A new highly sensitive assay for breath acetaldehyde: Detection of endogenous levels in humans

AYIII.YTICAI.

114,

BIOCHEMISTRY

A New

l-7

Highly

(1981)

Sensitive

Detection R.

JOHN Depurlmenl

Assay

of Endogenous

DANNECKER,

JR.,

of P.rychiarrl,. Alcohol Connecticut 06032; and

G.

EDWARD

Kesearch Diraision

Levels

Centrr, oj’ Infernal

Received We describe a new technique out the volatile components specimen by gas chromatography. AcH/liter.

of

Endogenously

in concentrations and convenient

and

metabolism

in man.

4cH

October

generated

subjects, reproducible,

levels

Acetaldehyde ( AcH)’ is the first metabolic product of ethyl alcohol and may be toxic to humans (I ). Abnormalities in the metabolism of AcH have been implicated in the etiology and pathogenesis of alcoholism (1,3). Genetic and racial differences in the metabolism of ethanol to AcH have also been reported (4,5 ). Research into the metabolism of AcH has been fraught with conflicting results and controversy, largely due to technical difhculties in measuring AcH in blood in an accurate and reproducible fashion. Methodologies in current use are open to criticism, since AcH continues to be generated and metabolized Irn the blood even after the specimen has been collected in vitro (6 8). In an effort to circumvent these problems. many researchers have attempted to measure the concentration of AcH in the breath (5.9 11). Since AcH readily penetrates cell membranes and equilibrates between capillary blood and alveolar air, it seemsreasonable to assume that the concentration of 03510-03.

chromatography;

detector.

FID.

dual-flame

ionization

23.

gc,

MICHAEL

of Connecticut Georgetown

Health Unrwrsit~

Center. Ho~piful.

Furmington.

I980

of AcH

ranging from to use: it offers

’ Supported by NIAAA Grant I P50 AA Center for the Sludy of Alcoholism. ’ Abbreviations used: AcH, acetaldehyde;

Ptjtt.Ltps

AND

D. C. 20007

for measuring acetaldchyde breath in liquid nitrogen The method is uniquely

nonalcoholic accurate.

Acetaldehyde:

in Humans’

SHASKAN,

Univrrsiry Medicine.

Washington.

pmol

for Breath

gas

(AcH) in the breath. by freeftng then assaying this concentrated sensitive, detecting as little as IO were

detected

in

the

breath

of

1-t

0.7 to 11.0 rig/liter. This technique is a new approach to the study of ethanol

AcH in breath reflects its concentration in arterial blood ( IO, 12). Direct experimental evidence for this relationship between breath and blood levels was recently demonstrated in human subjects by Finnish workers, using an isotonic semicarbazide method ( 12). Since semicarbazide derivatization methods may include a temperature-dependent step and numerous intermediate steps which can be sources of error, we experimented with a technique to capture and concentrate volatile components of breath and analyze them directly. Early in our studies, we found that this method was sufficiently sensitive to measure endogenous (i.e., nondrinking) levels of AcH and ethanol. which gave us further encouragement to continue development of this novel technique. MATERIALS

AND METHODS

Brearh sampling procedure. The principle employed in this procedure involves freezing out and trapping those volatile components of breath that have a freezing point greater than the temperature of liquid nitrogen. The device is shown in Fig. I. The subject inhales and places his mouth tirmly around a Z-cm-

2

DANNECKER.

FIN;. I, Human piece: (b) pump; containing bath, 37°C;

liquid (g)

breath sampling (c) sampling nitrogen; Erlenmeyer

(i) I in. (o.d.) plastic (k) bypass: (A) sidearm C: (D) stopcock and Methods.

apparatus: loop: (d)

(a) Dewar

mouth tlask

(e) glass beads; (f) water flasks: (h) Tygon tubing;

tubing; (j) Wright A: (B) sidearm

D. Detailed

SHASKAN.

