Acetaldehyde metabolism in mammals : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand

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

the permission of the Author.

ACETALD.l::HYDB MZ'l'ABOLI31.: IN J!AJ,fl,!ALS

A the sis presented in partial fulfilment of the requirement s

for the degree of

Doctor of Philo sopny

in Biochemi stry at

Massey University New Zealand

All.::m Robert STO','f..::LL

1977

;,BJTJ.ACT

'l'o determine the pharmacological importance of acetalclehyde in the actions of ethanol, this study ^1•1.3 planned to J.efine tbe levels of free acetaldehyde occurrint.; in human blood duri.ne the oxidation of ethanol in the bodJ.

Peripher^al venous blood. acetal<lehy<le levels v1ere detE.:r:ninecl by direct assay ^v.hile pub10nary blood lL:vel:.> weru estir;.·ttt.<l from breath acci,l.luehyde levels by employint; tLe blood:uir partition ratio for a^cetaldehyde at

37°C ^of 1 39 .:!:. 16. Pulmmnry blood �tcetalu.ehy<.l.e levtls were de:terw.ined to obtain values for (a) acetal<lehy<.l.e ou'vput from the liver and. (b)

acetaldehyde levels in blood. likely to ^be reaching the brain.

Sensitive enzymic metl!od.J for the determination of ace^talJ.^eh^{yde in} human blood and breath samples were J.ev.slope<l, ^allo\ing levels of

acetalJ.ehyde as low as 0. 5 Jlmoles

/

^{1 and} approximately o. 2 nmoles/1 OOml to be measured in blood and brenth samples respectively, using either yeast or sheep-liver aldeLyde dehydrogena.Jes. The methods were developed

to be opernted in semi- ^{or fully} auto:rnted modes and involved continuous­

flow distillation of samples -.vi ^th fluoror.1etry.

Two �nethodolot;ic.J.l problems associated. with the direct as3ay of acetaldehyde in blood were ::Jtud..i.bd. These were (a) the production of acetaldehyde durinG the deproteinization of ethanol-containinG blood with nerchloric acid and. (b) the r_apid disappen.rance of acetaldehyde in blood samples.

It was found that over 90;-o of the a^ce^taldehy^{de p}roduced durine the processinG of ethanol-containing human blood for assay originates frou reactions occurring when red blood cells, as distinct from plasma, are treated with perchloric acid.

The disappearance of acetaldehyde nr-..i.ch had been added to human blood saraples wns found to result fror:1 thtl rapid metabolism of acetaldehyde to acetate by red cells. By contrast, acetaldehyde formed frol!l in vivo

ethanol metabolism did not appear to be metabolized significantly in blood samples. It was sugcested that acetaldehyde formed in vivo may ^{be bound} to blood components.

ilbile hwnan subjects were metabolizing standard 1

g/ke

^doses of ethanol, breath aceta^ldehyde concentrations were found to range from 0.5 to

10.0

iii

nmoles/100ml while periph8rnl whole blood acetaldehyde levels ranged

from

0

^{- 12}

p1!

^and peripheral plasma levels ranc;ed from 0 - .3 J.ll.�.

Estimated pulmonary blood and hepatic venous blood levels of acetaldehyde

fell within

the ranges 0.9

^-

19

^{J-l1·l and 1,.5 - 95 pH} respectively. The changes in the acet

a

^ldehyde concentrations of blood and breath durins the metabolism of ethanol did not follow any identifiable pattern. ., LOwever, the results obtained suceesteU. that there was no free acetaldehyde pr^esent in the peripheral venous blood of h1unans metabolizinG moderate doses of

ethanol and the ^impo_rtance of acE:taldehyde in the ^effects of ethanol in peripheral tissues may be negligible. The esti^mated levels of acetaldehyde in blood passing to the ^brain way be sufficient to exert sit:;nificant

pharwacolot:;ical effects on the brain be1t further ^study of the ^binding of

ac�::taldehyde to tissue:. is requireU. before a fuller understanding of the toxic potential of acetalU.eh.yJe ^{can b8 G:J.ineJ.}

i .... ra^t-live^r pe^rfu_sion systeJ,;, S8t up ^to study hepatic ethallol me_tabolism, was used to dete1·mine tht! ^nature of hepatic acetalJe^hyU.e

