Ethanol and acetaldehyde metabolism in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry

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Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

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ETHANOL iJ'.JD llCETi.IDEHYDE l'1ET.ABOLISM IN SHEEP

A. the s is presented in partial fulfilment of the requirements

for the deg ree of D octo r of Philosop�

in Biochemistry at

Massey University, New Zealand.

J

ane HENDT Ll�SS

1973

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Ethanol and acetaldehyde metabolism in sheep has been studied in three different types of experiments:in purified enzyme systems� in liver homogenates and

in

the intact animals. Particular emphasis has been placed on the aldehyde oxidase enzyme from sheep liver, a molybdo

flavoprotein with a broad specificity which includes aldehydes� quinines and N1-methyl nicotinamide. This thesis describes a method for prepar

ing an enzyme solution in which sheep liver aldehyde oxidase constitutes 85% of the total protein present. Investigations of its physical and kinetic properties show that the sheep liver enzyme differs from the aldehyde o�idases previously prepared from pig and rabbit livers. In addition,

an

antibody to sheep liver aldehyde oxidase has been prepared from rabbit serum and has been shown to act as a specific� competitive

inhibitor of the enzyme. This has been used to assess the contribution that aldehyde oxidase makes to acetaldehyde oxidation in sheep liver homogenates under several different conditions.

The effects of steroids on ethanol and acetaldehyde metabolism has been investigated, special interest being taken in the effects of progesterone. Progesterone stimulates sheep liver aldehyde oxidase activity

in

vt�-� and inhibits sheep liver aldehyde dehydrogenase.

Administration of progesterone to castrated sheep in vivo increased the rates of ethanol and acetaldehyde oxidation, and aldehyde oxidase has been identified as a factor in decreasing acetaldehyde concentrations in the homogenates of livers from these animals during the metabolism of exogenous ethanol. Low endogenous ethanol concentrations in

peripheral venous blood of sheep are positively correlated with high progesterone levels in sheep due to its experimental administration, and to pregnancy and the oestrus cycle.

Studies of the effects of disulphiram on ethanol and acetaldehyde metabolism have shown that the compound inhibits sheep liver aldehyde oxidase and aldehyde dehydrogenase enzymes �vitro, increases

endogenous concentrations of acetaldehyde

in

peripheral venous blood, and causes acetaldehyde accumul�tion during ethanol metabolism

in

vivo.

When diazepam

;:�·l?

present together with disulphiram it provides

protection from all but one of the effects shown by disulphiram alone.

It does not alter the disulphiram inhibition of sheep liver aldehyde dehydrogenase. Amitryptyline is an inhibitor of both aldehyde o�idase and aldehyde dehydrogenase enzymes. It seems to increase the aldehyde

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oxidase response to disulphiram, and its in vivo administration causes acetaldehyde ac cumulation in peripheral blood during and in the absence of metabolism of exogenous ethanol.

Investigations into the effects of ethanol on ethanol and acetalde

hyde metabolism in sheep have shown that ethanol increases the activity of aldehyde oxidase in vitro and its chronic administration accelerat�s acetaldehyde oxidation in vivo. A supplementary study of the inter

relationships between the relative conc entrations of NADH and NAD+, and ethanol and ac etaldehyde metabolism shows that aldehyd e oxidase participation in acetaldehyde oxidation is dependent on the NAD+

c oncentrations, and that acetaldehyde oxidation can account for much of the NADH ac cumulation that oc curs during ethanol metabolism in vivo.

Acetaldehyde oxidation during ethanol metabolism in sheep can be diverted through the aldehyde oxidase catalyzed pathway, avoiding

dependence on the NAD+-linked aldehyde d ehydrogenase enzyme. The results

in

this thesis have shown that aldehyde oxidase can catalyze up to two

thirds of acetaldehyde oxidation in sheep liver when NAD+ is limited,

and that the pathway is dependent on the endocrine state and the

pattern of ethanol consumption of the animal.

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ACiiliOWLEDGEi�NTS

The author wishes to thank her supervis ors, Prof . R .D . Batt and Dr . R . M .Greenway , for their help and advice throughout t he c ourse

of this s tudy .

In

addition she expresses sincere apprec iat ion of the help, adv ice and technical as sistance rendered by other members of the

staff of the Department of Chemistry and Biochemistry , Massey Univer sity; in particular, thru1ks are d ue to Mis s L . Burson who willingly performed many duties outside her usual sphere of work .

The staff of the Animal Physiology Unit , especially rtr. L .Robertson, were generous in giving freely of their time and knowledge

in

experiments involving live animal s .