description

respirometer; B; (C) stopcock under

Materials

diameter polypropylene mouthpiece. Two seconds later an air pump (DeVilbliss, No. 561, Somerset, Pa.) is turned on, pulling the subject’s breath through the sidearm (B) of the sampling device at a rate of approximately IO liters per minute. Teflon threeway T-shaped stopcocks (C,D) are positioned to allow breath passage through the sampling loop (c). Sidearms A and B and the bypass are constructed from Pyrex glass tubing (%-in. o.d.). The bottom third of the sampling loop is constructed of -‘/x-in. (0.d.) Pyrex glass tubing which contains small glass beads serving to increase the cooled surface area inside the loop. Since the sampling loop is submersed in liquid nitrogen prior to and during the breath collection period, all volatile components of the breath freeze out onto the surface of the glass beads. The remainder of the breath exits the sampling device through A, travels through the pump, and is returned to body temperature by passing through a series of Ehrlenmeyer flasks submersed in a water bath at 37°C. The breath sample then flows to a Medishield Wright respirometer (BOC International LTD, Harlow. England) via 1 in. (0.d.) tubing for volume measurement. Sam-

AND

PHILLIPS

pling time takes approximately 9 s for 1.50 liters of breath. After turning the pump off, stopcocks are rotated to the bypass position. The sampling loop is removed from the apparatus and stored in liquid nitrogen until time of analysis. Once the breath sample has been frozen in the sampling loop very few factors will affect the sensitivity of the method. given standardized gc conditions. To maintain this sensitivity we find it necessary to have scrupulously clean glassware by detergent and acid washing, followed by copious distilled water rinsing. Sample anaivsis. The principle of analysis is to first remove the sample from the sampling device and collect it at the head of the cooled gas chromatography (gc) column. In order to accomplish this reproducibly. an analytical apparatus was designed as depicted in Fig. 2. Prior to the connection 01 the sampling device, the carrier gas is allowed to flow through the line bypass for gc baseline stabilization (Fig. 2). Lines G and H are an extension of the carrier gas line prior to entering the gc injection block.

FIG. 2. Breath sampling device connected to gas chromatography for sample analyses. (a) Sampling loop; (b) line bypass; (c) tube bypass: (d) gas chromatograph: (A) sidearm A; (B) sidearm B: (C) stopcock C: (D) stopcock D: (E) stopcock E: IF) stopcock F; (G) and (F) extention of gc carrier gas lint. Detailed drscnption under Materials and Methods.

ENDOGENOUS

BREATH

ACETALDEHYDE

These tubes are constructed of ‘G-in. (o.d.) Teflon tubing and all connections are made with Teflon fittings. While the sampling loop is still immersed in liquid nitrogen. the Sampling device is connected into the analytic circuit. Stopcocks E and F are rotated, allowing the carrier gas to flow through the tube bypass. After purging the tube bypass. the oven temperature of the gc is lowered from 140 tL) 35°C. Stopcocks C and D are then rotated. allowing the carrier gas to pass through the sampling loop. The sampling loop is then removed from the liquid nitrogen. suspended in room air for 2 min. then submersed in an oil bath (120°C). where it remains for 5 min. During this period, the sample is Hushed from the sampling tube and deposited at the head of the gc column. At the end of this period, stopcocks E and F are rot:rted so that the carrier gns again flows through the line bypass. (ius rhronlarographic procedures. With ;I sample at 1he head of the column, the column oven is rapidly heated to 90°C and allowzd to remain at this temperature for 8 min. The oven temperature is then programmed at a rate of 32°C per minute to a find1 temperature of 14O”C, where it remains for 8 min. A Perkin -Elmer 3920 gas chromatograph, equipped with dual-flame ionization delectors (FID) was used. It contained ;I six foot glass column (%-in. o.d. and A-mm i.d.) packed with 80/100 mesh Poropak Q (Supelco, Bellefonte, Pa.). The helium gas flow rate was 40 ml per minute. Air and hydrogen llou rates for the FID were approximately 300 and 30 ml per minute. respectively. Injection port and detector temperatures were I75 and 25O”C, respectively. The signal from the FID was transduced onto an Omniscribe chart recorder. curves were constructed by Standard injecting ;I series of known quantities of t-edistilled AcH (ICN Pharmaceuticals, Plainview. N. Y.) and ethanol (Pharmaco Publicker, Inc.. Lintield, Pa.) into the sampling loop. follutved by analysis as described :~bo\,e. A daily standard of Acf-I was injected

