Disease-associated acrolein: A possible diagnostic and therapeutic substrate for in vivo synthetic chemistry

Article abstract of DOI:10.1016/j.bmc.2020.115831

Acrolein, a highly reactive α,β-unsaturated aldehyde, is a compound to which humans are exposed in many different situations and often causes various human diseases. This paper summarizes the reports over the past twenty-five years regarding disease-associated acrolein detected in clinical patients and the role acrolein plays in various diseases. In several diseases, it was found that the increased acrolein acts as a pathogenetic factor. Thus, we propose the utility of over-produced acrolein as a substrate for a promising therapeutic or diagnostic method applicable to a wide range of diseases based on an in vivo synthetic chemistry strategy.

Full text of DOI:10.1016/j.bmc.2020.115831

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Disease-associated acrolein: A possible diagnostic and therapeutic substrate
for in vivo synthetic chemistry
Kyohei Muguruma, Ambara R. Pradipta, Yudai Ode, Kazuki Terashima,
Hiroyuki Michiba, Motoko Fujii, Katsunori Tanaka
PII:
S0968-0896(20)30661-1
https://doi.org/10.1016/j.bmc.2020.115831
BMC 115831
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 October 2020
23 October 2020
24 October 2020
Please cite this article as: K. Muguruma, A.R. Pradipta, Y. Ode, K. Terashima, H. Michiba, M. Fujii, K. Tanaka,
Disease-associated acrolein: A possible diagnostic and therapeutic substrate for in vivo synthetic chemistry,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115831
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Disease-associated acrolein: A possible
diagnostic and therapeutic substrate for in
vivo synthetic chemistry
Leave this area blank for abstract info.
Kyohei Mugurumaa, Ambara R. Pradiptaa, Yudai Odea, Kazuki Terashimaa, Hiroyuki Michibaa, Motoko Fujiia
and Katsunori Tanakaa,b,c,
*
aDepartment of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 1-12-1, Ookayama,
Meguro-ku, Tokyo 152-8552, Japan
bBiofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
cBiofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia
Graphical Abstract
Overproduction
O
Disease
H
Acrolein
Pathogenesis
Progression

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Disease-associated acrolein: A possible diagnostic and therapeutic substrate for in vivo
synthetic chemistry
Kyohei Mugurumaa, Ambara R. Pradiptaa, Yudai Odea, Kazuki Terashimaa, Hiroyuki Michibaa, Motoko
Fujiia and Katsunori Tanakaa,b,c,

aDepartment of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 1-12-1, Ookayama, Meguro-
ku, Tokyo 152-8552, Japan
bBiofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
cBiofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Acrolein, a highly reactive ,-unsaturated aldehyde, is a compound to which humans are exposed
in many different situations and often causes various human diseases. This paper summarizes the
reports over the past twenty-five years regarding disease-associated acrolein detected in clinical
patients and the role acrolein plays in various diseases. In several diseases, it was found that the
increased acrolein acts as a pathogenetic factor. Thus, we propose the utility of over-produced
acrolein as a substrate for a promising therapeutic or diagnostic method applicable to a wide range
of diseases based on an in vivo synthetic chemistry strategy.
Available online
Keywords:
Acrolein
Disease-associated substrate
Diagnosis
2009 Elsevier Ltd. All rights reserved.
Therapy
In vivo synthetic chemistry
———
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: tanaka.k.dg@m.titech.ac.jp & kotzenori@riken.jp

1. Introduction
stress; therefore, acrolein could be a target substrate for disease
diagnosis and pathological staging, as well as treatments. This
review summarizes the reports available regarding disease-
associated acrolein which has been detected in clinical patients
over the past 25 years. We also highlight the utility of acrolein as
a substrate for therapy or diagnosis on the basis of an in vivo
synthetic chemistry strategy.
Acrolein is a highly electrophilic ,-unsaturated aldehyde
(Figure 1) to which humans are exposed in many situations when
it is produced by environmental sources such as engine exhaust,
cigarette smoke, and vapors of over-heated cooking oil. Acrolein
is formed during the combustion of organic substances.1,2 It has
also been reported that the ordinary intake of foods and beverages
such as fruits, vegetables, alcohol, and milk includes ppm levels
of acrolein exposure.3,4 In particular, acrolein is generated during
the cooking processes of fermentation or heating via the Maillard
reaction.