The author also thank s Mrs.E. Self for typing the manuscr ipt and remaining patient during inevitable delays .

Finally the patience and perseverance o f �tr . R . E . Hendtlass must be recognised , for without these this thesis would not have been.

Upon the advice of Prof . R .D . Batt, this the sis is presented in

the form of several paper s to facilitate it s later publication.

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LIST OF CONTENTS

Chapter

1

INTRODU:::.TION i1ETii.BOLISM OF f-.CETiJ.,DEHYDE

I) Enzymatic Synthesis of J�cetaldehyde in Mammals II) Enzymatic Degradation of Acetaldehyde in Mammals PHLRMilCOLCGY OF LCETi;LDEHYDE

OUTLINE OF THE WORK PRESENTED IN THIS THESIS

Chapter

2

ALDEHYDE OXIDLSE 1"1ETHODS

_i1ND

;.1.f,TERH.LS

I) Enzyme i�ssays with Various Hydrogen

_.f'^...

c ceptors II) Other Methods

RESULTS

I) Enzyme Purification: Sheep Liver Lldehyde Oxidase II) Physical Properties:Sheep Liver lUdehyde Oxidase III) Kinetic Properties:Sheep Liver l�dehyde Oxidase

IV ) Immunology DISCUSSION

Chapter

3

ESTIMATION OF ETH.iJiJOL

LND

liCETJ ... LDEHYDE

TI�

BIOLCGICLL FLUIDS DET LI

_IS

OF THE 111ETHOD

Collection of Blood Storage of Blood Samples

Estimation of Ethanol,

l ^...

cetaldehyde and Acetone in Blood Samples

Infusion of Ethanol or Acetaldehyde into Sheep Liver Homogenate Preparation and AnQlysis DIS:: USSION

Chapter

4

EFFECT OF PRCDESTERONE ON ETHJ.J'i!OL

_lJID

LCET,-.. LDEHYDE METi.BOLIS1'1

METHODS RESULTS

f...ldehyde Oxidase and Xanthine Oxidase

_J^..

ctivities in Sheep Livers

Steroid Effect on Sheep Liver J.ldehyde Oxidase

Steroid Effect on Sheep Liver Aldehyde Dehydrogenase

Page

1

1 4 8 9

11 12 12 16 17 17 20 24 32 39

40 47 47 50

50 54 54 57

59 59 61

61 61 64

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L.dministration of Progesterone to Sheep Liver Homogenates

Oestral Va�iation in Endogenous Levels of Ethanol and J:..cetaldehyde

}!-:ndogenous Levels of Ethanol and Acetaldehyde during Pregnancy

DISCUSSION

Chapter 5 EFFEQT OF DISULPHIRJ.M fiND OTHER COMPOUNDS ON

Page 64 70 73

73 76

ETH!liTOL AND. J:..CETiJ.JJEHYDE 1'1ETiJ30LISM ₇₇

METHODS 80

RESULTS 81

I) Disulphiram, Diazepam and i.mitryptyline 81 i) Effects of Disulphiram, Diazepam and f:..mitryptyline

Hydrochloride on Sheep Liver Enzymes in vitro 81 ii) Effects of Disulphiram, Diazepam and i.mitryptyline

J.dministration .in vivo on Endogenous .i:..cetaldehyde, Ethanol a'l.d J.cetone Levels in Peripheral Venous

Blood of Sheep 83

iii) Effects of Disulphiram, Diazepam and l�itryptyline on the Clearance of Exogenous Ethanol from Peripheral

Venous Blood of Sheep 88

I I ) Metronidazole 90

DISCUSSION 93

Chapter 6

METHODS RESULTS

OTHER J.SPECTS OF ETH.i.NOL i.ND J.CETM.DEHYDE .J'.-lET IJ30LISM

I) Effects of Chronic Ingestion of Dilute Ethanol Solutions on Ethanol and J:..cetaldehyde Metabolism .in ^__yivo

i) Voluntary Intake of Dilute Ethanol Solutions ii) Effect of Chronic Ingestion of Ethanol on Basal

L.cetaldehyde , Ethanol and Acetone Levels

in

Peripheral Venous Blood of Sheep

iii) Effect of Chronic Consumption of Ethanol on the Clearance of Exogenous Lcetaldehyde from the Peripheral Venous Blood of Sheep

98 99 100

101 101

101

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-vii-

II) Relationships between Relative Concentrations of Nl�H and Will+, and Ethanol and ll.cetaldehyde Metabolism in

