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Acrolein, an electrophilic α,β-unsaturated aldehyde, is present in foods and beverages, and is a product of incomplete combustion, and thus, reaches high ppm levels in tobacco smoke and structural fires. Exposure to acrolein is linked with cardiopulmonary toxicity and cardiovascular disease risk. The hypothesis of this study is the direct effects of acrolein in isolated murine blood vessels (aorta and superior mesenteric artery, SMA) are transient receptor potential ankyrin-1 (TRPA1) dependent. Using isometric myography, isolated aorta and SMA were exposed to increasing levels of acrolein. Acrolein inhibited phenylephrine (PE)-induced contractions (approximately 90%) in aorta and SMA of male and female mice in a concentration-dependent (0.01-100 μM) manner. The major metabolite of acrolein, 3-hydroxypropylmercapturic acid (3HPMA), also relaxed PE-precontracted SMA. As the SMA was 20× more sensitive to acrolein than aorta (SMA EC 50 0.8 ± 0.2 μM; aorta EC 50  > 29.4 ± 4.4 μM), the mechanisms of acrolein-induced relaxation were studied in SMA. The potency of acrolein-induced relaxation was inhibited significantly by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); and, 4) a TRPA1 antagonist (A967079). TRPA1 positive immunofluorescence was present in the endothelium. Compared with other known TRPA1 agonists, including allyl isothiocyanate (AITC), cinnamaldehyde, crotonaldehyde, and formaldehyde, acrolein stimulated a more potent TRPA1-dependent relaxation. Acrolein, at high concentration [100 μM], induced tension oscillations (spasms) independent of TRPA1 in precontracted SMA but not in aorta. In conclusion, acrolein is vasorelaxant at low levels (physiological) yet vasotoxic at high levels (toxicological)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The appropriate selection of quality marker (Q-marker) for performing the comprehensive quality evaluation of traditional Chinese medicines (TCMs) has much more significance. Wu-Wei-Wen-Tong Capsule (WWWTC), a TCMs prescription, is mainly utilized to treat rheumatoid arthritis (RA) in China. However, the comprehensive quality control for WWWTC has not been achieved because of lacking system analysis for the Q-marker. In this study, a dual wavelength, 203 and 270 nm, was selected based on the feature of 15 Q-markers, and a reliable UHPLC-UV fingerprinting approach was established, achieving the comprehensive quality evaluation of WWWTC. First, we identified 91 prototypes in rat plasma after administering a set amount of WWWTC by using UHPLC-QTOF/MS technique and selected them as the candidate Q-markers. Next, based on the "five principles" of Q-marker selection, 15 absorbed components among them including coumarin, cinnamic acid, cinnamaldehyde, cinnamic alcohol, and 2-methoxycinnamaldehyde derived from Monarch medicine of Cmnamomi Mmulus; epimedin C, icariin, baohuoside I, and anhydroicaritin derived from Monarch medicine Epimedii Folium; germacrone, the sesquiterpene compound in Minister medicine Rhizoma Wenyujin Concisum; pachymic acid, the tetracyclic triterpenoid acids in Assistant medicine Poria; baicalin, baicalein, wogonin, and wogonoside in Guide medicine Scutellariae Radix, respectively, were seriously chosen as the Q-markers, indicating preferable pharmacological effect on RA, characterization of transitivity and traceability as well as measurable components in WWWTC. The effective and meaningful strategy displayed a unique perspective for the exploration of Q-markers in the quality evaluation and further ensured efficacy and safety of the TCMs., (© 2021 John Wiley & Sons, Ltd.)

Objective: This study aimed to clarify whether plasma acrolein level actually increases in rheumatoid arthritis (RA) patients, and to elucidate whether any relationship exists between the levels and the RA background variables., Methods: Plasma levels of protein-conjugated acrolein (PC-Acro) in 84 patients (RA group) and 298 normal individuals (Control group) were measured by enzyme-linked immunosorbent assay procedures. The data were statistically analyzed with Wilcoxon rank-sum test, multiple logistic regression analyses and Spearman's rank correlation coefficient., Results: The RA group showed significantly higher PC-Acro levels than the Control group (median [interquartile range]: 80.5 [63.2-105.2] and 65.9 [58.9-78.1] nmol/ml, respectively). Of background factors giving influence to PC-Acro level in the combination of the two groups, 'diagnosis of RA positive' indicated strong correlation to high PC-Acro level (odds ratio: 2.96; 95% confidence interval: 1.54-5.71). These increases of PC-Acro in the RA patients did not correlate to their disease duration and/or inflammatory variables: PC-Acro level could elevate even in early RA patients showing negative inflammatory findings., Conclusion: Plasma levels of PC-Acro increased with RA, but the levels did not correlate with RA background variables. This report provides the basis for further studies of early diagnosis of RA as well as its pathogenesis.