In the human body, acrolein can be endogenously produced
through the enzyme-mediated oxidation of polyamines5 or
threonine6 and is also generated by the oxidation of unsaturated
fatty acids with reactive oxygen species (ROS).7 Polyamines are
known to be essential for cell growth and exist mainly as RNA-
polyamine complex in cells. Once RNA is degraded by ROS or
cell death, polyamines are released in their free form. Free
spermine, a representative polyamine, is metabolized by spermine
oxidase (SMO) producing spermidine and 3-aminopropanal,
which spontaneously decomposes to ammonia and acrolein
(Figure 2A). Threonine, which exists at up to 200 µM in plasma,8
is oxidized by hypochlorous acid that is produced by
myeloperoxidase (MPO), and further reactions including
decarbonation and dehydration produce acrolein (Figure 2B).6
Since MPO is released by the activation of phagocytes, acrolein is
produced in high concentrations at sites of inflammation. Another
source of acrolein is from unsaturated fatty acids via oxidation
with ROS.7 One production scheme from arachidonic acid, which
includes peroxidation and -cleavage, is shown in Figure 2C.
However, the oxidative lipid degradation is quite complex, so that
acrolein is not the exclusive product; the reaction also includes the
Figure 2. Endogenous acrolein production from A) spermine, B)
threonine, and C) arachidonic acid.
2. Intrinsic reactivity of acrolein for detection and in vivo
synthetic chemistry
production
of
malondialdehyde,
crotonaldehyde,
4-
hydroxynonenal (4-HNE), and others. Perhaps due to this
complexity, the amount of acrolein produced from lipid oxidation
is lower compared to polyamines.9 Based on these mechanisms,
acrolein levels in oxidatively stressed cells sometimes reach the
millimolar scale.
Acrolein shows various reactivities owing to both the ,-
unsaturated carbonyl and aldehyde structures. In the human body,
through the reaction of two acrolein molecules with a Lys residue
in the protein, 3-formyl-3,4-dehydropiperidine (FDP)-Lys15 is
produced via a doubly conjugated intermediate (Figure 3, arrow
1). FDP-Lys is gradually converted to 3-methylpyridinium (MP)-
Lys over an extended period of time.16-18 As a result of the reaction
with the guanine base in DNA, acrolein-guanine adducts (Acr-dG)
are generated as cyclic products with two possible isomers (Figure
3, arrow 2).19 On the other hand, the acrolein-glutathione (GSH)
adduct also obtained by a Michael addition of a thiol group of
GSH (Figure 3, arrow 3) subsequently undergoes cleavage of
amide bond and acetylation via the mercapturic acid pathway to
generate 3-oxopropylmercapturic acid (OPMA).20 Reduction or
oxidation of OPMA produces 3-hydroxypropylmercapturic acid
(HPMA) or carboxyethylmercapturic acid (CEMA), respectively.
An 8-membered ring compound is also produced by the reaction
with Lys or polyamines (Figure 3, arrow 4).21 Moreover,
endogenous acrolein can react with some administered
compounds in vivo. For example, additional thiol compounds,
such as N-acetylcysteine (NAC) and 2-mercaptoethanesulfonate
(Mesna), can capture acrolein via a Michael reaction (Figure 3,
arrow 5). Hydrazine or hydroxylamine compounds, such as
hydralazine and N-benzylhydroxylamine, are also effectively
trapped in the aldehyde structure of acrolein (Figure 3, arrow
Pathogenesis
Endogenous
acrolein source
H
Disease progression
Pathological condition
H2N
H2N
N
N
H
O
H2N
OH
O
oxidative stress,
inflammation,
cell death etc...
OH
H
Acrolein
(Overproduction)
Figure 1. Schematic illustration of disease-associated acrolein.
