The effects of dietary niacin and riboflavin on voluntary intake and metabolism of ethanol in rats

Pharmacology Biochemistry & Behavior, Vol. 1I. pp. 575-579. Printed in the U.S.A.

The Effects of Dietary Niacin and Riboflavin on Voluntary Intake and Metabolism of Ethanol in Rats LEENA

PEKKANEN

AND MAIJA RUSI

The R e s e a r c h L a b o r a t o r i e s o f the S t a t e A l c o h o l M o n o p o l y (Alko), B o x 350, S F O 0 1 0 1 H e l s i n k i I0, I'Tnland R e c e i v e d 1 A u g u s t 1979 PEKKANEN, 1.. AND M. RUSi. 77w elJ~'cts ~ff'dietary niacin and riboflavin on voluntary intake and metabolism of ethanol in rat.~. PHARMAC. BIOCHEM. BEHAV. 11(5) 575-579, 1979.--The effects of dietary deficiency and excess of niacin and riboflavin on voluntary drinking of 10% (v/v) ethanol were studied in male rats. The effectiveness of dietary deficiency and excess of both niacin and riboflavin on tissue levels of these vitamins was demonstrated by measurements of urinary Nt-methylnicotinamide and blood glutathione reductase (EC 1.6.4.21 activity. A high-niacin diet containing 75 mg niacin/kg food decreased ethanol intake by about 36% compared to the control diet containing 15 mg niacin/kg. Niacin or riboflavin deficiency and a high-riboflavin diet containing 40 mg riboflavin]kg did not affect significantly ethanol drinking. Changes in dietary levels of niacin or riboflavin did not influence on ethanol elimination rate or levels of blood acetaldehyde during ethanol oxidation. Therefore, blood acetaldehyde was not responsible for the decreased ethanol intake of rats fed with a high-niacin diet. It was concluded that the increased ethanol intake caused by dietary deprivation of B-vitamin complex found in earlier studies is not a result of deficiency of niacin or riboflavin but niacin may be involved in the decrease in ethanol drinking, which follows dietary B-vitamin complex supplementation. Voluntary ethanol intake Glutathione reductase

Niacin Riboflavin N ~-methylnicotinamide

Ethanol elimination rate

V O L U N T A R Y ethanol consumption has been shown to increase in experimental animals fed diets lacking B-vitamin complex and supplementation of the diets with the same complex appears to reverse this effect 12, 20, 291. It is not known which members of the B-vitamin complex participate in this effect. Of the single B-vitamins, thiamin, riboflavin, niacin, pyrodoxine and pantothenic acid seem to modify ethanol intake in the same way as B-vitamin complex but the results have not been consistant in all studies [1, 3, 20, 23, 281. One explanation for the contradictory results could be that in the earlier studies the effects of the experimental diets on the tissue metabolism of the vitamin in question have not been studied. Therefore, in the present experiment the effect of dietary deficiency and excess of niacin and riboflavin on voluntary ethanol intake in rats was studied, and the effectiveness of the experimental diets was estimated by measuring the niacin metabolite, NJ-methylnicotinamide (N-MNA) in urine and the activity of riboflavin-enzyme, glutathione reductase (EC 1.6.4.2), at the end of the study. Since niacin and riboflavin are precursors for coenzymes essential in ethanol oxidation and ethanol drinking of animals has been assumed to be ',fffected by their capacity to oxidize ethanol [10, 241, the effects of dietary deficiency and excess of niacin and riboflavin on ethanol elimination rates were studied. It has also been hypothetized that ethanol drinking is regulated by blood concentration of the first oxidation product of ethanol, acetaldehyde, which as a toxic substance

C o p y r i g h t :,7 1979 A N K H O

Blood acetaldehyde

could cause aversion to ethanol 17, 14, 25, 26J. Therefore, the relationship between vitamin intake and blood acetaldehyde concentrations was also studied during ethanol metabolism. METHOD

Animals and Apparatus The animals were 3 month old males of a mixed strain derived from cross-breeding Sprague-Dawley, Wistar and Long-Evans rats in our laboratory [8]. Animals were individually housed in galvanized cages with a mesh bottom. The experiment was carried out in a room under a 12 hr light/12 hr dark cycle with temperatures maintained between 2224°C. In the free-choice situation 10% (v/v) ethanol, tap water and food were freely available for the animals. Two 100 ml graduated cylinders were used for delivery of fluids, the position of them being interchanged once a week to avoid a possible effect of place affinity on fluid intake.

