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J Occup Health 1999; 41: 112–114
Journal of
Occupational
Health
Possible Effects of Drinking and Smoking Habits on Hippuric Acid
Levels in Urine of Adults with No Occupational Toluene Exposure
Edna Maria ALVAREZ-LEITE, Alessandra DUARTE, Marcia Martins BARROCA and
Josianne Nicacio S
ILVEIRA
Department of Clinical Chemistry and Toxicology, College of Pharmacy, Federal University of
Minas Gerais-UFMG, Brazil
Abstract: Drinking and Smoking Habits and Urinary
Level of Hippuric Acid in Adults not Exposed to
Toluene: Edna Maria A
LVAREZ-LEITE,
et al.
Department
of Clinical Chemistry and Toxicology, College of
Pharmacy, Federal University of Minas Gerais-
UFMG, Brazil—Hippuric acid (HA) is still the biomarker
most used for monitoring exposure to toluene, but it is
produced by the body even in the absence of this
solvent, and has the disadvantage of showing
significant variation in and between individuals,
depending on environmental factors and individual
characteristics. A number of studies have reported the
influence of individual drinking and smoking habits on
toluene metabolism, but the effect on urinary excretion
of HA is still controversial. This study was conducted
in an attempt to examine whether these individual
habits also affect HA excretion in individuals not
exposed to toluene. Urine sample from 195 people
(99 women and 96 men), ranging in age from 17 to 46
years old, were collected. The individuals were
classified in groups according their drinking and
smoking habits. The data from the current study
indicate that these two social habits, either separately
or combined, do not influence basal urinary HA levels
in this study group.
(J Occup Health 1999; 41: 112–114)
Key words: Urinary hippuric acid, Smoking habits,
Drinking habits
The peripheral biomarker of exposure depends,
amongst other things, on the basal values for individuals
who have not been exposed. With the progressive
improvement in working environments, at least in the
majority of countries, levels of biological markers are
becoming closer to their background levels, a fact which
Received June 23, 1998; Accepted Feb 9, 1999
Correspondence to: E.M. Alvarez Leite, Faculdade de Farmacia
da UFMG, Av. Olegario Maciel 2360 -30180-112—Belo Horizonte,
MG, Brazil
emphasises the pressing need for an understanding of their
base levels and the factors which influence them
1)
.
Hippuric acid (HA) is still the biomarker most used
for monitoring exposure to toluene, but it is produced by
the body even in the absence of this solvent, and has the
disadvantage of showing significant variation in and
between individuals, depending on environmental factors
and individual characteristics.
A number of studies have shown that toluene
metabolism can vary as a function of such as genetic
polymorphism, diet, alcohol consumption, cigarette-
smoking, as well as body surface
1–9)
. The effect of ethanol,
especially, has been the subject of numerous studies.
It is known that alcohol interacts with toluene
metabolism
3, 10, 11)
, and has both stimulating (chronic
consumption) and inhibiting (acute consumption) effects
on the solvent metabolism
12, 13)
. This is a possible
explanation for the controversial effects of alcohol
consumption on urinary excretion of HA in humans
exposed to toluene, reported by different studies. Dossing
et al.
12)
and Baelum et al.
13)
recorded a reduction, whereas
Bavazzano et al.
14)
recorded an increase, in the elimination
of hippuric acid into urine as a result of alcohol
consumption. Hjelm et al.
15)
found that the excretion of
this metabolite is reduced when alcoholic drinks are
combined with a carbohydrate restricted diet.
Another factor which plays an important role in toluene
metabolism is cigarette-smoking. According to Inoue et
al.
16)
, the combination of alcohol and cigarettes reduces
the HA in the urine of workers exposed to toluene, and
this association would be a confounding factor in the
separate evaluation of the effects of the two habits on
toluene metabolism.
These studies indicate the need for a careful evaluation
of the effects of smoking and alcohol consumption when
monitoring occupational exposure to toluene with urinary
HA as a biomarker. The important question addressed
here is whether these habits affect HA excretion into urine
113Edna Maria ALVAREZ-LEITE, et al.: Individual Habits and Urinary Hippuric Acid Level
from non-exposed individuals i.e. basal HA excretion,
increasing as such individual variation in the levels
recorded.
Materials and Methods
Sampling Procedure
Urine samples were obtained from 195 people (99
women and 96 men), ranging in age from 17 to 46 years
old, who had not been exposed to toluene. They were all
residents of the metropolitan region of the city of Belo
Horizonte, Minas Gerais, Brazil. The selected individuals
were asked to reply to a toxicological protocol at the time
when the samples were taken. There were five exclusion
factors: renal, hepatic or pulmonary illness of any sort,
use of medicines, and the ingestion of preserved foods
involving the use of benzoic acid or benzoate within the
previous 24 h. The individuals were divided into the
following groups: control group (C) - did not smoke or
drink alcohol (n=92); experimental group 1 (E
1
) - drank
alcohol but did not smoke (n=44); experimental group 2
(E
2
) - smoked cigarettes but did not drink alcohol (n=30);
experimental group 3 (E
3
) - smoked cigarettes and drank
alcohol (n=29).