Chemically, acrolein is highly reactive and has become
recognized as a real hazard to human health. The toxicity and
function of acrolein are well-summarized in some reviews.2,10-12
In particular, the intrinsic reactivities in biological settings often
lead to many problematic alkylations of proteins or nucleobases
by reacting mainly with amino, thiol, and hydroxyl groups.
Indeed, acrolein is known to be more toxic to cells than ROS13
such as H2O2 or hydroxyl radicals, which are major oxidative
stress factors associated with a variety of disorders, and inhibitors
of several specific enzymes such as glucose and glutamate
transporters.14 Therefore, it is considered that acrolein is
associated with many kinds of diseases and pathologies.
6).22,23 Furthermore,
a
highly substrate-specific [2+3]
cycloaddition between acrolein and phenyl azide provides an
unstable 1,2,3-triazoline compound without a catalyst (Figure 3,
arrow 7).24 It is noteworthy that these reactions are found under in
vivo conditions.
On the other hand, acrolein was derivatized to some detectable
compounds, on the basis of its unique reactivity, by pre-treatment
Recently, some clinical studies have investigated and analyzed
the presence of endogenous acrolein or its metabolites in diseases
(Table 1) and its influence on pathological conditions. Acrolein is
upregulated in many diseases through the effects of oxidative

of patient specimens (Figure 4). Similar to the in vivo reaction, the
aldehyde structure can change to stable hydrazone or oxime by
treatment with 2,4-dinitrophenyl (DNP) hydrazine25 and O-
pentafluorobenzyl (O-PFB) hydroxylamine.6 In the presence of
ammonium acetate, two molecules of 5,5’-dimethyl-1,3-
cyclohexanedione (DCHD)26 react with acrolein to produce the
tricyclic compound by a Hantzsch reaction. In addition, the
reaction with 3-aminophenol under reflux conditions generates 7-
hydroyquinoline, which shows fluorescence emission.27
Rheumatoid arthritis (RA) is an autoimmune disease often
involving two kinds of autoantibodies, rheumatoid factor (RF) and
anti-cyclic citrullinated peptide antibody (ACPA). Although the
pathogenesis of RA is not completely understood, it is known that
the arginine residue in protein is changed to citrulline by
peptidylarginine deiminases, which are recognized by ACPA as
causing the immune response. In addition, several
epidemiological studies have proposed an association between
RA and oxidative stress.31
Plasma FDP-Lys levels in patients with RA and normal
subjects were measured by ELISA (Table 1).32 The RA group
showed significantly higher FDP-Lys levels than the control
group. The FDP-Lys level increased even in patients with early
RA without any inflammatory findings, and there is no correlation
between the FDP-Lys level and disease duration and/or degree of
inflammation. In addition, other clinical studies revealed that the
3. Disease-associated acrolein produced in patients
3.1. Sjögren's syndrome
Primary Sjögren's syndrome (pSS) is a systemic autoimmune
disorder mainly affecting the salivary and lacrimal glands.28 The
cardinal symptom is dry mouth and eyes as a result of decreased
salivary and lacrimal secretions caused by the destruction of these
glands. Although the etiology is not clearly understood,
autoantibodies recognizing Sjögren's syndrome A (SSA) and
Sjögren's syndrome B (SSB) were frequently found in the sera of
pSS patients.
Two clinical studies were performed for female patients with
pSS and healthy women or women with dry eyes and/or dry mouth
as control (Table 1).29,30 The concentration of FDP-Lys, in
particular as albumin or immunoglobulin-conjugated acrolein, in
saliva and plasma was measured by ELISA or western blotting.
The FDP-Lys level was significantly increased in saliva from
patients with pSS compared to controls, while there was no
difference in plasma FDP-Lys. In addition, FDP-Lys in saliva was
inversely correlated with the flow rate of saliva, which was used
as an index of the severity of salivary gland destruction.
NO2
NO2
NO2
NH
NO2
H2N
HPLC/MS
detection
NH
H
DNP hydrazine
H2SO4 aq.
