CLIN. CHEM.40/8, 1485-1488 (1994)
#{149}
Drug
Monitoring
and Toxicology
Pentane and Isoprene in Expired Air from Humans: Gas-Chromatographic Analysis
of Single Breath
Shanthi
Mendis,”2
Paul A. Sobotka,’
and David E. Euler”3
Both pentane and isoprene are excreted in human breath.
Although pentane is considered an index of lipid peroxidation, the significance of isoprene is unknown. Having a
similar boiling point, these two hydrocarbons are difficult
to separate by gas chromatography.
We separated pentane from isoprene on both a Poraplot 0 and a Poraplot U
column, injectingsingle-breath samples directly into a gas
chromatograph. The breath samples were pressurized to
800 mmHg to increase the amount of sample volume
delivered to the column. In a group of 43 healthy volunteers, the concentrations of end-expiratory pentane and
isoprene were 0.57 ± 0.3 and 7.05 ± 3.53 nmol/L, respectively. There was a significant linear correlation (r =
0.57, P <0.0001)
between age and pentarie concentration in expired air; isoprene showed no correlation with
age or pentane concentrations. The age-related increase
in pentane production suggests that oxidative stress may
play a role in the aging process in humans. The method
described should allow for rapid, inexpensive, serial measurement of expired pentane and isoprene.
Because the amount of pentane
in expired
air is less
than the detection limit of most flame-ionization
detectors (FIDs), it is often necessary
to concentrate pentane
before analysis. This has been accomplished
by passing
large volumes
of expired air through
cold- or ambienttemperature
traps containing
hydrocarbon
adsorbents
(6-10). Some previously reported
methods also included
a rebreathing
circuit as part of the apparatus so as to
concentrate
the pentane
in the lungs and reduce the
volume of expired
air required
for analysis
(2, 7).
Recently, a technique
for the analysis of pentane from
a single-breath
sample injected directly into a gas chromatograph
has been described (11). Here, we report the
use of porous-layer
open-tubular
columns to separate
isoprene from pentane in combination
with a singlebreath sampling
technique. Using this method, we investigated
the pentane and isoprene concentrations
of
expired air in normal volunteers
and the variation of
the concentration
of these hydrocarbons
in relation to
subject age.
Indexing Terms: lipid peroxidation/hydrocarbons/freeradicals/respiratolygases
Materials and Methods
The formation
of free radicals
and lipid peroxidation
has been implicated
in many conditions that lead to cell
injury. The measurement
of breath alkanes,
especially
ethane
and pentane,
has been used as a noninvasive
index of lipid peroxidation.
Although both gases have
been measured
by gas chromatography
(GC) in the
breath of experimental
animals,
studies in humans are
usually limited to the measurement
of pentane
(1, 2).
For measurement
of breath pentane
to be widely accepted as a diagnostic
indicator of cell injury, one must
recognize
some of the limitations
of the methodolor
used to perform the assay. Several studies
have identified isoprene as the major hydrocarbon
of human breath
(1, 3-6), and two reports describe the coelution of pentane and isoprene on most GC columns (1, 6). However,
isoprene was separated
from pentane on a porous-layer
open-tubular
column (Poraplot U) (1, 6).
‘Department
of Medicine/Cardiology, Loyola University Medical Center, Maywood, IL 60153.
2Department
of Medicine, Faculty of Medicine (Peradeniya),
and Institute of Fundamental
Studies, Sri Lanka.
3Mdress
correspondence to this author at: Section of Cardiology, Rm. 0730, Loyola University
Medical Center, 2160 S. 1st
Ave., Maywood, IL 60153. Fax 708-216-9433.
Received March 7, 1994; accepted April 29, 1994.
We collected end-expiratory
breath samples (125 mL)
from 43 healthy volunteers
(20 men, 23 women) by use
of a modified Haldane-Priestly
tube (12). The volunteers were nonsmokers
between ages 22 and 75 years.
Subjects breathed into a polyvinylchloride
tube [1.2 cm
(i.d.)] with a disposable
mouthpiece
at one end and a
one-way
valve
at the other.
Breath
samples
were
aspi-
rated into 60-mL polyethylene
syringes
(Fortuna
Syringe; Aldrich Chemical Co., Milwaukee,
WI) through a
stopcock
inserted just proximal
to the one-way valve.
Samples were collected
in the morning, 3 h after the last
meal, and then analyzed within
3 h. All subjects were
breathing
room air for at least 1 h before the breath
sampling.
Procedures
followed were in accordance
with
the Helsinki
Declaration
of 1975, as revised in 1983.