production. Acetaldehyde production by perfused rat livers wa.s

characterized by a peak ^of acuti<lde: ,yJ.e, of variable magnitude, appearing in the hepa^ti^{c v}enous pel'fU3nt^{e in} the ^{first .30 min} of the p^erfusion

with medium containinG eth^�cnol. After the ^pea.k in acetaldehyde production, the metabo1isu1 of ethanol b.f the perfused livers cave negligible amounts

of acetaldehyU.e in the perfus^ate^s,

ACKNO','JLEDG J:l,�ENTS

I wish to thank my supervisors, Professor R.D.Batt and Dr R.M.

Greenway for the help they have given me throughout the course

of this stu�. I am also pleased to acY�owledge the help and advice received from all the members of the alcohol research

group, especially !.:r Y..Couchman and Dr E.E.Crow. Thanks are also

extended to Dr O.Batt for her me^dical supervision and Mr T.J.Braggins

and !.:rs K.!.:.Hewland for their expert technical assistance.

Abstract

Acknowledgements List o� contents List of �igures

COIJTENTS

SECTIOH 1

Tim<.ODUCTIOH

SECTIOH 2

DEVELOPMEnT OF AN AUTOI.:ATED ErJZYl.!IC ASSAY FOR ACETALDEHYDE lli Hill.W'I BLOOD SAi ^lPL:ZS

2-1 ^• Introduction

2-2. Reagents

2-3. Testing the automuteu distillation manifold

2-3-1. Distillation and calorimetric analysis of

Paee ii

iv V X

1

5

5

7

acetaldehyde 8

2-3-1 -1 ^• Optir:ri. zation of reaction conditions 8 2-3-1 _-2. ;.utoma tion of the I.:BTH method 1 0 2-3-2. Distillation and spectrophotometric assay of

acetaldehyde 1 0

2-4. Distillation a�d enzymic assay of acetaldehyde 17

2-h-1

^• Initial experiments 1 7

2-4-2. Semiautomated assay of acetaldehyde 17 2-4-2-1 • Phasing of sample collection 1 9 2-4-2-2. Development of fluorescence 19

2-4-2-3. Reproducibility 20

2-4-2-h. Characteristics of the distillation

manifold 20

2-4-2-5. Standard curves 23

· 2-4-2-6. Specificity of the semiautomated assay 23 2-4-2-7. Use o� yeast aldehyde dehydrogenase

with the semiautomated assay 2-4-3. Fully automated acetaldehyde assay

2-4-3-1. The manifold

2-4-3-2. Operation o� the rnani�old

2-4-4. Recovery of acetaldehyde from human blood

2-4-4-1 • Uethods

2-4-4-2. Results

26 28 28 28 30 30 32

V

2-4-5. Assay of blood samples containing ethanol 2-5. Discussion

SECTION 3

FORJ.!ATION OF ACEI'ALDEIIYDE IH DLOOD SJ�J!PLES COHTAIHTITG .i:.'TE/IJWL Dtra:C:G DEPRO'r.c.I!TIZATION

3-1

^• !ntroJ.uction 3-2. ll�ethods

3-2-1 ^• Sample r1repa1·a tion 3-2-2. Controls

3-2-3. Bla'1ks 3-2-4. Standards

3-3. Results

3-3-1.

^{Acetalder�d}e production in controls

3-3-2. Acetaldef\yde production in whole blood and plasma usine a 50i� dilution

3-3-3. Effect of thiourea on acetaldehyde production using a 5q� _dilu^ti_on of blood

3-3-4. Effect of blood dilution on acetaldehyde production

3-3-5. Effect of thiourea on acetaldehyde production

Paee

34 34

36

36 37 37 38 38 38 39 39 39

44

using ^a

1

01� dilution of blood 46 3-3-6. Affect of preincubation of ethanol vdth whole

blood on acetaldehyde form�tion 46 3-3-7 ^{• .}\cetaldehyde production in supernatants after

deproteinization

3-3-8. 3ffect of distillation ter.1perature on acetaldehyde level

3-3-9. Acetaldehyde production in ox blood 3-4. Discussion

SBCTION 4

DISAPPL.Al11JTCE OF AGE.l'ALDEIIYDE IN ^BLOOD SAJ,:PLES 4-1 • Introduction

4-2. Materials and methods

4-2-1 ^• Reacent s

4-2-2. Determination of radioactivity 4-2-3. Blood samples

4-2-4. Sample trea�ent and assay methods 4-2-5. Use of 14c-labelled acetaldehyde

4-3. Results

46

49 49 51

55 55 56 56 56 56

57

58 59

4-3-1. Comparison of the rates of disappearance of acetaldehyde in human whole blood and plasma

