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Biol. Pharm. Bull.

39, 915–919 (2016)

Vol. 39, No. 6915

Current Topics

Recent Advances in Research on Bioactive Ingredients in Cigarette Smoke

Recent Progress in Analytical Methods for Determination of Urinary

3-Hydroxypropylmercapturic Acid, a Major Metabolite of Acrolein
Kyohei Higashi,a Kazuei Igarashi,a,b and Toshihiko Toida*,a
 Graduate School of Pharmaceutical Sciences, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba
260–8675, Japan: and b Amine Pharma Research Institute, Innovation Plaza at Chiba University;
1–8–1 Inohana, Chuo-ku, Chiba 260–8675, Japan.
Received December 16, 2015

3-Hydroxypropylmercapturic acid (3-HPMA), a major metabolite of acrolein in urine, has been recog-
nized as a noninvasive biomarker of exposure to cigarette smoke. Since acrolein is formed endogenously from
polyamines and is also formed during oxidative stress and aggravates tissue damage by changing protein ac-
tivity through its conjugation in pathological lesions, it is thought that the urinary 3-HPMA level is useful as
a biomarker to monitor the severity of several diseases related to acrolein. To study the correlation between
3-HPMA and disease severity, it is important to understand the properties of analytical methods for deter-
mination of 3-HPMA. In this article, we summarize the analytical methods for determination of urinary
3-HPMA and discuss the utility of 3-HPMA as one of the biomarkers for the diagnosis of brain infarction.
Key words acrolein; 3-hydroxypropylmercapturic acid (3-HPMA); brain infarction; LC/MS/MS; GC/MS

1. INTRODUCTION fecal excretion was very low (less than 2%).27) There were
no differences in the excretory patterns between males and
Acrolein (CH2=CHCHO), the strongest electrophile among females. Furthermore, compositions of urinary metabolites
the 2-alkenals, is produced during the combustion of fossil were analyzed; N-acetyl-S-2-carboxy-2-hydroxyethylcysteine
fuels, including engine exhaust, wood, and tobacco and during (5.4–10.4%), N-acetyl-S-2-carboxyethylcysteine (2.6–18.3%),
the heating of cooking oils in the environment, indicating that N-acetyl-S-3-hydroxypropylcysteine (3-HPMA: 52.5–73.8%)
monitoring acrolein is of significant importance to delineate and 3-hydroxypropionic acid (5.2–33.2%) were found in
the pathogenesis of various diseases related to exposure to ac- urine from rats at 4–24 h after intravenous administration
rolein.1) Acrolein is also endogenously generated through poly- of [2,3-14C]acrolein.28) These compounds were quantified by
amine (especially spermine) oxidation by amine oxidase,2,3) gas chromatography/mass spectrometry (GC/MS) and liquid
myeloperoxidase-catalyzed threonine oxidation4) and, at least chromatography/mass spectrometry (LC/MS). When [2,3-
in part, lipid peroxidation5,6) in pathological lesions in which 14
C]acrolein was taken orally, 3-HPMA (22.5–41.2%) and
DNA, protein and lipids are attacked by acrolein directly and significant amounts of oxalic acid (16.8–34.9%), N-acetyl-S-
these functions are changed or abolished through the conju- 2-carboxy-2-hydroxyethylcysteine (7.8–18.1%), N-acetyl-S-
gation with acrolein. Protein conjugated acrolein (PC-Acro) 2-carboxyethylcysteine (3.1–11.7%), and 3-hydroxypropionic
containing Nɛ-(3-formyl-3,4-dehydropiperidino)lysine (FDP- acid (5.6–30.8%) were produced in rat urine. Based on the
Lys) and Nɛ-(3-methylpyridinium)lysine (MP-Lys) in particular above results, 3-HPMA has been recognized as a major uri-
is increased in pathological lesions and plasma in patients nary metabolite of acrolein.
with various diseases,7–19) PC-Acro has been increasing in 3-HPMA data has become increasing popular as a biomark-
popularity as a reliable biomarker for the quantification of er to maintain human health because the noninvasive nature
acrolein (Table 1). In general, determination of PC-Acro can of measuring urinary 3-HPMA allows long-term monitoring
be achieved using a commercially available enzyme-linked of cigarette smoke containing acrolein. Investigation of the
immunosorbent assay (ELISA) kit with a murine monoclonal severity of acrolein exposure has been performed by liquid
antibody 5F6 (mAb5F6) which recognizes FDP-lys and MP- chromatography/mass spectrometry tandem mass spectrometer
Lys, respectively.23,24) (LC/MS/MS) and it was concluded that the urinary 3-HPMA
On the other hand, acrolein is generally scavenged by glu- level in smokers was higher than that in nonsmokers.29–33)
tathione (GSH) and metabolized to produce 3-hydroxypropyl- However, there is no report of acrolein metabolism in urine
mercapturic acid (3-HPMA) as one of the major and stable within 12 h after the onset of cigarette smoking. Watzek et
metabolites in urine (Fig. 1A). In the 1970s, urinary 3-HPMA al. determined acrolein content in commercially available
was found in rats as a metabolite of acrolein together with potato crisps/chips (26.5±2.1 µg/kg, mean±standard deviation
allyl alcohol, allyl formate, allyl propionate, allyl nitrate and (S.D.)) and monitored urinary 3-HPMA levels in volunteers
cyclophosphamide using paper chromatography and gas chro- after their consumption.34) As a result, urinary 3-HPMA
matography.25,26) Parent et al. reported that the major path- levels were increased exponentially within the first 4 h and a
way of [2,3-14C] acrolein excretion was via the kidney into gradual decrease was observed at 8 h after consuming the po-
the urine (66–69%) in intravenously dosed animals whereas tato crisps. In light of this, 3-HPMA produced by the liver and
 To whom correspondence should be addressed.  e-mail:
© 2016 The Pharmaceutical Society of Japan
916 Biol. Pharm. Bull. Vol. 39, No. 6 (2016)