N
DNP hydrazone
F
F
F
F
F
F
F
F
GC/MS
detection
O
F
H2N
O
O
H
F
O-PFB hydroxylamine
N
pyridine, 65ºC
H
acrolein
O-PFB oxime
O
The activities of autoantibodies recognizing SSA and SSB in
saliva from patients with pSS were approximately 3- to 5-fold
higher than those from control subjects.30 Interestingly, the
activity of the autoantibodies increased with the acrolein treatment
of saliva from control subjects. MS/MS analysis of
immunoglobulins from patients with pSS showed some acrolein
conjugation with cysteine, histidine, and lysine residues, of which
modification would affect the antigen recognition ability. Thus,
the changed antigen recognition ability of immunoglobulins due
to acrolein conjugation is at least partially associated with
autoimmune diseases.
O
O
O
HPLC/MS/MS
detection
DCHD
NH4OAc, H2O, 60ºC
N
H
DCHD-Acr
HO
NH2
HO
N
Fluorescence
detection
3-aminophenol
HCl aq., reflux
7-hydroxyquinoline
Figure 4. Derivatizations of acrolein in vitro (under non-
physiological conditions) to analyze the acrolein level of
specimen from clinical patient. The structure derived from
acrolein is shown in red.
3.2. Rheumatoid arthritis
O
N
OH
O
N
N
R3
N
N
or
N
N
N
R1
R1
S
CHO
N
R2
N
N
H
N
OH
MP-Lys
FDP-Lys
H
R2
Ac
N
CO2H
Acr-dG
H
O
R3 = CHO;
OPMA
CHO
R3 = CO2H; CEMA
R3 = CH OH;
N
NH
2. nucleic base
in DNA
HPMA
2
N
N
NH2
CHO
N
R2
R1
CHO
S
1.
O
H
N
O
3. GSH
HO2C
CO2H
Lys
R5
O
N
mercapturic acid
metabolic pathway
H
H
7.
N
HN
NH2
4. Lys or polyamine
O
H
N
R5
acrolein
1,2,3-triazoline
N3
R4
N
R4
N
X
6.
Thiol compounds
5.
S
hydralazine,
hydroxylamine
(NAC, Mesna, etc)
N
R4
N
R4
N
N
X
CHO
8-membered ring
O
NaO3S
O
N
NH
S
H
N
Ac
N
CO2H
H
H
H
Figure 3. Acrolein-related reactions which proceed under in vivo (physiological) conditions. The structure derived from acrolein is
shown in red.

level of many kinds of lipid peroxidation products, including
acrolein, significantly increased in the plasma and urine of
patients with RA.33,34 These clinical studies suggest that the
relationship between lipid peroxidation and the development of
RA and FDP-Lys levels could be applied in the diagnosis of early
RA. Moreover, smoking history was associated with a high value
of ACPF and RF in patients with RA,35 and acrolein is possibly
one of the pathogenetic factors in RA, similar to its role in pSS.
COPD
acrolein
(DNP-
hydrazone)
HPMA
FDP-Lys
HPLC/MS
plasma,
37
homogenized
lung tissues
urine
AAH
DM
UPLC/MS
ELISA
39
42,
43
44
urine
HPMA,
CEMA
acrolein
(DCHD-Acr)
acrolein(O-
PFB-oxime)
FDP-Lys
UPLC/MS
HPLC/MS/MS
GC/MS
urine
AD
homogenized 48
brain tissue
homogenized 49
brain tissue
homogenized 49
brain tissue
3.3. Chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is a common,
complex disorder that is characterized by progressive airflow
obstruction due to varying degrees of bronchitis and
emphysema.36 It is thought that cigarette smoke including high
levels of acrolein is involved in the pathogenesis of COPD.
Acrolein could induce neutrophil activation, resulting in enhanced
systemic acrolein production, as acrolein can absorb via lung
tissue due to its membrane permeability.
