Annals of Clinical & Laboratory Science, vol. 31, no.

4, 2001 399

Research Note:
Urine Trans,trans-muconic Acid as a Biomarker for Benzene
Exposure in Gas Station Attendants in Bangkok, Thailand
Viroj Wiwanitkit,1 Jamsai Suwansaksri,2 and Paweena Nasuan3
1 Department of Laboratory Medicine, Medical Faculty, Chulalongkorn University, Bangkok
2 Department of Clinical Chemistry, Allied Health Science Faculty, Chulalongkorn University, Bangkok
3 King Rama VII Hospital, Chantaburi Province, Thailand

Abstract. The toxicity of benzene, a chemical used in many industrial processes, involves bone marrow
depression and leukemogenesis and is associated with damage to multiple classes of hematopoietic cells and
hematopoietic functions. Environmental exposure to benzene causes an increased body burden, which is
reflected in several biomarkers, eg, urine trans,trans-muconic acid (ttMA). Associated with the
industrialization of Thailand, a developing country in Southeast Asia, workers in many occupations have
acquired substantial risks of benzene exposure. In this study, benzene exposure was monitored by high-
performance liquid chromatography (HPLC) of urine ttMA in 79 persons, including 49 controls and 30
gas station attendants. In controls, urine ttMA concentration averaged 0.12 (SD ± 0.03) mg/g creatinine;
in gas station attendants, urine ttMA concentration averaged 4.00 (SD ± 12.49) mg/g creatinine (p <
0.05). Based on these findings, wider use of urine ttMA determination is recommended as a biomarker for
occupational exposure to benzene. (received 31 July 2001; accepted 5 August 2001)

Keywords: benzene, gas station attendants; occupational and environmental health, biomarker

Introduction significant exposures to benzene, to estimate benzene

Benzene is an important chemical agent that is used
in many industrial processes. Its toxicity involves
bone marrow depression and leukemogenesis caused
by damage to multiple classes of hematopoietic cells
and various hematopoietic cell functions [1].
Potential mechanisms whereby benzene can damage
cellular macromolecules include (a) covalent binding
of reactive metabolites, and (b) the capacity of
benzene metabolites to induce oxidative damage [2].
Environmental or occupational exposure to
benzene results in increased body burden, which is
reflected in several biomarkers of benzene exposure.
These include the urinary levels of benzene and its
metabolites, as well as adducts of reactive benzene
metabolites with cellular constituents. Assays of such
biomarkers can be used to identify populations with
Address correspondence to Viroj Wiwanitkit, M.D., Department
of Laboratory Medicine, Faculty of Medicine, Chulalongkorn
University, Bangkok 10330, Thailand; tel 662 256 4136; fax
662 218 3640; e-mail viroj.w@chula.ac.th.
exposure levels, and to delineate differences in
benzene metabolism [3,4].
Trans,trans-muconic acid (ttMA) is a minor non
-phenolic metabolite of benzene that is excreted in
urine. Assay of urine ttMA has been recommended
as a biomarker for benzene exposure [5-7]. The urine
metabolites that might serve as indices of occupa-
tional or environmental exposures to benzene
include phenol, hydroquinone, ttMA, and S-phenyl-
mercapturic acid. Of these, urine ttMA concent-
ration is increasingly recognized as a reliable bio-
marker that is relatively convenient to measure [8,9].
Due to the recent industrialization of Thailand,
a developing country in Southeast Asia, workers in
many occupations appear to be at high risk for
benzene exposure. We previously provided the initial
evidence that Thai mechanics have high exposure
to benzene, based on monitoring their urine ttMA
concentrations [10]. Among the other occupations
with high risk for benzene exposure are gas station
attendants, who are the focus of this investigation.