Table 1. Correlation between Acrolein and Several Diseases

Diseases Sample Substrate Method Compared with control subjects References

Atherosclerosis Arterial tissue Protein conjugated Immunostaining Increase 7, 8)

acrolein (FDP-Lys)
Chronic renal failure Plasma 9)
Brain infarction 10)
Silent brain infarction Protein conjugated 11, 12)
acrolein (FDP-Lys) ELISA Increase
Alzheimer’s disease 13, 14)
End-stage renal disease 15)
Diabetic retinopathy Haemoglobin 16)
Sjögren’s syndrome Saliva Protein conjugated Western blotting Increase 17)
acrolein (FDP-Lys)
Type1 and Type2 diabates Urine Protein conjugated ELISA Increase 18, 19)
acrolein (FDP-Lys)
Brain infarction Urine 3-HPMA LC/MS/MS Decrease 20)
Alzheimer’s disease 21)
Cardiovascular disease risk Urine 3-HPMA GC/MS Increase 22)

Fig. 1. Measurement of 3-HPMA and Cre in Urine Collected from Control Subjects and Stroke Patients20)
A. Metabolic pathway of acrolein. Four enzymes shown in the figure are involved in the formation of 3-HPMA from acrolein.25) B. Measurements of 3-HPMA and Cre
in urine were performed twice. Average value of two experiments is shown with median (horizontal line). C. Data with the same age group (60 to 79 years) were analyzed
like (B). The p value was calculated using the Wilcoxon rank sum test.

kidney is rapidly excreted into urine,25,28) suggesting that the urinary 3-HPMA analysis for the diagnosis of brain infarction
urinary 3-HPMA level quickly reflects the degree of acrolein is also discussed.
exposure and consumption of GSH in the body. However, few
reports have monitored the urinary 3-HPMA level in patients 2. ANALYTICAL METHODS OF URINARY 3-HPMA
with several diseases. To further investigate the relationship
between 3-HPMA and disease, it is important to understand 2.1. Paper Chromatography Paper chromatography is
the analytical methods established thus far. It is also thought an analytical technique for separating dissolved materials by
that development of a less costly and more reliable analytical taking advantage of their different rates of migration across
method will further facilitate the study of diseases related to sheets of paper. In the 1970s, the detection of 3-HPMA in
acrolein. urine was performed by paper chromatography. At first, a
In this review article, we introduce several analytical meth- sample pretreatment procedure that included liquid–liquid
ods including paper chromatography, gas chromatography/ extraction, fractionation with Amberlite CG 400 column (for-
mass spectrometry and liquid chromatography/mass spectrom- mate form) and desalting with a Zeo-Karb 225 column (H+
etry for 3-HPMA determination. Furthermore, the utility of form) was performed before paper chromatography.25,26) After
Biol. Pharm. Bull.
Vol. 39, No. 6 (2016)917