Diets The basal diet was made from vitamin-free casein (ICN Pharmaceuticals Inc., Cleveland, Ohio, USA), rice starch and maize oil so that 20°,4. of the total energy was derived from protein, 65% from carbohydrate and 15% from fat. The control diet contained 4 mg of thiamin hydrochloride (Hoffmann and LaRoche, Basle, Switzerland), 8 mg of riboflavin (Merck, Darmstadt, Germany) and 15 mg of

I n t e r n a t i o n a l 1nc.--0091-3057/79/110575-05501.00/0

576

P E K K A N E N A N D RUSI

nicotinamide (Hoffman and LaRoche) in each kg of fresh diet. which were the highest levels listed in the nutrient requirement tables fc,r rats [5, 15, 16, 27l. The riboflavindeficient diet (RD) contained no added riboflavin and the niacin-deficient diet (ND) no added nicotinamide. According to our analyses the basal diet contianed 0.2 mg of riboflavin and 0.4 mg niacin,:kg of fresh diet. The high-riboflavin diet (RH) contained 40 mg of riboflavin.kg diet and the highniacin diet (NH) 75 mg of n i c o t i n a m i d e k g diet, which were five times the control diet level. The exact content of other vitamins and minerals in the basal diet has been described previously by Pekkanen and c o w o r k e r s [221.

1 ABI,E I A C I I V ] I Y o l - ( I I , U I A I t l I O N E RI-DLCTASIi A N D A C I I V A ' I I O N COEFFICII'LN r I:OR FLA'VlN AI)ENINI- I)INU('I,I.I()TIDI.~

Grotlp

2.76

' 11.21

1.12

" 0.04

E - R H Ig~

3.71

• 0.49

1.20

- 0.04

R D (91

1.77

-

1.58

'

('(8i

2.37

- 0.32

1.34

• 0. ll

RII(8~ R I ) 18~

3.39 2.117

,

• 0.45 1136

1.30 • ().07 1.611 • (I. 14

I'i

-

WW

Test groups of g-9 animals given a free-choice between I/Y;~ {wv) ethanol and water were: ethanol-high-niacin diet (E-NHJ. ethanol-niacin-deficient diet I F - N D ) , ethanolhigh-riboflavin diet (E-RH), ethanol-riboflavin-deficient diet (E-RD) and ethanol-control diet (E-C/. The corresponding water IW) groups were W - R H . W - R D , W - N H . W - N D and W-C. The water-groups were included to dertermine the effects of the test-diets on growth rates, energy and water consumption and ethanol metabolism. For 3 weeks all animals received the control-diet and tap water and the test diets were given during the next 4 weeks. The first of these test diet weeks was an ethanol habituation period lbr the ethanol free-choice groups, when the ethanol groups had 10'7,: (v:v) ethanol as their only drinking fluid. For the latter 3 weeks they had a choice between ethanol and water. The fluid intake was recorded daily, food consumption and body weight twice a week.

Amdytical :'~h'lltods The ethanol elimination rates and blood acetaldehyde concentrations were measured in all animals, first, before the test diets were presented, then after the 1st, 2nd and 4th weeks on the test diets. The rats were injected with 1.5 g..kg ethanol IP as 1(Y22 (w/v) solution in saline, and the concentrations of ethanol and acetaldehyde were measured in tail blood haemolyzed with water 30, 10(1, 140. 180 and 2211 rain after the injection by head-space gas chromatography [ I I]. At the end of the study the blood samples for e n z y m e assay were taken from the hearts of the Nembt, tal"-anaesthetized animals. Glutathione reductase activity was meastired from whole-blood haemolysates according to the method of Glatzle and c o w o r k e r s [12l. Activities att'e reported as the change in absorbance at 334 nm per rain per g hemoglobin. Activation coefficients to describe the e n z y m e saturation with F A I ) were calculated as a quotient of the activity with added FAD,activity without added F A D . Hemoglobin was determined by the c y a n m e t h e m o g l o b i n method [131. Urine was collected from animals kept for 24 hours in metabolic boxes free of food and ethanol. For analysis of N - M N A the urines of all rats in each group were pooled and N - M N A was determined thtorimetrically [4] in a commercial laboratory (Technical Reasearch Centre of Finland. Food Research Laboratory, Espoo, Finhmdl. R E S U t . IS AND I ) I S ( ' t ' S S I ( ) N