Gas Chromatography
To analyze the hydrocarbons
in expired air, we used a
gas chromatograph
(5890 series II; Hewlett-Packard,
Naperville,
IL) equipped
with a gas sampling
valve, a
10-mL sampling loop, and a Fifi operating in at 1 nAJV.
The sample loop was flushed with 40 mL of the breath
sample and manually
pressurized
to 800 mmHg with
the last 20 mL of the sample by use of a digital manometer (IJM 2000/200; Netech, Hicksville,
NY). The hydrocarbons were eluted by passing the sample through a 10
CLINICAL CHEMISTRY, Vol. 40, No. 8, 1994
1485
To verilSr that the Fl]) responses to pentane
and isom x 0.53 mm Poraplot Q capillary column (Chrompack,
prene were equivalent,
we vaporized
50 jL of liquid
Raritan, NJ) with helium as the carrier gas at a flow
isoprene and pentane
in a Tedlar
bag and diluted
as
rate of 4 mL/min. The temperature
of the sampling
loop
was 70#{176}C,
of the injector 90#{176}C,
and of the detector 225#{176}C.
above. The response of the FIB to isoprene was 9%
To minimize
peak broadening
from the large sample
greater than for pentane. Because we felt that a 9%
volume,
we held the column temperature
at 30#{176}C
for 2
difference was within the range of experimental
error,
mm to concentrate
the hydrocarbons
at the head of the
we considered these responses to be equivalent.
column. The column temperature
was then increased by
Results
20#{176}C/mm
to 95#{176}C
and held for 1 mm. Thereafter,
the
Isoprene and pentane could not be detected in room
column
temperature
was increased
by 0.5#{176}C/mm
to
air. The CVs for measuring
pentane
and isoprene
in
103#{176}C,
held for 1 min, and then increased
to 200#{176}C
at
repeated breath sampling
of a single subject every 2 mm
20#{176}C/min
and held for an additional
2 miii.
For comparison,
we also analyzed the breath samples
over a lO-min period were 20.5% and 17.0%, respecof 17 subjects on a 10 m x 0.53 mm Porapiot U capillary
tively (n = 5). Pressurization
of the sample loop to 800
column (Chrompack),
using helium at a flow rate of 3
mmHg doubled the area of individual peaks but did not
alter the peak widths or retention times. Injection of
mL/min. The temperature
was held at 30#{176}C
for 2 min
hydrocarbon-free
air samples between runs showed no
and increased
by 20#{176}C/mm
to 90#{176}C.
Thereafter
the column temperature
was increased
by 5#{176}C/min
to 115#{176}C carryover
of any component from one run to the next.
and by 20#{176}C/mm
to a final temperature
of 190#{176}C,
which
The elution order of breath hydrocarbons
on the Powas held for 3 mm.
raplot Q column was acetone (14.2 mm), isoprene
(19.6
Chromatogram
data acquisition,
storage,
and peak
miii), and pentane (20.5 min) (Fig. l#{192}).
On the Poraplot
area integration
was done with a 386 PC and PeaksimU column the elution order was ethanol (10.9 mm),
pie II software (SRI Instruments,
Menlo Park, CA). Linpentane
(11.1 min), isoprene (11.8 min), and acetone
ear regression
analysis
was performed
to examine the
(12.3 min) (Fig. 1B). When a breath sample from the
relation between variables.
Results are expressed as the
same subject was run on both columns (Fig. 1), the
mean ± SD. Statistical
analyses were carried out with a
concentration
of pentane obtained
with the Q column
PC version of Arcus Professional
Statistical
Analysis
Software (Arcus, West Lancs, UK).
16
Acetone
Calibrations
Calibrator
gas mixtures
were prepared
by using a
commercially
available
mixture
of hydrocarbons
(C2C6) at a concentration
of 100 41L (Alitech
Associates,
Deerfield, IL). An aliquot of the C2-C6 gas (1.0-10.0 mL)
was drawn into a gas-tight syringe
(Hamilton
Co., Reno,
NV) and injected into a Teflon bag that had been flushed
with hydrocarbon-free
air. Using either a 500- or
1500-mL gas-tight
syringe
(Hamilton), we injected various amounts of hydrocarbon-free
air into the bag to
achieve the desired dilution. To mix the commercial
gas
and air in the bag, we repetitively
withdrew the entire
contents of the bag into a 1500-mL syringe
and then
refilled the bag. The diluted calibrator gas mixtures
were injected into the gas chromatograph
in the same
manner as the breath samples. Calibration curves were
plotted for four pentane concentrations:
0 nIIL, 20 nlIL
(0.82 nmol/L), 50 nL/L (2.04 nmolJL), and 100 nL/L (4.09
nmolIL). The CVs (n = 5) for the latter three calibrators
were 6.5%, 4.1%, and 5.8%, respectively.