The individuals comprising E
2
and E
3
smoked between
5 and 30 cigarettes a day, with a mean of 12. The blood
alcohol level was not determinated in the members of
groups E
1
and E
3
but all of them were occasional alcohol
drinkers and they had drunk alcoholic beverage at least
once during the 24 h before the sample collection.
Statistical analysis
The statistical analysis of the results was carried out
with the Jandel SigmaStat
®
package for Microsoft
Windows
®
. The Kolmogorov-Smirnov test was used to
check for normal distribution of the results. The Mann-
Whitney Rank Sum Test was used to compare mean
values for hippuric acid in the control group and the
experimental groups. The effect of gender, age and
number of cigarettes smoked a day on HA excretion were
examined by multiple linear regression on the log values
of HA (gender scored: 0- male, 1- female).
Hippuric acid analysis
Hippuric acid was determinated in urine samples by
gas chromatography by the method of Kira
17)
, modified
by Alvarez-Leite et al.
18)
After methylation with
trimethylphenylammonium hydroxide the samples were
analysed by gas-chromatography with an ionizing flame
detector (FID) (Instrumentos Cientificos CG LTD,
Brazil).
Results
The GC method used in this work has resulted in a
detection limit of 0.10 g/l, CV (intra and inter assay) of 8
and 14% respectively and a rate of recovery from spiked
urine sample of 89.1%.
The urinary hippuric acid level in the total group of
samples (n=195) varied from <0.10 to 1.61 g/l (mean
0.37 g/l).
Table 1 shows the means, median and minimum and
maximum values found for urinary hippuric acid in the
control and experimental groups 1, 2 and 3. HA values
showed a non normal distribuition and log transformed
values were used to study the effect of gender and age on
metabolite urinary excretion. The urinary HA level was
not significantly influenced by gender or age (p>0.05) in
either control or experimental groups.
No significant difference was found between urinary
HA values in groups C and E
1
(p>0.05) and C and E
2
(p>0.05). The mean hippuric level of E
3
(smokers who
drank alcohol) was the lowest (0.34 g/l) in the four groups
analysed, but was still not significantly different from the
control group (p>0.05), from the experimental group 1
(non-smokers who drank alcohol) (p>0.05) nor from the
experimental group 2 (smokers but did not drink alcohol)
(p>0.05). Multiple linear regression did not show any
significant correlation between HA levels and the number
of cigarettes smoked a day either in group E
2
or E
3
.
Table 1. Urinary hippuric acid in the control group and experimental groups
Hippuric acid (g/l)
1
Groups
CE
1
E
2
E
3
n 92 (54 F–38 M) 44 (23 F–21 M) 30 (13 F–17 M) 29 (9 F–20 M)
age 33.0 ± 13 30.9 ± 11.2 33.7 ± 9.5 33.9 ± 7.9
Mean 0.37 0.40 0.38 0.34
Median 0,28 0,28 0,29 0,30
Min
2
<0.10 0.12 0.10 0.13
Max
3
1.61 1.35 1.98 0.95
F=female, M=male. E
1
=drink alcohol but do not smoke, E
2
=smoke but do not drink alcohol,
E
3
=drink alcohol and smoke.
1
The limit of quantification was 0.10 g/l.
2
Lowest concentration
of HA recorded in the group.
3
Highest concentration of HA recorded in the group.
Discussion
Taking into account that
the basal level of HA plays
an important role in the
interpretation of the results of
biological monitoring of
exposure to toluene, we
attempted to evaluate if the
effects of individual smoking
and drinking habits affect the
urinary excretion of HA in
adults not exposed to solvent.
Both positive and negative
effects of drinking alcohol or
cigarette smoking on urinary
114 J Occup Health, Vol. 41, 1999
HA excretion have been recorded by various authors,
but in almost all cases they were particularly examining
the effects of tobacco and alcohol on toluene
metabolism
2–4, 6, 10–13, 15, 16, 19, 20)
not on background HA
levels. The comparisons between the control group and
the experimental groups were made independent of
gender and age, as these had previously been shown to
have no effect on basal urinary HA levels.
The data from the current study indicate that the two
social habits of drinking and smoking do not,
independently, influence basal urinary HA levels. The
mean urinary HA level in group E
1
(drinkers but not
smokers) was the highest among the groups analysed,
but it was not significantly different from that of the
control group. It was also found that there was no
significant correlation between urinary HA and the habit
of smoking (control group and experimental group 2) even
when the number of cigarettes smoked a day was
considered. These results agree with the findings of
Bazzano et al.
14)
who also reported no effect of drinking
or smoking on the basal level of urinary HA.
Inoue et al.