Rib,dlavin .Slal,s In the present study dietary levels of ribollavin had

A eli,, at ion coefficient

E - ( ' * (8)

W-

Procedlll'e

(Hutathionc red uctase act(', it.,, A ming hemoglobin

-

0.18:i:

VHllleS :.ire e x p r e s s e d ax illealp, .~.[-'.m. a n i m a l s g r o u p in p a r e n t h e s c x . 'See text for exphmation of abbre\ iations. : / ' 0.(X)l c o m p a r e d to I.i • C g r o u p . :!:/,- (I.(11 c o m p a r e d to I" - (" g r o u p .

,,,,ith t h e

11.07;

number

of

affected the status of the vitamin in the rat tissue. The results in l-able I show a typical c o n s e q u e n c e of riboflavin deficiency which is a decreased activity of the FAI)-requiring e n z y m e , glulathione reductase, and high activation coefficient, which indicates the low saturation of the e n z y m e with F A D iJl vivo. In the E-RI) group Student's l-test showed a significant decrease in glutathione reductase activity and a significantly higher activation coefficient c o m p a r e d to the E-(" group. 1115~.-3.72. p. (1.01: I{15)--5.0(1. p" 0.IX)I. respectively. In the W - R D group the corresponding differences were not statistically significant. 1(141-0.60. p- I).55: l( 14! 1.69, p. 0. I I. indicating that ingestion of ethanol had potentiated riboflavin deficiency. Correspondingly. there was a trend, even though non-significant, for the highribollavin rats to show an increase in glutathione rednctase activity both in the E-RH group. I( 141- 1.80. p-- 0.09. and in the W-RH group, ; ( 1 4 1 1.87. /~-0.08. c o m p a r e d to the corresponding controls. Also the growth rate and food intake of the ribolla',indeficient groups were slightly but not significantly lower than in the other groups. The consumption of water wits equal in all groups. .\.'ia~i n .~lalIi s The urinary outptlt of the niacin metabolite, N - M N A . varies with the degree to which tissue stores are saturated

with niacin when urine is collected in fasted animals. Even though it was measured in pooled samples of each group N - M N A tended to be clearly lower in the urine of the I--NIt and W - N D groups and higher in the urine of the E - N H group compared to the control groups (Table 21. In the W - N H group the high-niacin diet had not increased niacin stores c o m p a r e d to the W-C group. This could be explained if the le,,el of niacin in the control diet was high enough to saturate tissues maximally, ft,llher accumulation of niacin by feeding a high-niacin diet thus being impossible. H o w e v e r . the high-niacin diet had effected tissue stores in the E-NH grot, p: in the control group ethanol ingestion decreased niacin stores ( N - M N A excretion in the E-C group was 35%: lower than in the W-C group) more than in the high-niacin group ( N - M N A excretion in the E-NH group was 13eL lower than in the W-NH group) indicating that in the E-NH group a

DIETARY NIACIN AND RIBOFI.AVIN

577

TABLE

2

EXCRETION OF N'-METHYI.NICOI'INAMIDE IN URINE PER GROUP OF 8 RAI'S PER DAY Group

/xg:grotip:day

E - C* E - NH E - ND W -C W - NH W - Nf)

193 255 51 299 294 83

*See text for explanation of abbreviations. TABLE

3

I-I:I:ECTS OF DIFIARY RIBOFI.AVIN AND NIACIN ON FREE-CHOICI.I CONSUMIrIION OF ETHANOL DURING 3-WEEK E'II.tANOL-CHOICE PERIOD Ethanol intake.d g.kg