For clinical measurements of acrolein in COPD, plasma and
homogenized lung tissue were obtained from patients with COPD,
current smokers without COPD, and non-smokers.37 Acrolein
concentrations in the supernatants of homogenized tissue were
measured by HPLC/MS analysis after derivatization of acrolein
by DNP hydrazine. As predicted, acrolein was expressed strongly
and differently in the lung tissues of patients with COPD and non-
COPD smokers compared to non-smokers, presumably due to
direct exposure to acrolein from cigarette smoke. On the other
hand, plasma concentrations of acrolein were significantly higher
in patients with COPD than in non-COPD smokers. Therefore, the
increased acrolein levels in plasma may be derived not only from
cigarette smoke but also endogenous acrolein production and
would be associated with the pathology of COPD through
interference in the balance of oxidative stress versus antioxidant
potentiality.
Dot-blotting
FDP-Lys
ELISA, Western plasma,
blotting
50,
cerebrospinal 51,
fluid
urine
53
52,
53
54,
55
55,
56
17
59
62
HPMA
FDP-Lys
HPMA
HPLC/MS/MS
ELISA
Stroke
plasma
urine
HPLC/MS/MS
MP-Lys
FDP-Lys
FDP-Lys
ELISA
ELISA
ELISA
plasma
plasma
plasma
SBI
Renal
failure
Acrolein(7-
hydroxy-
quinoline)
Acr-dG
FDP-Lys
HPMA
Fluorescence
measurement
plasma
62
Tumor
Dot-blotting
ELISA
LC/MS/MS
Immunohistoch- tissue
emical staining
tissue DNA
plasma
urine
63
63
63
64
FDP-Lys
Acrolein
Fluorescence
microscope
tissue
67
3.4. Acute alcoholic hepatitis
Excessive intake of alcoholic beverages leads to acute alcoholic
hepatitis (AAH) which involves increased inflammation,
oxidative stress, lipid peroxidation, and hepatocytes death,38
although the exact mechanisms remain unclear. It is known that
free radicals generated by alcohol consumption and metabolism
can result in oxidative stress, leading to the production of acrolein
by peroxidation of unsaturated fatty acids.
Urinary HPMA was assessed by UPLC/MS in patients with
severe or non-severe AAH and healthy subjects.39 The HPMA
levels in patients with severe AAH were increased compared to
healthy controls, but the non-severe cases showed similar results
with controls. In addition, the involvement of acrolein in liver
injury is supported by the correlation between hepatocyte death
markers and HPMA combined with proinflammatory cytokines,
which contribute to hepatic injury. Indeed, the protective effects
of hydralazine, a known anti-hypertensive drug and acrolein
scavenger (Figure 3, arrow 6), for hepatic cells was proven in
animal models.40 Thus, the HPMA level can be used for diagnosis
of disease severity in AAH and treatment assessment in severe
ALD. Acrolein scavenging has therapeutic potential for liver
injury in ALD.
3.5. Diabetes mellitus
Diabetes mellitus (DM) is a metabolic disorder that induces
hyperglycemia through the breakdown of the blood glucose
regulatory system for various reasons. There are a number of
hypotheses of its etiology which are intricately connected.41 DM
is classified as two types, type 1 and type 2, based on the failure
mode of the blood glucose regulatory mechanism. In both types,
blood glucose is not stored as glycogen in the liver or adipose
tissue, leading to hyperglycemia. It is known that the metabolism
of polyol, advanced glycation end products, and the TCA
(tricarboxylic acid) cycle induce oxidative stress and are a
common pathology of DM.
Clinically, an association between both types of DM and
acrolein metabolites has been reported by several studies. FDP-
Lys from the urine of patients with type 1 or type 2 DM was
measured by ELISA (Table 1).42,43 FDP-Lys levels in both studies
were significantly increased compared to control subjects. The
other study showed an association of the other urinary
metabolites, HPMA and CEMA, with type 2 DM and insulin
resistance.44 Furthermore, it was suggested that acrolein has been
associated with the dysregulation of glucose transport in human
endothelial cells, diabetic nephropathy,45 and diabetic
retinopathy.46 Accumulation of FDP-Lys involved the
abnormalization of Müller glial cells occurring in the early stages
of diabetic retinopathy.46
Table 1. List of clinical investigations of disease-associated
acrolein.