extraction of 3-HPMA from urine, chromatograms are devel- reaction monitoring (MRM) mode through monitoring the
oped overnight on 3MM paper by the descending method at following transitions: m/z 220 [M−H]− to 91 [HO(CH2)3S]−.
room temperature with one of the following solvent mixtures: The MRM mode has been proven to be one of the most sensi-
A, butanol–water–acetic acid (12 : 5 : 3, v/v/v); B, butanol–pyri- tive and specific methods used in tandem mass spectrometry
dine–3 mol/L NH3 solution (4 : 3 : 3, v/v/v); C, propanol–water– in which an ion of particular mass and an ion product of a
NH3 solution (sp. gr. 0.88) (80 : 19 : 1, v/v/v). The Rf values of fragmentation reaction of the precursor ion are selected, re-
the reference compounds in these three solvent mixtures are spectively.
0.76–0.78, 0.49–0.52 and 0.47–0.48, respectively.25,26) After In 2001, Mascher et al. were the first to develop an LC/
development, the chromatograms are dried and the 3-HPMA MS/MS system with a positive ESI mode.29) In this report,
spot is visualized by dipping the papers in a platinum reagent positive ion m/z 222→163 was monitored and the limit of de-
(H2PtCl6). The detection range of sulfur-containing com- tection (LOD) was found at 50 µg/L. Sample preparation was
pounds using platinum reagent was reported to be 12–24 µg.35) performed by SPE using an ENV+ cartridge (Separtis GmbH,
The paper chromatography method is relatively easy, however, Grenzach-Wyhlen, Germany). In 2007, Carmella et al. devel-
a large amount of urine was required due to the consumption oped the LC/MS/MS system with a negative atmospheric pres-
of urinary sample during the extraction step and low sensitiv- sure chemical ionization (APCI) mode.30) In this system, mon-
ity of the detection system. For this reason, there was a ten- itoring of negative ion m/z 220→91 was performed and LOD
dency to change the analytical method to LC/MS/MS. was found at 0.9 µg/L. Sample preparation was performed by
Consequently, a conventional pretreatment procedure of SPE using an Oasis MAX (Waters). In 2008, an LC/MS/MS
urine samples with a column of solid phase extraction (SPE) system with a negative ESI mode was subsequently developed
was established for the analysis of LC/MS/MS or GC/MS.29–31) and LOD was 5 µg/L.31) SPE was performed with an ENV+
SPE is a technique designed for rapid, selective sample prepa- cartridge (Separtis GmbH, Grenzach-Wyhlen, Germany). In
ration and purification prior to chromatographic analysis. Ac- 2010, direct analysis of urinary samples using the LC/MS/
cording to the method of Eckert et al., the recovery rate of MS system without the SPE step during sample preparation
3-HPMA using Isolute ENV+ (Biotage, Grenzach-Wyhlen, was developed (LOD: 22 µg/L).32) In 2014, Zhang et al. re-
Germany) was approximately 80%.36) It was also reported that ported high sensitive analysis of 3-HPMA (LOD: 0.049 µg/L)
mercapturic acids including phenylmercapturic acid (PMA) using an API5500 triple quadrupole mass spectrometer (Ap-
and benzylmercapturic acid (BMA) were sensitive to porcine plied Biosystems, Foster City, CA, U.S.A.) without the SPE
acylase I, and o-phthalaldehyde derivatization of deacetylated step during sample preparation.33) Detection ranges of µg
compounds was achievable.37) The fluorescence detection lim- 3-HPMA/g Cre in urine from nonsmokers and smokers were
its of PMA and BMA were 0.5 µg/L. If 3-HPMA is sensitive 37–730 and 132–5345 µg/g Cre, respectively.
to porcine acylase I, it is possible to establish an easier and