The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.

It was found recently that acrolein (CH 2 =CH-CHO), mainly produced from spermine, is more toxic than ROS (reactive oxygen species, O 2 -· , H 2 O 2 , and ·OH). In this review, we describe how the seriousness of brain infarction, dementia, renal failure, and Sjӧgren's syndrome is correlated with acrolein. In brain infarction and dementia, it was possible to identify incipient patients with high sensitivity and specificity by measuring protein-conjugated acrolein (PC-Acro) in plasma together with IL-6 and CRP in brain infarction and Aβ 40/42 in dementia. The level of PC-Acro in plasma and saliva correlated with the seriousness of renal failure and Sjӧgren's syndrome, respectively. Thus, development of acrolein scavenger medicines containing SH-group such as N-acetylcysteine derivatives is important to maintain QOL (quality of life) of the elderly.

Purpose: Cigarette smoke produces a high level of acrolein, which is thought to be pathogenically involved in the development of chronic obstructive pulmonary disease (COPD). The present study investigated the pathological role of acrolein in the development of COPD., Patients and Methods: Acrolein concentration was measured in plasmas obtained from 47 patients with COPD and 18 current smokers without COPD, and in supernatants of homogenized lung tissues obtained from 10 never-smokers, 8 current smokers, and 8 patients with COPD by high-performance liquid chromatography. Oxidant status and antioxidant activity were measured using derivatives of reactive oxygen metabolite (d-ROM) and bio-antioxidant power (BAP), respectively, in the Free Radical Elective Evaluation FRAS4 system. In addition, immunohistochemistry was used to evaluate the over-presentation of acrolein in lung tissues of patients with COPD., Results: Plasma concentrations of acrolein were significantly higher in the patients with COPD than the non-COPD smokers ( P <0.001), which significantly correlated with the oxidant status in patients with COPD (R=0.69, P <0.05). Similar pathological alterations in acrolein concentrations were found in the lung tissue supernatants of patients with COPD, which significantly correlated with the oxidant status in patients with COPD. Furthermore, acrolein was strongly expressed in the lung tissues of patients with COPD., Conclusion: The increased acrolein concentrations were highly involved in the pathogenesis of COPD through interference in the balance of oxidative stress versus antioxidant potentiality., Competing Interests: The authors report no conflicts of interest in this work.

It is thought that tissue damage at advanced age is mainly caused by ROS (reactive oxygen species, O 2 - , H 2 O 2 , and ·OH). However, it was found that acrolein (CH 2 =CH-CHO) is more toxic than ROS, and is mainly produced from spermine (SPM), one of the polyamines, rather than from unsaturated fatty acids. Significant amounts of SPM are present normally as SPM-ribosome complexes, and contribute to protein synthesis. However, SPM was released from ribosomes due to the degradation of ribosomal RNA by ·OH or the binding of Ca 2+ to ribosomes, and acrolein was produced from free SPM by polyamine oxidases, particularly by SPM oxidase. Acrolein inactivated several proteins such as GAPDH (glycelaldehyde-3-phosphate dehydrogenase), and also stimulated MMP-9 (matrix metalloproteinase-9) activity. Acrolein-conjugated GAPDH translocated to nucleus, and caused apoptosis like nitrosylated GAPDH. Through acrolein conjugation with several proteins, acrolein causes tissue damage during brain stroke, dementia, renal failure, and primary Sjögren's syndrome. Thus, development of acrolein scavengers with less side effects is very important to maintain QOL (quality of life) of elderly people.

Exposure assessment is a fundamental part of the risk assessment paradigm, but can often present a number of challenges and uncertainties. This is especially the case for process contaminants formed during the processing, e.g. heating of food, since they are in part highly reactive and/or volatile, thus making exposure assessment by analysing contents in food unreliable. New approaches are therefore required to accurately assess consumer exposure and thus better inform the risk assessment. Such novel approaches may include the use of biomarkers, physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry, and/or duplicate diet studies. This review focuses on the state of the art with respect to the use of biomarkers of exposure for the process contaminants acrylamide, 3-MCPD esters, glycidyl esters, furan and acrolein. From the overview presented, it becomes clear that the field of assessing human exposure to process-related contaminants in food by biomarker monitoring is promising and strongly developing. The current state of the art as well as the existing data gaps and challenges for the future were defined. They include (1) using PBK modelling and duplicate diet studies to establish, preferably in humans, correlations between external exposure and biomarkers; (2) elucidation of the possible endogenous formation of the process-related contaminants and the resulting biomarker levels; (3) the influence of inter-individual variations and how to include that in the biomarker-based exposure predictions; (4) the correction for confounding factors; (5) the value of the different biomarkers in relation to exposure scenario's and risk assessment, and (6) the possibilities of novel methodologies. In spite of these challenges it can be concluded that biomarker-based exposure assessment provides a unique opportunity to more accurately assess consumer exposure to process-related contaminants in food and thus to better inform risk assessment.

Chronic kidney disease (CKD) is characterized by the progressive reduction of glomerular filtration rate and subsequent retention of organic waste compounds called uremic toxins. While patients with CKD are at a higher risk of premature death due to cardiovascular complications, this increased risk cannot be completely explained by classical cardiovascular risk factors such as hypertension, diabetes mellitus, and obesity. Instead, recent research suggests that uremic toxins may play a key role in explaining this marked increase in cardiovascular mortality in patients with CKD. While spermine, a tetra-amine, has previously been hypothesized to act as an uremic toxin, the following review presents a summary of recent literature that casts doubt on this assertion. Instead, acrolein, an oxidative product of spermine and the triamine spermidine, is likely responsible for the toxic effects previously attributed to spermine.

Plasma, urine and cerebrospinal fluid (CSF) were examined for biochemical markers of dementia. Protein-conjugated acrolein (PC-Acro) and the amyloid-β (Aβ)40/42 ratio in plasma can be used to detect mild cognitive impairment (MCI) and Alzheimer's disease (AD). In plasma, PC-Acro and the Aβ40/42 ratio in MCI and AD were significantly higher relative to non-demented subjects. Furthermore, urine acrolein metabolite, 3-hydroxypropyl mercapturic acid (3-HPMA)/creatinine (Cre) and amino acid-conjugated acrolein (AC-Acro)/Cre in AD were significantly lower than MCI. It was also shown that reduced urine 3-HPMA/Cre correlated with increased plasma Aβ40/42 ratio in dementia. The Aβ40/PC-Acro ratio in CSF, together with Aβ40 and Aβ40/42 ratio, was lower in AD than MCI. Increased plasma PC-Acro and Aβ40/42 ratio and decreased urine 3-HPMA/Cre correlated with cognitive ability (MMSE). These results indicate that the measurements of acrolein derivatives together with Aβ and Cre in biologic fluids is useful to estimate severity of dementia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of cinnamaldehyde, cinnamic acid, and 2-methoxy cinnamic acid in rat whole blood. It was the first time to study the pharmacokinetics of 2-methoxy cinnamic acid in rat whole blood. Samples were processed by a one-step protein precipitation with acetonitrile-37% formaldehyde (90:10, v:v). Chromatographic separation was performed on a Thermo Scientific C18 column (2.1mm×50mm, 1.9μm) at room temperature. The total run time was 4min. The detection was accomplished by using positive and negative ion electrospray ionization in multiple reaction monitoring mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The lower limits of quantification (LLOQ) were 0.1ng/mL for cinnamaldehyde, 5.8ng/mL for cinnamic acid, and 10ng/mL for 2-methoxy cinnamic acid, respectively. To our knowledge, this was the first time that the LLOQ for cinnamaldehyde in validated methods for biological samples was as low as 0.1ng/mL. Intra- and inter-day precision and accuracy were within ±9% for all of the analytes during the assay validation. Assay recoveries were higher than 80% and the matrix effects were minimal. The half-life were 8.7±0.7h for cinnamaldehyde, 1.0±0.5h for cinnamic acid, and 1.4±0.4h for 2-methoxy cinnamic acid, respectively. The validated assay was firstly applied to the simultaneous quantification of cinnamaldehyde, cinnamic acid, and 2-methoxy cinnamic acid, especially for 2-methoxy cinnamic acid in rat whole blood after oral administration of 15mg/kg essential oil of Cinnamoni Ramulus. It was observed that the Cmax and AUC of 2-methoxy cinnamic acid (0.01% in essential oil of Cinnamoni Ramulus) were greater than those of cinnamaldehyde (83.49% in essential oil of Cinnamoni Ramulus), which implied that 2-methoxy cinnamic acid might be the major bioactive constitutes in essential oil of Cinnamoni Ramulus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment., (Copyright © 2015 John Wiley & Sons, Ltd.)

Acrolein, a cytotoxic α,β-unsaturated aldehyde and disease biomarker, was determined in plasma by means of a novel tethering strategy using Michael addition of the compound to a fluorescent dye, followed by immobilization of the product on microbeads via the aldehyde moiety. Elevation of blood acrolein was detected in mice treated with an anticancer agent cyclophosphamide, which releases acrolein upon activation. This method should be suitable for high-throughput diagnostic and clinical application.

In this study, a headspace, solid-phase dynamic extraction method coupled to gas chromatography-tandem mass spectrometry (HS-SPDE-GC-MS/MS) method was developed for the simultaneous determination of four volatile compounds, namely, isoborneol, borneol, muscone and cinnamaldehyde, in rat plasma after oral administration of Shexiang Baoxin Pill (SBP) using naphthalene as an internal standard (IS). The target compounds were extracted using an SPDE needle device coated with a poly (dimethylsiloxane) (PDMS) phase. The detection was achieved by GC-MS/MS in multiple reaction monitoring (MRM) mode. The optimised mass transition ion pairs (m/z) for quantitation were 95.1/67.1 for isoborneol and borneol, 85.0/67.0 for muscone, 131.0/77.0 for cinnamaldehyde and 128.1/102.1 for the IS. The parameters that affect the extraction ratio, such as the pre-incubation time, extraction temperature, number of extraction cycles, desorption volume and pH, were also optimised. The method was thoroughly validated with respect to specificity, linearity, precision, accuracy, recovery and stability. A sufficiently sensitive HS-SPDE-GC-MS/MS method was first developed in this study to determine the pharmacokinetics of volatile compounds found in rat plasma following oral administration of SBP. The method developed uses a simple procedure for plasma sample preparation and could be a promising tool for the analysis of complex volatile samples, such as traditional Chinese medicine (TCM)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: Brain white matter hyperintensities can be divided into periventricular hyperintensity (PVH) and deep-and-subcortical white matter hyperintensity (DSWMH); the former contributes more to cognitive dysfunction and infarction risk. We conducted the present investigation to define the relationship between PVH and DSWMH., Design: Cross-sectional study., Setting: University hospital., Participants: We prospectively enrolled 228 healthy Japanese volunteers with relative risk values (RRVs) >0.5., Primary Outcome Measures: We investigated whether it is possible to use the RRV to predict PVH and DSWMH., Results: Among 228 volunteers, 103 (45.1%) and 157 (68.8%) exhibited PVH and DSWMH, respectively. Age, body mass index and PVH were significant independent determinants of RRV. A significant OR for PVH was noted in the highest RRV tertile compared with the lowest, after adjusting for potential confounding factors. A significant OR for high predicted PVH risk was found for RRV levels as well., Conclusions: Elevated RRV levels were significantly associated with increased predicted PVH, suggesting that measuring the plasma protein-conjugated acrolein, interleukin 6 and C reactive protein levels may be useful for identifying Japanese at high risk for PVH.

Rationale: Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR., Objectives: To study the effect of cigarette smoke on extrapulmonary CFTR function., Methods: Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy., Measurements and Main Results: Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke-exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein., Conclusions: Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.