16)
, studying workers exposed to toluene, found
a marked reduction in HA elimination in those who
habitually smoked and drank alcohol. The same result was
not found among individuals not exposed to toluene, as
reported here. Although the mean hippuric level of E
3
(drinkers and smokers) was the lowest (0.34 g/l) among the
four groups analysed, there was no significant difference in
urinary HA levels from those of the control group.
In conclusion, the results presented here showed that
the individual habits of smoking and drinking, either
separately or combined, did not significantly alter urinary
HA levels, in this study group not exposed to toluene.
Nevertheless, conclusion should be interpreted in the light
of the sample population studied, and further examination
is necessary to generalize the results.
Reference
1) Kawamoto T, Koga M, Oyama T, Kodama Y. Habitual
and genetic factors that affect urinary background levels
of biomarkers for organic solvent exposure. Arch
Environ Contam Toxicol 1996; 30: 114–120.
2) Kawamoto T, Matsuno K, Kodama Y, Murata K,
Matsuda S. ALDH2 polymorphism and biological
monitoring of toluene. Arch Environ Health 1994; 49:
332–336.
3) Huang MY, Jin C, Liu YT et al. Exposure of workers
to a mixture of toluene and xylenes I: Metabolism.
Occup Environ Med 1994; 51: 42–46.
4) Nise G. Urinary excretion of o-cresol and hippuric acid
after toluene exposure in rotogravure printing. Int Arch
Occup Environ Health 1992; 63: 377–381.
5) Baelun J, Lundqvist GR, Molhave L, Andersen NT.
Human response to varying concentrations of toluene.
Int Arch Occup Environ Health 1990; 62: 65–71.
6) Nakajima T, Wang RS, Murayama N. Immunochemical
assessment of the influence of nutritional, physiological
and environmental factors on the metabolism of
toluene. Int Arch Occup Environ Health 1993; 65:
S127–130.
7) Nakajima T, Wang RS, Katakura Y, et al. Sex-, age-
and pregnancy-induced changes in the metabolism of
toluene and trichloroethylene in rat liver in relation to
the regulation of cytochrome P 450IIE1 and P450IIC11.
J Pharmacol Exp Ther 1992; 261: 869–874.
8) Kawamoto T, Murata K, Koga M, Hattori Y, Kodama
Y. Distribution of urinary hippuric acid concentrations
by ALDH2 genotype. Occup Environ Med 1994; 51:
817–821.
9) Baelum J. Human exposure factors influencing the
pharmacokinetics and acute toxicity. Hobro: Institute
of Environmental and Medicine, University of Aarhus,
1990; 36 p.
10) Takahashi S, Kagawa M, Inagaki O, Akane A, Fukui
Y. Metabolic interaction between toluene and ethanol
in rabbits. Arch Toxicol 1987; 59: 307–310.
11) Wang RS, Nakajima T. Effects of ethanol and
phenobarbital treatments on the pharmacokinetics of
toluene in rats. Br J Ind Med 1992; 49: 104–112.
12) Dossing M, Baelum J, Hansen SH, Lundqvist GR.
Effect of ethanol, cimetidine and propranolol on toluene
metabolism in man. Int Arch Occup Environ Health
1984; 54: 309–315.
13) Baelum J, Molhave L, Honore-Hansen S, Dossing M.
Hepatic metabolism of toluene after gastrointestinal
uptake in humans. Scand J Work Environ Health 1993;
19: 55–62.
14) Bavazzano P, Perico A, Li-Donni V, Colzi A.
Esposizione professionale e fattori individuali che
condizionano l’eliminazione urinaria di acido ippurico.
G Ital Med Lav 1994; 16: 57–61.
15) Hjelm EW, Lof A, Sato A, Colmsjo A, Lundmark BO,
Norstrom A. Dietary and ethanol induced alterations
of the toxicokinetics of toluene in humans. Occup
Environ Med 1994; 51: 487–491.
16) Inoue O, Seiji K, Watanabe, T, et al. Effects of smoking
and drinking on excretion of hippuric acid among
toluene-exposed workers. Int Arch Occup Environ
Health 1993; 64: 425–430.
17) Kira S. Measurement by gas chromatography of urinary
hippuric acid and methylhippuric acid as indices of
toluene and xylene exposure. Brit J Ind Med 1977; 34:
305–309.
18) Alvarez-Leite EM, França LS, Monteiro RB, Cioletti
AG, Barroca MM. Liquid chromatography
determination of urinary hippuric acid after trimethyl
phenylamonium derivation. Toxicorama 1994; 6: 5–8.
19) Hjelm EW, Naslund PH, Wallen M. Influence of
cigarette smoking on the toxicokinetics of toluene in
humans. J Toxicol Environ Health 1988; 25: 155–163.
20) Kawamoto T, Koga M, Murata K, Matsuda S, Kodama
Y. Effects of ALDH2, CYP1A1, and CYP2E1 genetic
polymorphisms and smoking and drinking habits on
toluene metabolism in humans. Toxicol Appl
Pharmacol 1995; 133: 295–304.