Disease Detected
acrolein
Detection
method
Specimen
Ref
product
pSS
RA
FDP-Lys
Western blotting, saliva, plasma 29,
ELISA
30
3.6. Alzheimer’s disease
FDP-Lys
ELISA
plasma
32
acrolein (O- GC/MS
PFB-oxime)
plasma, urine 33
Alzheimer's disease (AD) is a neurodegenerative disorder
characterized by progressive impairment of cognitive function.

There is some hypothesis of the pathogenesis, although the exact
mechanism is still unclear. The accumulation of amyloid β peptide
(Aβ) in the brain and increased oxidative damage is a process
thought to be involved in the development of AD.47 Before AD is
identified symptomatically, A begins to aggregate. This stage is
referred to as preclinical AD (PCAD), and the disease progresses
to AD via mild cognitive impairment (MCI).
The acrolein level in homogenized brain tissues
[hippocampus/parahippocampal gyrus (HPG), superior and
middle temporal gyrus (SMTG), and cerebellum (CER)] of MCI,
early AD, and control subjects were analyzed by LC/MS/MS after
derivatization with DCHD.48 Acrolein levels in all three brain
regions of patients with early AD and MCI were significantly
increased in comparison with control subjects, while there were
no statistical differences between MCI and early AD. More
recently, the levels of FDP-Lys and extractable acrolein in brain
tissues (HPG, SMTG, and CER) from patients with PCAD were
evaluated by dot-blotting and GC/MS, respectively.49 The
extractable acrolein in the HPG of patients with PCAD
significantly increased compared to control subjects, contrary to
the decrease in CER. There were no significant differences in the
levels of FDP-Lys in the HPG, SMTG, or CER of patients with
PCAD compared to control subjects.
was significantly higher,17 FDP-Lys correlated with disease
severity and prognosis of patients who have suffered a stroke. In
contrast, urinary HPMA was decreased in patients compared to
control subjects, due to the depletion of GSH, which is supported
by the decrease of plasma GSH levels in patients.56,57 The change
of HPMA in patients is found in both types of stroke, CI and CH,
with no statistical difference.57 A mechanistic study suggested that
the acrolein is produced via activation of the NF-kB pathway
during ischemia,56 and acrolein elicited a vicious cycling of
oxidative stress resulting in stroke-related neuronal damage.
Therefore, acrolein has been suggested as the primary culprit of
neuronal damage in patients who have suffered a stroke. Of note,
NAC or N-benzylhydroxylamine effectively prevented
neurotoxicity by scavenging toxic acrolein in an animal model
(Figure 3, arrow 5 and 6).56,58
Increased acrolein production was not only seen in severe
strokes but also silent brain infarctions (SBI).59 Measurements of
FDP-Lys in the plasma of SBI and normal subjects revealed that
the levels were significantly higher in patients with SBI. Analysis
of FDP-Lys levels along with other factors including interleukin-
6 (IL-6), C-reactive protein (CRP), and age can predict SBI with
89% sensitivity and 91% specificity in a receiver operating
characteristic curve.
To evaluate the utility of acrolein metabolites for AD
diagnosis, FDP-Lys levels in plasma50 or cerebrospinal fluid51 of
patients with AD were evaluated by ELISA or western blotting,
because homogenized brain tissue was not suitable as a diagnostic
specimen. FDP-Lys levels were increased in patients with AD and
MCI compared to control subjects. In contrast, other studies
showed that urinary HPMA was decreased in patients with AD
and MCI compared to the control,52,53 which is likely attributed to
the depletion of GSH. These studies cannot distinguish MCI and
AD in patients solely by acrolein metabolite levels.
Moreover, we reported that acrolein smoothly reacts with
polyamines, such as spermidine and spermine, to obtain an 8-
membered ring compound (Figure 3, arrow 4) that can suppress
the toxic aggregation of A (Figure 5).21 This reaction is likely an
innate biological defense mechanism of polyamines and could
possibly be applied as a therapeutic strategy against AD through
the utilization of acrolein as a treatment modality.
3.8. Tumor
A tumor is a proliferative lesion that invades surrounding
normal tissue and is often fatal. Cancer cells are known to utilize
ROS,60 such as H2O2, and polyamines61 to drive proliferation and
other events required for tumor development. Therefore, cancer
cells are believed to produce a significant amount of acrolein
associated with a high concentration of an acrolein source,
polyamines, and conditions conducive to oxidative stress.