Page

Sheep 104

i)

Metabolism of Exogenous Ethanol in Sheep Liver

Homogenates 104

ii) Changes in the Relative Concentrations of Pyruvate and Lactate

in

during li.cetaldehyde arrl Ethanol Metabolism in vivo 107

DISCUSSION 1 07

Chapter 7 CONCLUSION 11 0

il.ppendix I 1�IDEHYDE DEHYDRCGENJ"'§� 113

REFERENCES 119

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FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

FIGURE 6

FIGURE 7

FIGURE 8

FIGURE 9

LIST OF FIGURES

Standard Curve for the Determination of Changes in Oxidised Methylene Blue Concentration

Standard Curve for the Emission Spectrographic Estimation of Holybdenwn

Electrophoresis of a Purified Sheep Liver Aldehyd e Oxidase Preparation

Diagrams to show the Patterns obtained when Sheep Liver Aldehyde Oxidase and Xanthine Oxidase were separated by Gel Electrophoresis and Stained with a Hethylene Blue Activity Stain

Sedimentation Pattern of a Purified Sheep Liver Aldehyde Oxidase Preparation in the Analytical Ultracentrifuge

Absorption Spectra of Oxidised and Reduced Sheep Liver Aldehyde Oxidase

Determination of the Optimum pH for Sheep Liver Hdehyde Oxidase Activity using J.cetaldehyde as Substrate

Determination of the Michaelis Constant of Sheep Liver Aldehyde Oxidase for Acetaldehyde as Substrate Effect of Various E-l:.hanol Concentrations on the Rate of Oxygen Uptake catalyzed by Sheep Liver Aldehyde

Oxidase

FIGURE 1 0 Irnmunoelectrophoresis of Aldehyde Oxidase Prepar·-

FIGURE 11

ations from Ewe and Wether Livers and a Xanthine Oxidase Preparation

Oxygen Uptake catalyzed by Sheep Liver Aldehyde Oxidase in the presence of Anti-Aldehyde Oxidase

Page

15 15 21

22

23 26

27 30

34

36

·{

-Globulin or Blank

;{

-Globulin Preparations 38 FIGURE 12 Effect of Varying the

'(

-Globulin Concentration

on the Rate of Oxygen Uptake catalyzed by Sheep Liver Aldehyde Oxidase

FIGURE 13 Effect of Anti·-Aldehyde Oxidase (-Globulin Preparation on Ethanol and Acetaldehyde Levels in Sheep Liver Homogenates during Metabolism of Exogenous Ethanol

38

40

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FIGURE 1 4

F IGURE 1 5

-ix-

Postulated Sequence of Sites of Electron Egress and Inhibition of Electron Transport within the Sheep Liver Aldehyde Oxidase Molecule

C ollection of Blood from the Left Jugular Vein of a Sheep

F IGURE 1 6 Typical Variation in Endogenous Acetaldehyde , Ethanol and Acetone Levels in Sheep Blood

Page

43 48

(Left Jugular ) throughout the Day 51 FIGURE 1 7 Variations in Acetaldehyde and Ethanol C oncentrations

in a Typical Blood Sample during Storage at 4°C 52 F IGURE 1 8 Typical Traces of the Gas Chromatographic Separation

of Volatile Compound s in Standard Aqueous Solution and in a Sheep Blood Sample

F IGURE 1 9 C alibration Curves for the Gas Chromatographic

FIGURE 20

FIGURE 21

Estimation of Ethanol , Acetaldehyde and Acetone Concentrations

Histogram to show the Distribution of Aldehyde Oxidase l�ctivities in the Livers of Individual Sheep

Effect of varying the Progesterone C oncentration on Sheep Liver 1ldehyde Oxidase Activity

F IGURE 22 C learance Pattern of Acetaldehyde from the Peripheral Venous Blood of Sheep following its F IGURE 23

FIGURE 24 FIGURE 25

Intravenous Infusion

C learance Pattern of Ethanol from the Peripheral Venous Blood of Sheep fo llowing its Intravenous Infusion

Metabolism of Ethanol in Homogenates of Livers from Progesterone-Treated Wethers

Metabolism of Ethanol in Homogenates of Livers from Progesterone-Treated \rJethers in the presence