more sensitive paper chromatography method by combining 3. CORRELATION BETWEEN URINARY 3-HPMA
SPE and fluorescence detection. LEVEL AND SEVERITY OF STROKE
2.2. GC/MS In 2009, Conklin et al. established a quan-
titative analysis method for 3-HPMA using GC/MS.38) In this One of the main endogenous sources of acrolein is amine
system, a urine sample was initially treated with SPE (Oasis oxidase-mediated degradation of both spermine and spermi-
MAX; Waters, Milford, MA, U.S.A.). After extraction, sam- dine in pathological lesions.2,3) Tomitori et al. reported that
ples were separated by HPLC with UV detection and 3-HPMA FDP-Lys content and spermine oxidase (SMO) and acetylpoly-
fractions were collected. 3-HPMA can be detected by UV de- amine oxidase (AcPAO) activities in blood plasma were good
tection (210 nm).39) In order to achieve an efficient ionization markers for human stroke.10) If the urinary 3-HPMA level is
of the 3-HPMA, hydroxyl and carboxyl groups were deriva- correlated with the severity of brain infarction, it must be pos-
tized with bistrimethylsilyltrifluoroacetamide for 1 h at 70°C. sible to develop a noninvasive biomarker of acrolein for the di-
The resulting product (m/z 366) data were acquired in positive agnosis of brain infarction. For this reason, we have measured
chemical ionization mode using a gas chromatograph equipped urinary 3-HPMA from patients with brain infarction using the
with an HP-5 capillary column (50 m×0.2 mm i.d.×0.5-µm LC/MS/MS system using negative ESI mode.20)
phase thickness). DeJarnett et al. measured urinary 3-HPMA To begin with, the urinary 3-HPMA levels of smok-
using GC/MS and reported that the urinary 3-HPMA level ers (n=16) and nonsmokers (n=74) were measured to judge
was correlated with cardiovascular disease (CVD) risk.22) whether our analytical condition was correct or not. The me-
The median values of µg 3-HPMA/g Creatinine (Cre) in dian values of µg 3-HPMA/g Cre in smokers (n=16) and non-
the low CVD risk category (n=43) and high risk category smokers (n=74) were 1635±517 µg/g Cre (7.40±2.34 µmol/g
(n=168) were 181.5±30 µg/g Cre (0.821±0.135 µmol/g Cre) and Cre) and 493±232 µg/g Cre (2.23± 1.05 µmol/g Cre), respec-
453.7±46.7 µg/g Cre (2.05± 0.211 µmol/g Cre), respectively. In tively. These median values of urinary 3-HPMA obtained
the case of nonsmokers, the median values of µg 3-HPMA/g from smokers and nonsmokers are very similar to results
Cre in the low CVD risk category (n=28) and high risk cat- reported previously.
egory (n=99) were 105.2±23 µg/g Cre (0.476±0.104 µmol/g We next determined the urinary 3-HPMA level from pa-
Cre) and 220.9±26.7 µg/g Cre (1.00±0.121 µmol/g Cre), respec- tients with brain infarction. Unexpectedly, we found that the
tively. 3-HPMA level in urine from stroke patients was lower than
2.3. LC/MS Most 3-HPMA analysis has been carried that in control subjects, unlike the increased level of FDP-Lys
out by LC/MS/MS using a triple quadrupole mass spectrom- content in plasma from stroke patients (Fig. 1B). The median
eter equipped with an electrospray ionization (ESI) inter- values of µmol 3-HPMA/g Cre in 90 control subjects (age 40
face. The 3-HPMA data were generally acquired in multiple to 79) and 78 stroke patients (age 42 to 96) were 2.83 µmol/g
918 Biol. Pharm. Bull. Vol. 39, No. 6 (2016)

Cre (625.4 µg/g Cre) and 1.56 µmol/g Cre (344.8 µg/g Cre), interest.
respectively. This result was observed in terms of both the
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Conflict of Interest The authors declare no conflict of
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