0 0

samples at 4 C anti 37 C

4-3-2. Individual variations in blood ucetaldehytie disappearance rates

4-3-.3. Effect of haemolysis on acet^aldehyde disappearance in human blood

4-3-4. Disn!Jpearance of acet^aldeLyde in human ^blood which has been frozen and tl awed

4-3-5. Disappearance of endoGeno�s acetaltielvde in blood

vii

59

59 G2 62

samples taken frora subjects lll^et^abolizing ethanol 62 4-3-G. The effect of ethanol ^and acetate on the

disappearance of acetaldehyde atided to blood

in vitro 66

4-3-7.

Disappearance of added acetaldel�de from blood

samples taken frow subjects metabolizing ethanol 68

4-3-8. Fate of acetaldehyde adtied to human blood 68 4-3-9. Disappearance of ac0taldehyde in washed _{red blood}

cell suspension�

4-3-10. Disappearance of acetaldehyde in ox blood

4-4.

Discussion

4-4-1. Disappeo.rance of acetaldehyde added to blood

71 71

saoples 71

4-4-2. Stability of acetalde 1yde for,acd in vivo from

ethanol 76

3�'riO!� 5

D:E\TELOPJ,Jl:-7 01:<' A

J,J:.""THOD FOR

^THE D1'T:U"'U.ImATION OF

ACErALDilliYDE IN lfUJIAH ^BI:Rf..TH SAPPLES

5-1 ^• Introduction

5-2. Methods and results

5-2-1. Assay of acetaldehyde in simulated breath

acetaldehyde traps

5-2-2. _Trap^ping of acetaldehyde present in simulated breath samples

5-2-2-1 ^• Acetaldehyde trappin& efficiency 5-2-2-2. Rep�oducibility and linearity of

simulated breath samples

5-2-.3. Determination of the efficiency of trapping acetaldehyde from actual ^{breath samples}

79 79 80

80

83 85 86 86

5-2-4. Reproducibility of actual breath samples 5-2-5. A^utom^ation of sa.11ple acidification

5-2-6. Fully automated breath [\cetaldehyd.c ^assay 5-2-7. ^{Stability of samples}

5-2-8. Determination of the n:n·ti tion ratio of

acetaldehyde distributed bct\'/een blood and air

Page 89 90 90 91

at equilibriu:.1 $'1

5-2-9. ::Jependence of tl,e water:air partition ratio for

acetaldehyde on c�uilibration tumperuture 93 5-2-1 0. ^Detennina^{tion of t}he variation of breath

acetD.ldeb.yde concentr^ation with the uepth of'

expired _{air 96}

5-2-11 • Le^ve^{l of} assayuLle alcleiJiYde!j in spirits conm.u:,ed

by human volunteers 99

5-3. Discussion 100

Sl_l;'l'IOU

6

J .Gi:.'T,:J.DZI�YDE L.:;:;.r,;,J..� ^IIJ 'l'lili llili::.','J.'H, ·�;IIOLl� V.i:l�OUS BLOOD /12TD I'L.�.J:.�� OF _{II1H.:J��} J1JBJ�'l':J I.:.�:."l'ABOLlZTI�G A STAJJJ:Jill

6-1. Introduction

6-2. _l.:ethods

DOSE OF E"riUu:OL

6-2-1. IIur:w.n nt<bj^ect.s

6-2-2. Adrnini strati on of etll<..nol to voltmteers 6-2-3. Blood samples

6-2-h. '.thole blood nmi ^plasme. assays

6-.2-5. J.�easuremt:nt of acetal^a.ehyd_e n.lld ethanol in breath sanple:;

6-3.

^Results

6-3-1 • Bredh acetaldehyde cor.�.centrations after the ir.t.;ention of ethanol in do:>e� of o. 5 and 1 .o

Gikc

102 102 103 103 1 Ol.,

104 104

105 106

bo� weight 106

6-3-2. Intra-lndividual vari^ation in the levels of breath acetald.e!zyde deteroi.ned after a standard.

do se ^{of ethanol} 1 08

6-3-3. Inter-individual variation in the levels of ' breutL acetaldehyde determined after a standard

do�e of ethanol 108

G-3-4. Breath aceta.ldehyde ll:lvels in n.lcoholics 110

6-3-5. _Acetalde�de in plas��, whole venous blood

and breath of hwnan subjects metabolizing a standard. ^{dosu of} e^thunol

6-3-6. Levels of acetald.^ehy^de ⁱⁿ peripheral blood

immediately after U.ridd.nc 6-4. :::li::.cus::don

S:SC'i.'ICN 7

THE PRODUCTIOI'T OF ACETJ,LDEIIYDE BY P�::R.FUSED RAT

1 1 0

11 ₇ 124

LIV'L..lS !::J:.'TABOLIZD�G l:.".i'HAJ\01 1 .31

7-1 • Introduction 1 31

7-2. Hethods 1 j1

7-2--1. Experimental anirr.al::; and operative technlque 1.31

7-2-2. Perfusion system 1₃1

7-2-3 ^{• .}m^alytic_al methods 132

7-3. Results and discussion 133

7-.3-1. Ethanol oxid^{ation rates 13.3}

7-.3-2. _Perfusate col!centru.tions of lactate and pyruvate 13.3

7-3-3. Bile production 1.36

7-3-4. Acetalde�ue ^production by the perfused livers 136

APPAilDIX I

PRODUCTION PlW J70R.AGE OF 1 -1 _4c LC.i:.'TALDEIIYDE

I^-1 • Introduction I-2. _!:ethod.s

I-2-1 • Production of acotaldehyde semicarbazone fror;1 etbmol

I-2-2. eceneration of QCetCi.ldchyde from its semicarbazone

I-3. Results

I-3-1. Oxidation of et^hanol

I-3-2. Regeneration of 1 -1 4c acetaldehy^de

APPENDIX II

ABBIU.VIATIOHS

142 142

142

1 43 1 44 144

14h

147

ix

Figure

2-1.

2-2.

2-3.

2-4.

2-5.

2-6.

2-7.

2-8.

2-9.

2-1 o.

2-11 •

2-12.

2-13.

2-14.

2-15.

2-16.

2-17.

3-1.

3-2.

LIST OF }'IGUI<.l!:3

Acetaldehyde standard. curve-J.:BTH ^method 9 Analytical rna.nifold U:3ed to autcr.mte the

MBTH assay method 9

Conbined d_istillat_ion and analytical manifolds used ^to automate the J.mTH assay

method 11

Analytical manifold for the uutoruated

spectrophotometric assay of acetaldehyde 1 2 Reproducibility and linearity of peaks

obtained by assayiut acetaldehyde standards

u::>ing the r:J.anifold illu�tr�ted in figure 2-l�. 1 2 Co4lbined distillation and ftnalytical manifolds for the spectrophotometric assay of

acetaldehyde 1 4

Reproducibility of peaks obtajr.ed froo acetaldehyde standards processed by the

manifold illu^strated in ^ficure 2-6. 1 5 Standard curve obtaiz'ed usinG the combined

distillation aml analyticnl r'lll.nifol:ds shown

in ^fieure 2-G. 16

Bi'fect of distillati^on t.er.perature on recovery

of acetaldehyde 16

Analytical manifold for tLe automated enzymic assay of acetald^{ehJue 1 8}

:!::ffect of nitrogen flow-rate on recovery of

acetaldehyd e 22

l!:ffect of distillation t er'lperature on recovery

of acetaldehyde 22

Acetaldehyde standard curve produced usint;

the sE:Iniautomated enzymic a ssay method 24

Acetaldel�de standard curve produced using

tte semiautomat_ed enzymic assay method 25 Interference of ethanol with the semi-

automated enzymic assay

Analy^tical manifold for the fully automated

enzyrnic acetaldehyde ^assay

Chart recordine of acetaldelzyde standards

assayed using the fully autorJated enzymic assay system

Production of acetaldehyde in control samples Production of acetaldehyde in whole blood deproteinized in the presence of ethanol by a

!JO;� ^dil^ution with H: PGA

27 29

31 40

41

Figure

3-.5.

3-4.

3-5.

3-6.

4-1.

4-2.

4-3.

4-4.

4-5.

4-6.

4-7^.

4-8.

4-9.

4-1 o.

4-11.

4-12.

5-1.

5-2.

5-3.

5-4.

Title

-

Production of acetaldehyde in plasma deproteinized in the presence of ethanol by a ^50;b dilution with 1 _{�.� PCA}

An acetaldehyde correction curve prepared by deprotcinizing whole blood containine different umounts of ethanol and employing a 1 q; dilution

43

of blood 47

The effect of preincubation on the production of acetaldehyne _in whole blood deproteinized

in the pre3ence of ethanol 48

.Acetaldehyde production ^{in PCA} supernatants of whole blood where ethanol has been added after deproteinization

Disappearance of acetaldehyde added to human whole blood _and plasma sLU:Jples at 4 °C and 37°C

Individual variations in acetaldehyde

disappearance in human blood samples at 37°C Semilogarithmic plot of the data presented in figure ^4-2.

The effect of haemolysis on the disagpearance

of acetaldehyde in human blood at 37 C

Disappearance of acetaldehyde in human blood which has been frozen and thawed

Dinappearance of acetaldehyde in fresh ox blood

Changes in the concentrations of endogenous acetaldehyde in blood s·-u:1ples taken from subjects metabolizinG ethanol

The effect of ethanol and acetate on the disappearance of acetaldehyde added to whole blood in vitro

The disappearance of added acetaldehyde fro:n human blood sample3 ta.'l.;:en fl'om a subject metabolizing ethanol

The metabolism of 1 -1

4c

acetaldehyde by human blood in vitro

Graph of haematocrit versus the half-life of acetaldehyde in human erthrocyte suspensions The disappearance of acetaldehyde in a human

erythrocyte suspension

Comparison of standard curves obtained by assaying simulated breath acetaldehyde traps and a·ueous acetaldehyde standards

Apparatus used to produce and trap simulated breath acetaldehyde samples

Reproducibility of simulated breath samples Sample pumping unit used for the automatic acidification of breath acetaldehyde samples

50 60 61

64

64

65

67

69 70 72 73

82

84 8 7 8 7

xi

Figure

5-5.

5-6.

6-1.

6-2.

6-3 a to e.

6-4.

6-5 a to i.

6-6.

7-1.

7-2.

7-3.

7-4.

7-5.

7-6.

Title Paee

Dependence of the water:air partition ratio

for acetaldehyde on equilibration temperature 95 Variation of breath acetaldehyde and ethanol

concentrations with the depth of expired air 98 Blood ethanol and breath acetaldeLyde levels

of two male subjects after the consumption

of ethanol loads of 0.5 anQ 1.0 g/ke 107 Variation in the brec.th accttlld.ehyde

concentrations of one individual, after three

standard 1. 0

g/kg

ethanol loads 109 Inter-individual variation in the levels of

breath acetaldehyde determined after a standard

dose of ethanol 111 -11 5

Breath acetaldehyde levels versus blood ethanol concentrations in alcoholic patients 116

Acetaldehyde in plasma, whole venous blood and pulmonary blood of human subjects

metabolizinG a stanciard do�e of ethanol 11 8-1 22

Levels of eth�nol and acetaldehyde in peripheral venous blood :immediately after

initiation of ethanol consumption 1 25 Perfusate lactate levels during the perfusion

of rat livers 134

Perfusate pyruvate levels

during

^{the perfusion}

of rat livers 134

Chance in the lactate:pyruvate ratio of the perfusion medium during the perfusion of rat

livers 135

Chanc;es in bile flow-retes during perfusion

of rat livers 137

The appearance of acetaldeliyde in the perfusate leaving rat livers metabolizing ethanol 138

Scatter diagram of pea,c acetaldehyde

concentration versus eth�.mol clearance rate

for perf'used. livers 140