53

56

63 67

69

69 72

of Anti-1Udehyde Oxidase �-Globulin Preparation 72 FIGURE 26

F IGURE 27

C omparison of the Variation in Endogenous

Acetaldehyde and Ethanol Levels with the Levels of Progesterone in Peripheral Venous B lood of a Non-Pregnant Ewe reported in the literature Mean Endogenous Acetaldehyde and Ethanol Levels in the Peripheral Venous Blood of a Ewe during

and

following Pregnancy

74

75

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FIGURE

28

Absolute Endogenous Acetaldehyde ru1d Ethanol Levels in the Peripheral Venous Blood of a Pregnant Ewe for

Page

Three Weeks before and after Parturition

75

FIGURE

29

Structures of some Compounds that affect Acetaldehyde

and Ethanol Metabolism

78

FIGURE

30

FIGURE

31

FIGURE

32

Inhibitor Constant for Disulphiram Effect on Sheep Liver Aldehyde Oxidase

Graph to show the Effect of Varying Diazep&�

Concentrations on Disulphiram Inhibition of Sheep Liver Aldehyde Oxidase

Inhibition of Sheep Liver Aldehyde Oxidase by li.mi tryptyline Hydrochloride with and without

82

Disulphiram Present

85

FIGURE

33

Effects of Disulphiram, Diazepam and l®itryptyline Hydrochloride Administration in vivo on Endogenous J.cetaldehyde, Ethanol and Acetone Levels in

87

FIGURE

34

Effects of Diazepam and fillritryptyline Hydrochloride J.dministration in vivo on Endogenous Acetaldehyde, Ethanol and fJ.cetone Levels in Peripheral Venous

Blood of Sheep

89

FIGURE

35

Effects of Oral Administration of Disulphiram, a nd Diazepam and Disulphiram together on in vivo

Clearance of Exogenous Ethanol from Peripheral Venous Blood of Sheep

FIGURE 36 Effects of Oral Adr..-,t,.nistration of Disulphiram, and i:..rnitryptyline Hydrochloride and Disulphiram together on in vivo Clearance of Exogenous Ethanol from

FIGURE

37

Effects of Oral li.dministratio.1 of i'1etronidazole on in vivo Clearance of Exogenous Ethanol from

91

92

95

FIGURE

38a

Voluntary Consumption of Dilute Ethanol Solutions

by Sheep I

1 02

FIGURE

38b

Voluntary Consumption of Dilute Ethanol Solutions

by Sheep II

1 02

FIGURE

3 9

Effect of Chronic Consumption of Dilute Ethanol Solutions on the li1 vivo Clearance of Exogenous

Lcetaldehyde from the Peripheral Venous Blood of Sheep 103

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-xi-

FIGURE 40 Effects of Varying the NAD+ Concentration on Ethanol and l�cetaldehyde Levels in Sheep Liver Homogenates during Metabolism of Exogenous Ethanol FIGURE 41 Effect of Anti-lldehyde Oxidase

{

^-Globulin

Preparation on Ethanol and Acetaldehyde Levels in Sheep Liver Homogenates during Metabolism of Exogenous Ethanol in the presence of Varying

Nl�+ Concentrations

FIGURE 42 Effect of in vivo Metabolism of Exogenous Ethanol or i1cetaldehyde on the Relative Concentrations of Lactate and Pyruvate in the Peripheral Venous Blood of Sheep

FIGURE 43 Hajor Pathways for Ethanol Metabolism in the Liver

Page

105

106

108 111

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a. m . by vol.

GoQ

D .6 DGI DM E.C

.No.

EDTJ.

Fi.D

£.

HCl K K.

l

K m

ID

50 M MeB mmoles mg%

N Ni.J/

N1.DH Ni.DP+

Nl.DPH

NE OD p p(

pHmax R Rf RNJ.

s

s SD

LIST

OF

i...BBREVIL.TIONS

ante meridian by volume coenzym6 Q

d iffusion constant change in

2,6-dichlorophenol indophenol dry matter

Enz yme Commission Number ethylenediamine-tetra-acetate flavin adenine dinucleotide gravity

hydrochloric ac id degrees Kelvin inhibitor constant Michaelis constant

lethal d o se for 5 0% of a population molecular weight

methylene blue millimole s mg/1 00cm3

n��ber of sample s

nicotinamide adenine dinucleotide

reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate reduced nicotinamide adenine dinucleo tide pho sphate

not estimated optical density relative density pro bability les s than optimum pH

universal gas constant

distance travelled relative to the front ribonucleic acid

sedimentation c oefficient Svedburg s

s tandard deviation

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SEM -8-S

tert.

T

vol.

w/v v/v

1zero1

-xiii-

standard error of the mean sulphydryl group

tert iary

absolute temperature p artial· specific volune v.olune

weight/volume by volume

taken at zero time