The calibration
slope for the Poraplot Q column was 0.62 ± 0.005 (n =
5); for the Poraplot U column, it was 0.59 ± 0.05 (n = 4).
For all calibration curve8, r >0.95.
To determine the retention times of isoprene, acetone,
and ethanol, we vaporized 50 tL of each reagent (Aldrich) separately and diluted each sample with hydrocarbon-free air. The concentration
of isoprene was estimated from the pentane
calibration
curves; these two
hydrocarbons
have the same number of carbon atoms,
and the FID response to hydrocarbons
is thought to be
predominantly
determined
by the carbon content (13).
1486
CLINICAL CHEMISTRY,Vol.40, No.8, 1994
A
a12
U)
C
a
U,
a,
Isoprene
C-
0
4)
U
a,
4-’
0
5
10
Time, mm
15
25.
20
Acetone
B
20,
a,
U)
C
a
15.
U)
10.
5.
a
3
4
6
Time,
1’2
mm
Fig. 1. Chromatogram of an expired breath sample from a single
subject, eluted on a Poraplot 0 capillary column (A) and on a
Poraplot U capillary column (B).
(0.38 nmol/L)
was similar to the pentane concentration
with the U column (0.42 nmol/L). Likewise,
the isoprene concentration
obtained with the Q column
(7.75 nmol/L) was similar to that obtained with the U
column (8.12 nmoIJL).
The mean concentration
of end-expiratory
pentane for
all 43 subjects included in the study was 0.57 ± 0.3
nmol/L; the mean isoprene concentration
was 7.05 ±
3.53 nmoIJL. To determine the influence of age on pentane concentration,
we assessed these two variables by
linear regression
analysis.
We found a direct relation
between age and pentane concentration
(r = 0.57, P
<0.000 1), but isoprene concentration
did not vary significantly with age (Fig. 2). Likewise, there was no significant relation between isoprene concentration
and
pentane concentration.
determined
Discussion
The mean pentane
concentration
in breath for 43
healthy subjects was 0.57 ± 0.3 nmol/L. It is difficult to
compare this value with previously published valueswhich range from 0.004 nmo]/L (9) to 5.4 nmol/L (14) in
adults-given
the absence
of a commonly
accepted
method for performing the analysis. The large variability in pentane concentrations
reported in the literature
L50
n=43, r0.57, p< 0.0001
A
125
#{149}
#{149}
I
1.00
.
#{149}#{149}
0.75
II
S
.
0.50
.
#{149}
I
#{149}
#{149}
#{149} #{149}#{149}
I
#{149}
.
025
20
30
40
50
Age.
years
1
60
18
70
80
n43, r0.09, pO.59
B
S
I
#{149}
I
12
I
S
I
#{149}#{149}.
I
#{149}
#{149}
#{149}
S
6
#{149}S
-----#{149}S
#{149}I
#{149}
#{149}
S
I
S
0
20
30
40
50
Age,
years
60
70
80
FIg. 2. Simple linear regression analysis of age vs (A) pentane and
(B) isoprenein expired air.
may be partly attributed to the techniques
used to determine the concentration
of hydrocarbons
in expired
air. The reproducibffity
and recovery rates of commonly
used trapping techniques
are often not documented.
Furthermore,
the use of a rebreathing
circuit in some
studies may have influenced the results: Pentane undergoes significant
hepatic metabolism
(2). Coelution
of
isoprene
and pentane
also represents
a significant
source of variability.
Because commonly used columns
fail to resolve isoprene from pentane (1, 6), many authors may have unknowingly
reported the combination
of isoprene and pentane as pentane.
In the present study, we obtained a clear separation of
pentane and isoprene on both Poraplot Q and U columns
(Fig. 1). Because we did not use mass spectroscopy, one
might argue that comparison
of retention
times with
those of the calibrators does not guarantee
reliable peak
identification.
However, the peak elution order and the
relative peak amplitudes we observed on the Poraplot U
column were identical to those reported by Kohimuller
and Kochen (1), using mass spectroscopy.
Although
these investigators
could not resolve pentane from isoprone on a Poraplot Q column, the temperature
ramp
they used may have been too steep to achieve adequate
resolution.
We were able to resolve pentane from isoprone on the Q column
only when we used a temperature ramp of 0.5#{176}C/min
above 95#{176}C.
Given that the concentrations
of pentane
and isoprene
were nearly
identical for analysis of breath samples from one subject
on the two different columns (Fig. 1), it is quite likely
that our peak identification
was accurate.