Group

Energy from ethanol if>,: total)

E-

C * (g)

2.2

0.6

10 * 2

E-

R H (8)

1.6 - 0 . 4

7 - 2

1.3 , 0.2 0.8 -_ 0.2 i 1.2 • (1.2

6 • I 4 • 1 5 • I

E - RD(8) E - NH (91 E- NI)(8)

,

I(Y'/; (v;v) ethanol intake (¢~ total fluid) 31 _+ 8

26 22 II 17

+_ 7 ,- 2 • 3,- 3

Values are expressed as means .~ S.E.M. with the number ofanimals:group in parentheses. *See text tbr explanations of abbreviations. ~p. 0.05 compared to the [" - C group.

high daily i n t a k e o f n i a c i n c o m p e n s a t e d f o r the l o s s o f t h e v i t a m i n k n o w n to be i n d u c e d by e t h a n o l i n g e s t i o n [6, 17]. T h i s n e g a t i v e i n t l u e n c e o f e t h a n o l d r i n k i n g o n v i t a m i n bala n c e c a n a l s o be s e e n in the niacin d e f i c i e n t - g r o u p ( N - M N A e x c r e t i o n in the E - N D g r o u p w a s 3 8 ~ l o w e r t h a n in the W-ND group).

~'o/t,ttarv Etha,ol I~lt~l~" A l t h o u g h d i e t a r y levels o f niacin a n d r i b o f l a v i n had afli~cted the s t a t u s o f t h e s e v i t a m i n s in this s t u d y t h e c h a n g e s in v o l u n t a r y e t h a n o l c o n s u m p t i o n o f the r a t s w e r e not d r a s t i c as s h o w n in T a b l e 3. O f all t e s t - d i e t s , o n l y the h i g h - n i a c i n diet had s o m e effect o n e t h a n o l intake: in the E - N H g r o u p e t h a n o l i n t a k e w a s s i g n i f i c a n t l y l o w e r w h e n e x p r e s s e d as g¢'kg b o d y w t , t(14) = 2 . 1 9 , p < 0 . 0 5 , a n d a s p e r c e n t o f total tluid i n t a k e , t ( 1 4 ) - 2 . 3 1 , t')<0.05, a n d a l s o slightly l o w e r w h e n e x p r e s s e d as a p e r c e n t a g e o f the total e n e r g y i n t a k e , t l l 4 ) = 1.98, p < 0 . 0 7 , c o m p a r e d to the c o n t r o l g r o u p . An i n c r e a s e d e t h a n o l c o n s u m p t i o n as a r e s u l t o f niacin defic i e n c y w a s not f o u n d in this s t u d y a n d t h e d i e t a r y level o f r i b o f l a v i n s h o w e d no effect at all o n v o l u n t a r y e t h a n o l consumption.

Blood Acetaldehyde a , d Ethamd E l im i, a li o , Rate Blood a c e t a l d e h y d e levels a f t e r the e t h a n o l t e s t - d o s e o f 1.5 g,kg a r e p r e s e n t e d in T a b l e 4. T h e m a t c h e d - p a i r t - t e s t s h o w e d a significant d e c r e a s e in b l o o d a c e t a l d e h y d e levels d u r i n g the s t u d y in t h e E - N H g r o u p , t ( 7 ) - 6 . 1 0 , p<-'0.001, a n d

TABLE 4 THE BI.OOD ACETALDEHYDE I.EVEI.S OF RATS 30 rain AI"I'ER THE IN'I RAPI'.'RITONEAI. INJECTION OF 1.5 g ETHANOl. PER kg BODY WEIGHT

Group

nmol.ml Before intr¢~lucing test-diets

After 4 weeks on test-diets

E - C ~18) E - RH (8) E - RD(9)

25 .-_ 4 25 + 4 21 _+ 2

19 + I 18 - 3 17 ~- 2

E-NH(8)

24

' 3

13 ' 3

E - NI)(7) W - C (8) W - RH (8) W - RD(8) W - NH (8) W - NI) (8)

16 20 24 23 22 26

~ + _' ± -' z.