3.8.1. Urothelial carcinomas from chronic kidney disease
It is known that urothelial carcinomas (UC) are highly
prevalent in patients with end-stage renal disease. Patients with
chronic kidney disease (CKD) have high plasma concentrations of
polyamines, as their ability to excrete these substances is
decreased. Along with this, free acrolein and FDP-Lys levels in
plasma have been found to be increased in patients with renal
failure.62 In patients with concomitant UC and CKD, plasma FDP-
Lys and urinary HPMA levels have been found to be significantly
increased compared to those of control subjects.63 Moreover, Acr-
dG levels in tumor tissues are statistically correlated with CKD
stages in patients with UC. Furthermore, several gene mutations
have been identified in the extracted p53 gene, a tumor suppressor
gene, from patients with UC. These results indicate that acrolein
acts as an endogenous uremic toxin and carcinogenic factor via
DNA damage and would contribute to UC formation in patients
with CKD.
3.7. Stroke
Stroke, including cerebral infarction (CI) and cerebral
hemorrhage (CH), is a sudden focal neurologic deficit caused by
vascular insult involving cell damage in the central nervous
system (CNS). Neurons contain high concentrations of
polyamines, which act as neuromodulators and are perturbed after
cerebral ischemia.54 In addition, the activity of polyamine
oxidases is considered to be upregulated by cell damage in the
CNS.
3.8.2. Colon cancer
Immunohistochemical distributions of FDP-Lys and p53
protein in colon tumors were evaluated using their respective
monoclonal antibodies (MAbs) (Table 1).64 These investigations
showed that the FDP-Lys levels increased along with the
incidence of colon carcinogenesis. Levels were found to be
moderate in tubular and villotubular low-grade adenomas and
abundant and diffuse in high-grade villotubular adenomas and
Dukes A carcinomas. FDP-Lys levels in cancer gradually
decreased as colon carcinomas progressed (Dukes B and C),
although FDP-Lys was found to be abundant in the non-malignant
colon epithelium of these patients. While there was no relationship
between acrolein adducts, FDP-Lys, and p53 distribution with
respect to protein levels, acrolein seems to be associated with the
transition from benign to malignant colon tumors.
Figure 5. Fibrillization of A40 peptide in the A) absence or B)
presence of an 8-membered ring compound.
Plasma FDP-Lys levels of patients who have suffered a stroke
were analyzed by ELISA (Table 1),55,56 and the levels were
increased compared to control subjects. While MP-Lys in albumin

3.8.3. Breast cancer
The CTS probe was applied to visualize acrolein in breast
cancer tissues (Table 1).67 The resected tissue specimens were
derived from cancerous tissue [invasive ductal carcinoma (IDC)
and ductal carcinoma in situ (DCIS)], normal breast gland (NBG),
and ductal hyperplasia (DH) from patients with breast cancer who
underwent breast-conserving surgery. The fluorescence images
are shown in Figure 6C. The fluorescence intensity of tissues from
cancers (IDC and DCIS) were statistically higher than that from
non-cancerous NBG and DH, indicating that cancerous (IDC and
DCIS) tissue could be distinguished from non-cancerous tissue
(NBG and DH) with high specificity and sensitivity. Importantly,
the CTS probe labeled the cancer tissues with similar fluorescence
intensity, regardless of phenotype status such as estrogen receptor
or human epidermal growth factor receptor 2. Moreover, the
fluorescent images of cancers are in agreement with those of H&E
staining (comparison of Figure 6C and 6D). Thus, this clinical
study succeeded in visualizing cancer morphology in live tissue
by utilizing cancerous acrolein as a diagnostic substrate.
Acrolein seems to be over-produced in tumors, but the
analytical method used thus far involving acrolein or acrolein
metabolites requires several specimen pre-treatment steps and is
time-intensive. Moreover, these values would not reflect the exact
acrolein level in real time for various reasons including the slow
formation of FDP-Lys.65 Therefore, a novel analytical method is
required in order to apply acrolein as a diagnostic substrate during
the intraoperative pathological diagnosis of the tumor.