Kohimuller
and Kochen (1), using total breath samples, reported lower concentrations
of pentane
(0.13 ±
0.11 nmol/L) and isoprene (1.62 nmol/L) than we observed with the single-breath
analysis.
However,
the
subjects in their study were all ages 20-30 years. The
ninesubjectsinthatagerangeinourstudyhadmean
±
SD pentane
of 0.28 ± 0.13 nmol/L and an isoprene of
5.54 ± 2.95 nmol/L. The higher concentrations
of hydrocarbons we observed may be due to the sampling
of
end-expiratory
air; Zarhng
and Clapper (11) found
higher hydrocarbon
concentrations
in end-expiratory
breath than in total breath.
Alternatively,
the isoprene
concentrations
that we determined
from our pentane
calibration
curves may have been systematically
biased
if the concentrations
of pentane
calibrators
were less
than the measured
isoprene concentrations.
The direct linear relation we observed between age and
pentane concentration
supports the hypothesis that aging
is associated with increased lipid peroxidation.
The aging
process may be accompanied
by increased production
of
free radicals and (or) a decrease in antioxidant
defense
mechanisms.
Our results are in agreement
with a prelimmary study in humans that suggested that pentane excretion was altered in older subjects (15). Plasma malondialdehyde,
another
index
of lipid peroxidation,
also
reportedly increases with age (16). Additionally,
pentane
production is directly related to age in rats (17), houseffies
(18), and honeybees
(19). In a study on the biochemical
correlates of longevity in the housefly, longer-lived strains
CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
1487
had higher
superoxide
dismutase/metabolic
rates
and
higher
activities of csltRlslse, glutathione
reductase,
and
thiotransferase,
supporting the view that longer-lived ffies
manifest lower levels of oxidative
stress than do shorterlived flies (20).
Kohimuller
and Kochen suggested
that oxygen-derived
free radicals might interact
with polyisoprenes
as well as
with membrane
lipids, so that both isoprene and pentane
are produced (1). However, we found no correlation
between isoprene and pentane
excretion,
which suggests
that polyisoprenes
may not be substrates for free-radical
attack.
Increased
concentrations
of pentane
in expired air
have been reported in various conditions,
e.g., exercise
(9,21),
rheumatoid
arthritis
(22), oxygen exposure
(23),
smoking (24), vitamin E deficiency (25), lipid infusions
(26), alcoholic cirrhosis
(27), endogenic
psychoses (16),
acute
myocardial
infarction
(28), Alzheimer
disease
(29), necrotizing
enterocolitis
(30), and multiple sclerosis (31). In view of the difficulties
in separating
pentane
and isoprene,
we recommend
reevaluating
the exhalation of pentane by humans
as an index of lipid peroxidation in health and disease.
References
1. Kohlmuller D. Kochen, W. Is n-pentane really an index of lipid
peroxidation
in humans and animals? A methodological
reevalu-
ation. Anal Biochem 1993;210:268-76.
2. Kneepkens CMF, Ferreira C, Lepage C, Roy CC. The hydrocarbon breath test in the study of lipid peroxidation; principles and
practice. Cliii Invest Med 1992;15:163-86.
3. Conkle JP, Camp BJ, Welch BE. Trace composition of human
respiratory gas. Arch Environ Health 1975;30:290-5.
4. DeMaster EG, Nagasawa lIT. Isoprene, an endogenous constituent of human alveolar air with a diurnal pattern of excretion. Life
Sci 1977;22:91-8.
5. Gelmont D, Stein RA, Mead JF. Isoprene-the
main hydrocarbon in human breath. Biochem Biophys Rae Commun 1981;99:
1456-60.
6. Cailleux
A, Allain P. Is pentane
a normal constituent
of human
breath? Free Radic Roe Commun 1993;18:323-7.
7. Wade CR, Van Ru AM. In vivo lipid peroxidation in man as
measured by the respiratory excretion of ethane, pentane and
other low molecular weight hydrocarbons.
Anal Biochem 1985;
150:1-7.
8. Morita 5, Snider MT, Inada Y. Increased n-pentane excretion in
humans as a consequence of pulmonary oxygen exposure. Anestheaiologr 1986;64:730-3.
9. Lawrence GB, Cohen G. Ethane exhalation as an index of in
vivo lipid peroxidation: concentrating
ethane from a breath collection chamber. Anal Biochem 1982;122:283-90.
10. Puncemail J, Camus G, Roesgen A, Dreezen E, Bertrand Y,
Lismonde M, et al. Exercise induces pentane production and
neutrophil activation in humans-effect
of propranolol. Eur J Appl
Physiol Occup Physiol 1990;61:319-22.
11. Zarling EJ, Clapper M. Technique for gas chromatographic
measurement of volatile alkanes from single-breath samples. Clin
Chem 1987;33:140-1.
1488
CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
12. Metz G, Gassull MA, Lees AR, Blendis LM, Jenkuns DJA. A
simple method of measuring breath hydrogen in carbohydrate
malabsorption
by end expiratory
sampling. Clin Sd Mol Med
1976;50:237-40.
13. Jorgensen AD, Picel AC, Stamoudis VC. Prediction of gas
chromatography
flame ionization detector response factors from
molecular structures. Anal Chem 1990;62:683-9.
14. Zarhng EJ, Mobarhan S, Bowen P, Sugerman S. Oral diet does
not alter pulmonary
pent.ane or ethane excretion in healthy
subjects. J Am Coil Nutr 1992;l1:349-52.
15. Kovaleva ES, Orlov ON, Bogdanova ED, Beliaev BS, Tentsul’kovskaia MIa. Study of the processes of lipid peroxidation in
patients with endogenous psychoses using gas chromatography.
Zh
Nevropatol Psikhiatr 1989;89(1):99-103
(in Russian).
16. Knight JA, Smith SE, Kinder VE, Anstall HB. Reference
intervals for plasma lipoperoxides: age-, sex-, and specimen-related variations. Clin Chem 1987;33:2289-91.
17. Sagai M, Ichinosi T. Age related changes in lipid peroxidation
as measured by ethane, ethylene, butane and pentane in respired
gases of rats. Life Sci 1980;27:731-8.
18 Sohal RS, Muller A, Koletzko B, Sies H. Effect of age and
ambient temperatures
on n-pentane production in adult housefly,
Musca domestica. Mech Ageing Dev 1985;29:317-26.
19. Young RG, Tappel AL. Fluorescent pigment and pentane
production by lipid peroxidation in honeybees, Apis meffifera. Exp
Gerontol 1978;13:457-9.
20. Sohal RS, Farmer KJ, Allen RG. Correlates of longevity in two
strains of the housefly, Musca domestica. Mech Ageing Dev 1987;
40:171-9.
21. Dillard CJ, Litov RE, Savin WM, Dumelin EE, Tappel AL.
Effects of exercise, vitamin E and ozone on pulmonary function and
lipid peroxidation. J Appi Physiol 1978;45:927-32.
22 Humad 5, Zarling E, Clapper M, Skosey JL. Breath pentane
excretion as a marker of disease activity in rheumatoid arthritis.
Free Radic Res Commun 1988;5:101-6.
23. Prilipko LL, Orlov ON, Kagan YE, Savov VM, Salt.anov Al.
Accumulation of gaseous lipid perozidation products in the expired
air of children during hyperbaric oxygenation. Bull Exp Biol Med
1983;96:24-6.
24. Hoshino E, Shariff R, Van Gossuni A, Allad JP, Pichard G,
Kurian H, et a].. Vitamin E suppresses increased lipid peroxidation
in cigarette smokers. J Parenter Enter Nutr 1990;14:300-5.
25. Lemoyne M, Van Gossum A, Kurian H, Jeejeebhoy KN.
Breath pentane analysis as an index of lipid peroxidation: a
functional test of vitamin E status. Am J Clin Nutr 1987;46:26772.
26. Van Gossum A, Shariff R, Lemoyne M, Kurian R, Jeejeebhoy
K. Increased lipid peroxidation after lipid infusion as measured by
breath pentane output. Am J Clin Nutr 1988;48:1394-9.
27. Moscarella S, Caranielli L, Mannaioni PF, Gentiluni P. Effect
of alcoholic cirrhosis on ethane and pentane levels in breath. Boll
Soc Ital Biol Sper 1984;60:527-33.
28. Weitz ZW, Birnbaum AJ, Sobotka PA, Zarling EJ, Skosey JL.
High breath pentane concentrations
during acute myocardial
infarction. Lancet 1991;337:933-5.
29. Fokin VF, Ponomareva NV, Orlov ON, et al. The relationship
of the electrical reactions of the brain to lipid peroxidation
processes in pathological aging. Biul Eksp Biol Med 1989;107:682-4
(in Russian).
30 Kuivalaunen
E, Pitkanen
OM, HalIman M, Anderson SM.
Ethane and pentane as potential indicators of necrotizing enterocolitis (NEC) in very low birth weight infants (VLBI) [Abstract].
Pediatr Res 1992;28:292.
31. Toshniwal PK, Zarling EJ. Evidence for increased lipid peroxidation in multiple sclerosis. Neurochem Bee 1992;17:205-7.