13 15 17 29 7 20

6 3 4 4 4 7

" 2 "- 3 ~3 _+ 4 ± I -' 3

Values are expressed as means , S.E.M. with the number of animals.grotJp in parentheses. *See text for explanation of abbreviations.

578

PEKKANEN

in the W - N H g r o u p , t ( 7 ) - 3 . 2 0 , p.-',O.05, a n d in the E-RH group, t ( 7 ) = 2 . 5 3 , p < 0 . 0 5 , and in the W - R H g r o u p , t ( 7 ) = 2 . 8 6 , p < 0.05. A non-significant d e c r e a s e in blood a c e t a l d e h y d e d u r i n g the s t u d y was also seen in the E-C group, t ( 7 ) = 1.62, p < 0.15, and in the W-C group, t(7)= 1.05, p < 0.32. T h e r e f o r e . the effects of the test-diet were tested statistically relative to the initial values using S t u d e n t ' s t-test for i n d e p e n d e n t variables, the individual c h a n g e s from the initial values in each g r o u p being c o m p a r e d with the c h a n g e s in the c o r r e s p o n d i n g control groups. T h i s s h o w e d no statistically significant d i f f e r e n c e s b e t w e e n the t e s t - g r o u p s and the control g r o u p s indicating that d e c r e a s e s in blood acetald e h y d e levels were not c a u s e d by dietary levels of niacin or riboflavin in this study. No c h a n g e s in e t h a n o l e l i m i n a t i o n rates during the e x p e r i m e n t were f o u n d to be c a u s e d by dietary deficiency or e x c e s s of niacin and riboflavin.

Gt, tttq'gt/ Commi'tlt,~

T h e d e c r e a s e d e t h a n o l intake in the g r o u p receiving five times more niacin in the diet t h a n the c o n t r o l group c a n n o t be e x p l a i n e d by c h a n g e s in e t h a n o l m e t a b o l i s m . This provides a f u r t h e r e v i d e n c e that a high level of a c e t a l d e h y d e in

AND RUSI

blood is not the only s u p p r e s s o r of v o l u n t a r y ethanol intake in a n i m a l s [18, 19, 22]. T h e B - v i t a m i n c o m p l e x e s , dietary d e p r i v a t i o n of which has i n c r e a s e d e t h a n o l c o n s u m p t i o n in the rat and m o u s e , h a v e c o n t a i n e d thiamin, riboflavin, p y r i d o x i n e , p a n t o t h e n i c acid, choline and niacin [2], biotin instead of niacin [201, or all the foregoing B - v i t a m i n s plus inositol, p - a m i n o b e n z o i c acid a n d folio acid [29]. S u p p r e s s i o n of e t h a n o l intake to the initial level h a s b e e n p r o d u c e d by a d m i n i s t r a t i o n of the B - v i t a m i n c o m p l e x used by Brady and W e s t e r f e l d [21 and Williams and c o w o r k e r s [29]. O f single B - v i t a m i n s , a defic i e n c y of thiamin has c o m m o n l y b e e n r e g a r d e d as an e l e v a t o r o f v o l u n t a r y e t h a n o l d r i n k i n g in e x p e r i m e n t a l animals. Also in o u r l a b o r a t o r y it has b e e n found that thiamin deficiency i n c r e a s e d ethanol intake of rats while dietary e x c e s s clearly d e c r e a s e d it e v e n in the rats without p r e v i o u s e x p e r i e n c e of deficiency [9,21] e m p h a s i z i n g the role of thiamin in the effects on e t h a n o l intake i n d u c e d by B-vitamin complex. T h e results of this report indicate that i n c r e a s e d ethanol c o n s u m p t i o n by deficiency of B - v i t a m i n c o m p l e x is not c a u s e d by the lack of niacin or ribot]avin but niacin could be i n v o l v e d in the d e p r e s s i o n of e t h a n o l intake by supplem e n t a t i o n with B - v i t a m i n c o m p l e x .

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DIETARY NIACIN AND RIBOFLAVIN

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