We have reported on the specific and rapid reactivity of
acrolein with phenyl azide to give the unstable 4-formyl-1,2,3-
triazoline derivative (Figure 3, arrow 7), which spontaneously
decomposes to a diazo compound.24 To apply this reaction as a
chemical probe for imaging of cancer, the TAMRA fluorophore is
attached to the phenyl azide, referred to as the click-to-sense probe
(CTS probe, Figure 6A). The probe can be internalized into cells;
if the CTS probe subsequently encounters intracellular acrolein,
the 1,3-dipolar cycloaddition and further tandem reactions
proceed and the resultant diazo intermediate reacts with the
nearest organelle to anchor the fluorophore via a covalent bond
(Figure 6B).66 The unreacted probe can be washed out to reduce
the background fluorescence as the probe has the ability to be
shuttled inside and outside of the cell. This reaction achieves the
visualization of cancer in live cells even at nanomolar levels of
acrolein. Herein, we propose a concept of “Transformative In vivo
Tandem reaction (TIT reaction)”, that efficiently converts the
reactive in vivo substances (such as acrolein) into certain
bioactive, diagnostic or therapeutic molecules through the tandem
reactions, directly in cells, plants, animals or human patients.
4. Perspective of disease-associated acrolein as a diagnostic
and therapeutic substrate
Acrolein and its metabolites were detected in many kinds of
diseases associated with oxidative stress, which sometimes
express complicated pathologies due to the local and systemic
influence of oxidative stress. Analysis of endogenously produced
acrolein would help to understand the unclear pathogeneses of
these diseases. Moreover, acrolein is considered to be a potent
substrate for use in the diagnosis or treatment of various diseases,
owing to the disease-specific overproduction of acrolein. In
Figure 7. Detection method of FDP-Lys on the basis of FDP-Lys
oxidation to MP-Lys analog mediated by 4-nitrophthalonitrile
and calcium ion. Reduced product, 4-aminophthalonitrile, can be
detected by fluorescence measurements (ex: 340 nm, em: 404
nm).
Figure 6. A) Structure of Click-to-Sense (CTS) probe. B) Cascade reaction in cancerous tissue. C) CTS stain and D) H&E stain of
tumor tissue from BCS. NG; Normal gland, DH; Ductal hyperplasia, DCIS; Ductal carcinoma in situ, IDC; Invasive ductal
carcinoma.

particular, clinical detection of acrolein could be a novel
diagnostic criterion of complicated diseases with no other
diagnostic methods.
K.T.) and JP18K14341, JP18K07265, JP18H05503 (to A.R.P.)
and JP19J00396 (to K.M.)]. Funding was also provided by the
Russian Government Program for Competitive Growth (to Kazan
Federal University).
The diagnostic application of acrolein metabolites may have
several limitations. As seen in patients with AD or stroke,52,53,56,57
urinary HPMA is decreased even if the acrolein level is increased,
due to GSH depletion. Moreover, the ratio of metabolites changes
easily depending on the strength of other metabolic pathways. In
addition, FDP-Lys production is considered to be slow65 and
gradually changes to MP-Lys, which is a better substrate of anti-
acrolein antibodies.16 Therefore, the signal from an acrolein-
conjugated protein with FDP-Lys would increase over time in a
Mab-mediated detection method, although we previously reported
an alternative FDP-Lys detection method without Mab. The non-
Mab method consists of the reduction of 4-nitrophthalonitrile by
FDP-Lys and subsequent fluorescence measurement of the
reduced product, 4-amino phthalonitrile (Figure 7).68 Detected
signal from metabolites did not reflect the exact acrolein levels.
The FDP-Lys level is in fact different from the extractable
acrolein level in patients with AD.49 From a different perspective,
if the patient already has an underlying disease involving
oxidative stress and acrolein production, we cannot separately
detect the acrolein from other concomitant disease processes.
Therefore, the diagnosis is somewhat difficult to interpret based
on this data.
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Declaration of interests
☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: