Ethanol metabolism in humans : a thesis presented in partial fulfillment for the degree of Master of Science in Biochemistry at Massey University

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ETHANOL METABOLISM IN ff(J}'Lfi.NS

A thesis presented in p~rtial fulfilment of the requirements for the degree of

Master of Science in

Biochemi str-J

at Massey University

Kenneth G. Couchman 197h

Alcohol metabolism in hurnt:ms has been studied by examining blood, urine and breath samples talrnn at f:cequent interve,ls for 3 hours after an alcohol loacl of 0. 5ml/kg in a fasting concli tion.

i

A gas chromatographic method was developed for the simultaneous estimation of acetaldehyclo, ethanol and acetone levels in blood and urine specimemJ and various column packings were investigated.

Porapak Q uas the most suitable material and the method finally adopted usecl the headspcwe gaD phase over urino or perchlorate precipitated blood specirn.ens to which had bGon acldecl sodium sulphate to displace the volatile components fro:~<. the aqueous phase. Protein precipitation uns necessary in onler to urevont the loss of acetnldehycle fro:1 the blood samples.

valve was fitted to eno.blo simila:::- determinationcJ in breath sai11ples but was not used in this study.

Assays by methods ~-rsTG developed for lactnte ₁ pyruvate, 13-hydroxybutyrate, glucose and glycerol utilisinc; the changes in concentration of Nl\DH uhich uas measured by fluorometry and. the merits of converting l'TAD+ to a fluorescent compound uas examined.

Twenty male and eight fomales volunteered for the study. Blood samples were obtained from ru1 intravenous catheter₁ a procedure supervised by a physician. Blood alcohol levels were monitored by breath tests with an electrochemical device, (0,n Alcolimiter) for detecting ethanol,

There were considerable variations in the peak alcohol levels and in the time ta_1rnn for equilibration in the body. E:stimates of

the rate of metabolism of ethanol and of body content were in agreement with those published in the literature. Breath testing was found to be a satisfactory means of e3timating bloocl alcohol concentration in the post-absorptive phase. Tho Alcolimiter gave readings that trnre on an averae;·o 1 OllG/1 OOml low, but thio could be corr·ected by recalibrn.tion. Blood acetnlclehyde levels roso to 0.1mg/100ml and occasionally to 0.2mg/100ml.

A fall in blood pyruvato level, which remained low throughout the test period, was seen to coincide uith the increase in blood alcohol. There ~·rns a tendaHcy for lactate to rise at the same time. This was not consiDtent, but the ratio of lactate to pyruvate increased 2 or 3-folcl in most cases uhich reflected the change in cytoplo..smj_c redox ratio.

Small increases were obse:t:'Vod in blood glucose even though the alcoholic drink uas free of sugar. Thero woro increases in

blood glycerol levels in all subjects and somo of these vrere quite large. '1¹hese findings 1-rere contrary to somo roports uhich have appeared in the literature.

The excretion of electrolytes was oxaminod in tlw urino but the results were difficult to interpret.

Alcohol concentrations in urino samples were measured and compared to the blood lovols and the diuretic effect of alcohol uas noted.

These findings, together with those repo:ctecl in the literature have boen discussec together '.ri.th their significance in

interpreting disturbances of motabol:Lsm when alcohol is consumed.

~Ioro assays o.ro thought to bo required including thoi3G for bloo<i Hcetato, blood triglycor:Ldos 1r:Lth froo fatty ncids o.ncl nom0 hormones. It is corn:iiderod_ ^.Ll ulW.T, ^: tho use of le.belled compounds could o.cld a nc;1J diBGnsio:n to ^·cho in vivo ir1v00 ti[s~a tio11s 011 htlD0.11

v-olu_:nteors.

ii.

ACKNOWLEDGJV".tENTS

I am indebted to my supervisor, Professor R. D. Batt for his continuing interest in the study and help with the preparation of the manuscript for the thesis. I am also grateful to Dr L. Bieder for his medical supervision of the work with volunteers and assistancG in formulating the protocol. Hy appreciation is due and acknowledged to Torry Braggim' for technical assistance 3 Dr R. Greenway both for advice and the assays of electrolytes, Dr R. Brooks for help with estimations using atomic absorption spectro- scopy; and to all the members of the team working on

alcohol re.search in the department for continual advice and discussion of the project.

The thesis was typed ancl copied by Ms Marion Trevor to whom I am indebted for her patience and care at all stages of preparing the final manuscript.

LIST OF CONTENTS

Chapter INTRODUCTION

Chapter 2 THE ESTIMATION OF ACETJ\.LDEHYDE₃ ACETONE A..llJD ETHANOL IN BREATH, URINE .!\ND BLOOD SJuVJPLES

2. 1 DEVELOPMENT OF GAS CHROMATOGRli.PHIC 2.1.1

2 .1 • 2 2 .1 .3

2.1.4 2. 1

.5

2.1.6 2.1.

7

2. 1 .8 2.1.9

l'flETHOD

Introduction

Sample preparation Gas chromatograph Integration

Syringos

Distilled water

Internal standardisation Standards

Deter~ination of optium operating conditions

2.2 COMPARISON OF COLUMN PACKINGS

5

:; r'

6 6

7

9 9 11 12 12

2.2.1 Porapak Q 14

2.2.2 Carbownx 1)00 (5%) on Chromosorb

W-DMCS 20

2 .2 .J Carbowax 400 ( 10%) on Chromosorb

W-DMCS 25

2.2.4 Carbowax 1540

(5%)

on Chromosorb

W-DMCS 26

2.2.5 Conclusions 27

2.3 APPLICA'rION TO BLOOD AND URINE 2. J.1

2.3.2

2.3.3

2.3.4

2.3.5

2.J.6 2.J.7 2.J.8

2.3.9

SAMPLES Introduction

Effect of blood volume Stability of blood sample Precipitated blood samples Headspace gas enrichment Gas chromatograph operation St2ndards

Urinary alcohol Conclusions

2B 29 31 33

35

36

37

39 39

2

.4

BREATH TESTING BY GAS CHRO:tvli1.TOGR!l.PHY

40 2.5 BREATH TESTING BY ELECTRO-CHEMICAL

ANALYSIS 41

Chapter

3 3.

1

3.2 3.3 3 .

.3 ^Q 1

3.3.2 3.3.3

3.4

3.6 J.7 3.8 3.9

3.10 3 .11 3.12 Chapter

4

4.1 4.

2

l+.

3

4.4

L1 .• 8

4.9 4

^.10 l+. 11

4.12

THE ESTIMATION OF NON VOLATILE CONSTITUE.l\JTS IN BODY FLUIDS INTRODUCTION

REAGENTS FLUORO:METRY Instrumentation Heasurem.ent of NAD~

Measurement of NA.D LACTATE

HYDROXYRUTYRATE PYRUVATE

YiR!jVATE BY DIRECT MEASUREMENT OF

NAD

ACETOACE'rii.TE GLYCEIWL GLUCOSE

CALCIUM .AND MAGNESIUM

SODIUM, POTASSIUM AND CHLORIDES RESULTS

SUBJECTS

EFFECTS OF ALCOHOL 1/ Absorption,

2/ Equilibration, a...~d

3/

Eliwination of alcohol BODY WATER .ESTIM.ll.TES

ll.CETALDEHYDE ACETONE 1/ Lactate

2/

Pyruvate_,

3/

f-hydroxybutyrate a.rid

4/

Redox ratios GLUCOSE

GLYCEROL

URINE 1U,COHOL LEVELS

DIURETIC EFFECT OF ALCOHOL

- f ++ ++ + +

46 47 48

49 50

51

5h 57 58 59 59

60

61 61

63 65 65

67

68 68 69 69

70

71 71

72 72

73

EXCHETION o Ca 9 Mg , Na ;) K

and Cl-.

75

BHE.I\TH VERSUS BLOOD ALCOHOL LEVELS 78

Pa~e

Chapter

5

DISCUSSION

5.

¹ EFFECTS OF J.\.LCOHOL 80 5.2 ABSORPTION, EQUILIBPJtTION .A.i"\JD

ELIMINATION OF ALCOHOL AND THE

ESTL\/fATION OF TOTfl.L BODY WATER 82

5.3 ACETil.LDEHYDE 86

5.4

KETOGENESIS

89

5.5

LACTA'l'E, PYRUVATE AND THE REDOX

RATIO

91

5.6

C.t.RBOHYDRATE METABOLISM 93

5,7

FAT METABOLISM ₉₄

5.8

^DIURESIS 95

5.9

THE ELIMINATION OF ELECTROLYTES

.1;-'\.ND MINERALS IN THE URINE 97 5.10 BLOOD ALCOHOL ESTil1ATES BY BREATH

TESTING 98

Chapter 6 CONCLUSIONS AND FUTURE WORK Appendix 1 A WORJIBOOK OF PROCEDURES Appendix 2 TABLES OF RESULTS

References

2. 1

2 " • c:

2.3 2.4

2.s

2.6

LIST OF FIGURES

A gas chromatogreJU of 0.5 ul. water on unpacked stainless steel columns before and after treatment with phosporic acid

fl gas chromatogram illustrating inte- grator function

Van Deemter plots of acetaldehyde and ethanol on Porapak Q

Gas chromatogrnm, 1

Jal.

acetaldehyde/

tithanol on Porapak Q showing tailing at high temperatures

Calibration curves for ethanol and acetaldehyde on Porapnk Q

Gas chromatogram of water and acetalde- hyde on

5%

Carbowax 1500 on Chromosorb Gas chromatogram.., headspace gas of acetaldehyde.9 ethyl methyl ketone and ethanol on

5%

Carbowax on Chromosorb

. . ^..

Effect of blood volume on equilibration of headspace gas

Effect of incubation at 55°c. on the levels of acetaldehyde.., ethyl methyl ketone and ethanol in tho headspace gas 2. 10 Calibration curves for acetaldehyde,

acetone ax1d ethanol. Headspace gas on

2.11

3.1

Porapak Q

Variation of ¹Alcoliwiter¹ readings with temperature on a standard 100mg/

1 OOml. simulated breath sample

Decay of fluorescence of Nil.DH in the assay of pyruvate

3.2

Effect of NA.DH concentration on the 3.3

4.1

pyruvate assay

Standard curve for pyruvate assay at various NADH concentrations

Graphs illustrating the variation of the rate of absorption and equilibration of alcohol

iv.

8

10 16

17

19 22

24

30

32

38

44 52 53

55

62

LIST OF TABLES

Table

4.

1 Th0 age, sex and drinking habits of the subjects with their inter- pretation of the effects of the

alcohol dose 41

4.2

Results of the application of the

Widmark formulae 66

4.J

The ratios, urine:blood alcohol levels during the absorptive and

postabsorptive phase

74

4.4

The average excr~tion+in ¥rine+~er

JO

^min~~es of Cl , Na , K ^J Ca ,

and Mg 76

4.5

Patterns of excretion of electrolytes

and minerals 77

Appendix 2 Tables:-

1 Blood alcohol 2 Blood acetaldehyde 3 Blood acetone

4

Blood lactate

c:: Blood pyruvate

"'

6 Blood J-hydroxybutyrate 7 Redox ratios

8 Blood glucose 9 Blood glycerol

10 Urine alcohol

11 Urine volume

12 Urine specific gravity 13 Urine sodium

14 Urine chloride

15

Urine potassium 16 Urine calcium 17 Urine magnesium 18 Alcolimiter readings

v.