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400 Annals of Clinical & Laboratory Science
During their daily work, gas station attendants
have direct contact with petroleum products, so
occupational exposure to benzene cannot be avoided.
In Thailand, most gas station attendants come from
rural areas where they receive scanty education about
personal health-care. The present report describes a
a pilot study to compare urine ttMA concentrations
in a group of gas station attendants versus those in a
comparable group of non-exposed control subjects.
Materials and Methods
Subjects. The study group included 30 male gas
station attendants who worked full-time at gas
stations in the Ladprao district of Bangkok,
Thailand. All of the workers were non-smokers in
compliance with the strict “no-smoking” rules of
gas stations. The stations were located in the business
area of Bangkok, where the monitored level of
atmospheric benzene is 0.76 - 4.14 ppm [11].
The control group included 49 healthy men
with low risk for benzene exposure. All were
villagers from rural areas without nearby factories.
All were non-smokers. These subjects were selected
as a reference group for our laboratory and they were
included in our previous study [10].
All of the 79 subjects provided informed
consent. A random urine sample was collected from
each subject on a workday between 2 pm and 7 pm,
about 4 to 8 hr after the subjects started to work.
Assay for ttMA in urine. Assays of ttMA
concentrations in the urine samples were performed
by HPLC, as previously described [12]. Briefly, 0.5
ml of urine was mixed with 2 ml of tris-HCl buffer
(pH 10) containing vanillic acid as an internal
standard. This mixture was percolated through a
preconditioned ion-exchange column (Dowex I,
100-200 mesh, 1 cm diameter, 10 cm height). After
the column was rinsed with phosphoric acid
solution, acetate buffer, and deionized water, the
analyte was eluted in 2 ml of a solution comprising
equal volumes of 1.5 M sodium chloride solution
and methanol. Of this, 10 µl was injected onto a
HPLC column (C-18 Lichrocart cartridge, 4 mm
diameter, 12 cm length, Merck Co). The mobile
phase consisted of 1% (v/v) acetic acid, 10% (v/v)
methanol, and 89% (v/v) 5 mM sodium acetate.
The flow rate was 1.2 ml/min. The effluent was
monitored with a UV detector (model LC-3A,
Shimadsu Co.) The analyte (ttMA) and internal
standard (vanillic acid) were eluted at 4.2-4.4 and
12.6-13.3 min, respectively. The detection limit was
0.05 mg/L. Urine ttMA concentration was reported
as mg/g creatinine.
Statistics. Results were expressed as means ± SD;
the p value was calculated by unpaired t-test; p <0.05
considered statistically significant.
Results
As listed in Table 1, urine ttMA levels in the group
of gas station attendants averaged 4.00 ± 12.49 mg/
g creatinine, which was significantly higher than the
levels in the control group (0.116 ± 0.027 mg/g
creatinine, p <0.05).
The group of gas station attendants included
one subject with an exceptionally high urine ttMA
level (64.2 mg/g creatinine). No explanation of this
high level was evident. However, even after exclusion
of this outlier, the urine ttMA levels in the gas station
attendants (1.93 ± 5.66 mg/g creatinine) was
significantly elevated in comparison to the controls.
Discussion
Because of its occurrence in mineral oil and its
formation in many combustion processes, benzene
Table 1. Concentrations of trans,trans-muconic acid
(ttMA) in urine specimens from gas station attendants
and non-exposed control subjects.
Group No. of Urine ttMA level*
subjects mg/g creatinine
Control group 49 0.116 ± 0.027
(0 - 0.40)
Gas station attendants 30 4.00± 12.49§
(0 - 64.2)
* mean ± SD, with range in parentheses
§ p <0.05 vs controls
 Urine trans,trans-muconic acid: a biomarker for benzene exposure 401
is a widespread environmental pollutant. Since
benzene is hemotoxic and has been classified as a
human carcinogen, it is important to monitor and
control exposure to benzene vapor in the environ-
ment and especially in workplace air [1,2].
In developing countries, there is growing
recognition of the public health hazards of benzene
exposure, but relatively few of these countries have
policies, regulations, and programs to combat the
problem. A main purpose of this study was to
document the increased exposure to benzene in a
population of low-skilled workers that is widely
distributed throughout the developing world.
Gas station attendants can be classified as an
occupation of low-skilled workers at high risk of
benzene exposure. They are constantly in contact
with petroleum products during their daily work.
Compared to mechanics, skilled workers who were
previously investigated [10], gas station attendants
generally have a lower socioeconomic status. Like
other migrants from the rural area to Bangkok [13],
they have a relatively low educational background.
Therefore, they need instruction about self-care and
personal hygiene in relation to their work with
hazardous chemicals. For these workers, protective
equipment, such as gloves and masks, is necessary
and should be provided. Specific policies and regul-
ations regarding benzene exposures are needed for
gas station attendants as well as for workers in other
forgotten high-risk occupations.
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