DETECTION

IN HUVAVS

3

into the gc to assess column viability and gc working parameters. This also generated a daily correction factor which was applied to minor shifts in the standard curves. In order to determine percentage recovery of AcH and ethanol. known quantities of redistilled AcH and ethanol were injected into the sample loop containing 3 fasting breath sample. Recordings were compared to those following injection of an equivalent amount of AcH or ethanol directly into the injection port of the gc. Mean percentage recoveries for ethanol and AcH were 95.39 (90.5 98.5%) and 93.8% (91 .O 95.0?+), respectively. Recoveries for methanol and acetone were not determined. Human studies: Etdogenous Ievrls in nonalcoholic suhje(‘ts. Fourteen caucasian subjects aging from I8 to 66, were recruited from the medical school community. Since AcH has been reported to be in cigarette smoke ( 13,14), nonsmokers were selected for these initial evaluations of the method. Similarly, because of issues of alcoholism, subjects were also selected according to the Feighner criteria ( 15) i.e., nonalcoholics with no first-degree alcoholic relative. These requirements were confirmed by ;I brief interview and a written questionnaire. Informed consent was obtained and instructions were given for an overnight fast. u hich included abstinence from all foods and beverages (except water) for 3 minimum of 8 h before arrival at the laboratory. Subjects were asked not to brush their teeth nor to use mouth washes. Upon arriving at the laboratory at approximately 8 AM, three breath samples (each of I.50 liters) were collected 5 min apart as described above. Humun studies: .4Icohol drinl\in,p sfurlies. For the subject conburning an oral dose of ethanol, the same overnight fasting schedule was observed. Thus, when the subject arrived at the laboratory, three breath samples were collected 5 min apart prior to consumption of an ethanol solution. The subject was then asked to consume I g of ethanol/kg of body wt, diluted fivefold with diet ginger ale. This

4

DANNECKFK,

SHASKAN.

chilled “cocktail” of 20?%,v/v ethanol was divided into three equal portions for consumption over three consecutive S min periods. Breath samples were collected, beginning 15 min after consumption of the final portion of the cocktail during which time constant rinsing of the mouth with water was done. This latter procedure was required to assure minimal contamination of breath samples by ethanol and AcH retained and/ or produced in the mouth. These conditions were previously determined by experiments in which a volunteer maintained a 20% (v/ v) solution of ethanol/diet ginger ale in his mouth for a 5-min period. This was followed by three 5-min periods, each consisting of continuous (nonstop) rinsing of the mouth with water. Baseline and intermittent breath samples were obtained as described above. RESULTS Endogenous

Levels

From fasting breath samples taken on all subjects. four elutants (methanol, AcH. ethanol, and acetone) were always present. These four elutants were identified on chro-

FIN;. 3. Representative chromatogram showing endogenous levels of volatile compounds in 1.50 liters of breath from a fasting human subject. (A) Methanol. h 8 attenuation: (B) acetaldehyde. ~8: (C) ethanol. x64: (D) acetone, ~256. For details of column packing and gc conditions, see Materials and Methods,

AND

PHILLIPS TABLE

I

Research subject number

Age

Sex

3506 3502

19 23

3501 3515

27 28

3505 3504

32 3-l

3503 3508

40 42

3510 3507

66 IX

3512 3511

20 21

3509 3516

49 55

M M M M M M M M M F F F F F

Acetaldehyde (w/l) 3.3

8.X 6.7 8.0

1,s 5.3

Il.0 0.5 1.0 4.0

I.0 4.0 0.7 5.0

“Subjects were nonsmoker, nonalcoholic Caucasian adults, fulfilling criteria outlined under Materials and Methods. Breath samples were taken after a minimum of an 8-h fast, as described in text.

matograms by retention time and by internal standards (Fig. 3). The order of elutants is as follows: methanol (5.0 min); AcH (7.5 min); ethanol ( 12.0 min); and acetone (I 5.0 min). A series of additional peaks were observed which showed considerable subject variability but these did not affect the resolution of the four identified elutants. Although other investigators have identified at least 100 volatile components in human breath ( 16), it was not our purpose to identify these additional peaks. Thus, concentrating on the AcH component of the breath sample, in a cohort of nonsmoking, nonalcoholic subjects endogenous AcH levels ranged from 0.5 to II.0 rig/liter (Table I). Given this small sampling of a Caucasian population, there is no apparent influence of age nor sex on endogenous AcH levels. Breath levels of ethanol, methanol, and acetone were also calculated for these sample individuals. Endogenous ethanol concentrations ranged from 60 to 200 rig/liters, while methanol and acetone ranged from 5

ENDOGENOUS

to I 10 and from spectively.

BREATH

ACETALDEHYDE

20 to 220 rig/liters.

re-

One experimental subject (female) was consecutively sampled at .5-min intervals for ;I total of seven breath samples. For these repetitive samples a mean AcH of 7.2 + I .O (SD) rig/liter was found, with a coefficient of variatior7 of 0.14. The same individual sampled over a 3-week interval gave the following values in nanograms per liter; 8.4, 6.0, 7.3. 7.3 (each value representing the mean of triplicate breath samples at each week). Two male subjects studied over similar weekly intervals displayed comparable degrees of \,ariability as did the female subject.

Within minutes of consuming a 20% (v/ v) ethanol cocktail (final dose = 1.0 g/kg), peak height:; of the principle volatile breath components were increased greatly above the endogenous levels (compare Figs. 3 and

Flc;. 4. Chromatogram showing levels of volatile compounds in 1.50 liters of breath from a human subject after ethanol consumption (1.0 g/kg) (A) methanol, Y 16 attenuation (B) acetaldehyde. X256; (C) ethanol, *1200; (D) acetone. X512.

DETECTION

IN HUMANS

5

4). Care was taken to assure minimal contamination of samples from ethanol and acetaldehyde retained and/or produced in the oral cavity. Thus, following the continuous rinsing procedure described under Materials and Methods, contributions from this possible source of error are negligible (Fig. 5). Up to 45 min after beginning ethanol consumption, a 23-year-old, male caucasian’s breath AcH increased at a steady rate and reached a plateau of 2.60 gg/liter. This value for AcH corresponded to a peak ethanol concentration of 650 &liter. DISCUSSION AcH is formed by enLymic oxidation of ethanol in the liver, then broken down to acetic acid and water by the action of aldehyde dehydrogenase (I 3). Hence, consumption of ethanol is promptly followed by a rise in the concentration of AcH in the blood and the breath. However, even in nonalcoholic individuals drinking no ethanol, a small amount of ethanol is detectable in the blood, probably resulting from bacterial fermentation in the intestine ( I ). Therefore, endogenously generated AcH should be constantly present in the blood, probably in amounts too small to be dependably detected by blood AcH assays in current use. One previous study reported the detection of endogenous AcH in breath ( 16) and precautions were also taken to limit ethanol intake beforehand. Thus, mean breath AcH levels of approximately 6 ng/ liter reported by Krotoszyski and co-workers ( 16) are similar to our values (Table I ). Our new technique reproducibly detected AcH in the breath with a higher degree ol sensitivity than any other method known to us. Using a l.50-liter breath sample. we could detect as little as 10 pmol of AcH. It should be noted that with this system there is no theoretical limit to the size of the breath sample collected and subsequently concentrated. Thus, the sensitivity of this method can still be increased severalfold.

