comporti, M., Burdino, E. & Raja, F. (1971) Life Sci. 10, 855-866
Di Luzio, N. R. (1963) Physiologist 6, 169
Di Luzio, N. R. (1964) Life Sci. 3, 113-118
Di Luzio, N. R. (1967) Prop. Biochern. Pharrnacol. 3, 325-342
936
BIOCHEMICAL SOCIETY TRANSACTIONS
Di Luzio, N. R . & Zilversmit, D. B. (1960) Amw. J . Phjviol. 199, 991-1006
Folch, J . , Lees, M. & Sloane-Stanley, G. H. (1957) J . Biol. Chem. 226, 497-509
Kalish, G . & Di Luzio, N. R. (1966) Science 152, 1390-1392
Krebs, H . A. (1968) Stoflwechsel der Isolierten perfundierten Rattenleber, pp. 21 6-224,
Springer-Verlag, Berlin
Lieber, C. S., Rubin, E. & Decarli, L. M. (1971) Gastroetiferology 60, 689
Mallov, S . & Bloch, J. L. (1956) Amer. J . Physiol. 184, 29-34
Slater, T. F. (1972) Free Radical Mechanisms in Tissue Injury, Pion Ltd., London
Slater, T. F., Sawyer, B. C. & Strauli, U. D. ( 1 9 6 4 ~Biochem.
)
J . 93, 267-270
Slater, T. F., Sawyer, B. C. & Strauli,U. D. (19646) Arch. In?.Physiol. Biochiin. 7 2 , 4 2 7 4 4 7
Van Handel, E. & Zilversmit, D. B. (1957) J . Lab. Clin. Med. 50, 152-157
Wooles, W. R. (1966) Life Sci. 5 , 267-276
A Preliminary Study on the Breakdown of Oxidized NicotinamideAdenine Dinucleotide by Rat Liver Microsomal Fraction
DOUGLAS J. VAN VENROOIJ and T. F. SLATER
Department of Biochemistry, Brirnel University, Uxbridge, M i d d x . UB8 3PH, U.K.
The enzyme-catalysed breakdown of NAD+ by rat liver microsomal fraction was described by Sung & Williams (1952). Jacobson & Kaplan (1957) have indicated that the
predominant type of cleavage is at the ribose-nicotinamide bond by the enzyme
NAD glycohydrolase (EC 3.2.2.5). However, an Mg2+-dependantpyrophosphatase can
compete with the glycohydrolase to split NAD+ into AMP and NMN. Kaplan (1966)
has suggested that the NAD+ concentration in the cell is partly controlled by hydrolysis
of free NAD+ by NAD glycohydrolase and that the remaining NAD+ is protected in
an enzyme-bound form not available for attack. This hypothesis has been tested by
Bernofsky & Pankow (1971) by using free NAD+ and NAD+ bound to glyceraldehyde
3-phosphate dehydrogenase in an incubation mixture containing NAD glycohydrolase.
These workers showed that substantial protection was afforded to the coenzyme by the
dehydrogenase, but dismissed Kaplan’s (1 966) hypothesis on the grounds that the
amount of bound NAD+ found in rabbit muscle was only 20% of the total and that the
remainder was therefore available for hydrolytic cleavage.
We are studying the importance of NAD catabolism in rat liver microsomal
fraction to the control of NAD+ concentration in rat liver, and here we present some
findings on the characteristics of the microsomal catabolic system. Microsomal
fractions were prepared from female rat livers by differential centrifugation of a rat liver
homogenate in 0.25~-sucroseand weighed amounts were stored at -20°C. The stored
pellets were resuspended in ice-cold 0.1 5 M-KCI immediately before use.
A series of breakdown curves were prepared for different concentrations of microsoma1 fraction in incubation mixtures containing 200pmol of phosphate (Na2HP04NaH2P04)buffer, pH7.4,40pmol of EDTA and 1mg of NAD+ made up to 9ml with
O . ~ ~ M - K CThe
I . reactions were performed at 37°C and started by the addition of 1 ml
of microsomal suspension. Samples (1 ml) were taken into 6 % (w/v) HC104 and assayed
for NAD+ content by the recycling procedure of Slater et al. (1964). Microsomal
protein was measured by the method of Lowry e t al. (1951). The amount of microsomal
protein in the incubation mixture and the initial rate of NAD+ breakdown were shown
to be highly correlated (P<0.001) over the range 0.09-1.04mg of microsomal protein/
ml of incubation mixture.
In a similar series of incubations a concentration of 500pg of NAD+/ml was used,
corresponding approximately to the concentration occurring in liver in vivo (see Slater
e t al., 1964). Samples (1 ml) were taken into 0.5ml of 12% (w/v) HCIO, and neutralized
with 0.6ml of ~ M - K H C O ,and
, NAD+ was reduced by an enzyme solution containing
1973