DANNECKER,

iI

3100

700

2

I 350

- 70

-60

;

500

-50

4oc

-40

t” 0 3 I I I

300

-30

600

4 z a E m 5

360

P

3000

= 2l d

SHASKAN. AND PHILLIPS

I

2oc

c

\ \ \

100

i

-20

\

\\

-IO

0

Time (minutes) FIO. a 20%

5. Time (v/v)

course

of ethanol

ethanol/diet

ginger

and ale

acctaldehyde solution

levels

in mouth.

before:. Prior

during.

to introduction

and

after of this

the

malntcnancc

solution

I .S-liter breath samples were taken for endogenous levels of ethanol (A - -- - A) and acetaldehyde (A -A). At zero time the ethanol solution was introduced. causing the ethanol (0 0) acetaldehyde (0 -~0) to go off scale. This Smin period was followed by continuous riming described in text (rinsing periods I. II. and III). Between one, 1.5-liter breath samples were taken for determination

It is evident that sensitivity and reproducibility also depend upon the human subject’s ability to consistently replicate breath sampling procedures. Accordingly, rigid experimental conditions were followed and all subjects were given typewritten instructions before breath sampling and allowed to practice the procedure until they were proficient. Thus with careful standardization of the technique of sample collection. mean AcH values for consecutive samples on the same

each of these four periods and of ethanol (a) and acetaldehyde

ol

in mouth.

after

and as

the last CO) levelc

day or over several weeks showed a variation of less than 155%. Using our breath assay method, we were able to routinely detect endogenously generated AcH and ethanol in the breath of nonalcoholic subjects abstaining from alcoholic beverages, readily measuring levels down to 0.5 rig/liter. In addition. we were able to replicate ( 12) the time course of rising breath AcH levels in a subject, drinking a dose of ethanol. without subjecting this

ENDOGENOUS

BREATH

ACETALDEHYDE

volunteer to venipuncture. Given the experimental conditions, the observed increases in breath AcH levels most likely reflect metabolic concersion of ingested ethanol rather than associated phenomenon (e.g., generation of AcH by microflora of the respiratory tract). These results are consistent with the findings of’ Stowell and co-workers (I 2) relating bre,jth AcH concentration to enzymatic met:abolism of ethanol. However, the validity of current techniques for measuring blood AcH levels is still in doubt: thus, quantitative comparisons between blood and breath AcH cannot yet be made with confidence. We conclude that this method offers a uniquely sensitive new approach to the study of AcH metabolism in man, free of the sources of (error afflicting assays of AcH in the blood.

DETECTION

2. Cederbaum.

sincere

thanks

Center

workers

and

friends

3. Korsten,

breath

sample.<

needed

to develop

Alcohol

Research

patience this

and

6. Stowell.

Kricka. istry

L. J., and of Alcohol

Horwood.

Ltd.

Clark. and Chichester,

A.

(I 977)

8.

C. J. P. ( 1980)

9. Couchman,

K.

10.

Forsander, 52,

I I.

E.rp.

Alcoholism, England.

Biochemp. 285.

Ellis

Kes. 0.

Christensen.

Eriksson, Stowell.

C.

M.

and

Batt.

R. D.

It?. 392-401. .3/c,.

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Sl,irnc,e

E.x-p.

207,

Crow. Sekki.

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P..

and

Pharmazol. A.

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Kr.5.

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(1979)

A. ( 1974)

Med.

Sippel. 26,

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.4/c. Biol

0..

in

15.

Fcighner, J. P.. Robins. E.. Gule. R.A.,Winokur,G.andMunoz.R.(1972).4n~h

16.

Krotoszyski,

-787.

Advances

C. M., and Smith. 9, 407-4 14.

26,

B.. Gabriel.

239m 244.

Salaspuro. In

York.

Sprince, H.. Parker. .4gents Acrions

P. A.

(1977)

(Israel, Y . Glascr, R. E.. Schmidt, W.. Vol. 4. pp I I I 176.

14.

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W.

29, 783

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and

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(1978)

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24 I-217. K.

Biochenr.

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dio,

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M. P. (I 980) 13.

and

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Biochem. 11.

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7. Eriksson, 22-29.

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Et@.

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(1979)

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REFERENCES I.

M.

Lieber.

7

HUMANS

1.. Lieber.

.4wh.

Plenum.

to fellow for their

A.

(1974)

ACKNOWLEDGMENTS We extend

IN

(1977)

57

G. E. ( 1979)

S. B.. Woodruff.

63. G., O’Neill. J.

Chronrcrtogr.

H.. and

Clau-

Sci.

15,