Fibrinogen is a key protein involved in coagulation and its deposition on blood vessel walls plays an important role in the pathology of atherosclerosis. Although the causes of fibrinogen (fibrin) deposition have been studied in depth, little is known about the relationship between fibrinogen deposition and reactive carbonyl compounds (RCCs), compounds which are produced and released into the blood and react with plasma protein especially under conditions of oxidative stress and inflammation. Here, we investigated the effect of glycolaldehyde on the activity and deposition of fibrinogen compared with the common RCCs acrolein, methylglyoxal, glyoxal and malondialdehyde. At the same concentration (1 mmol/L), glycolaldehyde and acrolein had a stronger suppressive effect on fibrinogen activation than the other three RCCs. Fibrinogen aggregated when it was respectively incubated with glycolaldehyde and the other RCCs, as demonstrated by SDS-PAGE, electron microscopy and intrinsic fluorescence intensity measurements. Staining with Congo Red showed that glycolaldehyde- and acrolein-fibrinogen distinctly formed amyloid-like aggregations. Furthermore, the five RCCs, particularly glycolaldehyde and acrolein, delayed human plasma coagulation. Only glycolaldehyde showed a markedly suppressive effect on fibrinogenesis, none did the other four RCCs when their physiological blood concentrations were employyed, respectively. Taken together, it is glycolaldehyde that suppresses fibrinogenesis and induces protein aggregation most effectively, suggesting a putative pathological process for fibrinogen (fibrin) deposition in the blood.

The objective of this study was to determine whether plasma levels of acrolein, a compound that causes cell damage, and amyloid-β (Aβ) are useful biochemical markers for Alzheimer's disease (AD). The study included 221 elderly subjects divided into 101 non-demented [33 healthy control and 68 non-demented subjects with white matter hyperintensity (nd-WMH)], 50 mild cognitive impairment (MCI), and 70 AD. Increases in both protein-conjugated acrolein (PC-Acro) and Aβ40/42 ratio were observed in MCI and AD patients compared with values in control subjects. When the combined measurements of PC-Acro and Aβ40/42 ratio were evaluated using the median value of the relative risk value for dementia, they were in the order AD (0.98) ≥ MCI (0.97) > nd-WMH (0.83) > control (0.35). The results indicate that measurements of PC-Acro and Aβ40/42 ratio not only detect MCI and AD patients but also nd-WMH subjects. Furthermore, both PC-Acro and Aβ40/42 ratio in plasma for 120 MCI and AD patients were significantly higher than those for 101 control and nd-WMH subjects, indicating that both values become useful biochemical markers for MCI and AD subjects.

The relationship between acrolein (CH(2) =CH-CHO) and brain infarction is the focus of this review. It has been found that acrolein is produced mainly within cells from polyamines by polyamine oxidases (PAOs), especially from spermine by spermine oxidase during cell damage, and that acrolein is more toxic than reactive oxygen species (ROS) in a cell culture system. Thus, the possibility that acrolein and PAOs are good biochemical markers of stroke was tested because there are no other reliable biochemical markers at the early stage of stroke. Levels of protein-conjugated acrolein (PC-Acro) and PAOs (acrolein-producing enzymes) were significantly increased in the plasma of stroke patients. The multiplied value of PC-Acro by PAOs was nearly parallel with the size of stroke. Furthermore, when the combined measurements of PC-Acro, interleukin-6 (IL-6) and C-reactive protein (CRP) were evaluated along with age using a receiver operating characteristic (ROC) curve, even silent brain infarction (SBI), which is a small brain infarction, was indicated with approximately 84% sensitivity and specificity. These findings clearly indicate that acrolein is strongly correlated with cell damage during brain infarction., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: We found previously that the measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to clarify how three biochemical markers are correlated to SBI, carotid atherosclerosis (CA) and white matter hyperintensity (WMH)., Methods: The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography., Results: A total of 790 apparently healthy volunteers were classified into 260 control, 214 SBI, 263 CA and 245 WMH subjects, which included 187 subjects with two or three pathologies. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age, using a receiver operating characteristic curve and artificial neural networks, the relative risk value (RRV), an indicator of tissue damage, was in the order SBI with CA (0.90)>SBI (0.80)>CA (0.76)>WMH with CA (0.65)>WMH (0.46)>control (0.14). RRV was also correlated with severity in each group of SBI, CA and WMH., Conclusion: The RRV supports the idea that the degree of risk to develop a stroke is in the order SBI>CA>WMH., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

A novel lanthanide probe was designed, synthesized, and employed for a sensitive and reliable assay of acrolein based on time-resolved luminescence measurement, which suppresses the background signal of serum.

Background: We recently found that an increased plasma concentration of protein-conjugated acrolein is a good biomarker for stroke. Therefore we determine whether the concentration of protein-conjugated acrolein is increased in saliva from patients with primary Sjögren's syndrome., Methods: Stimulated whole-mixed saliva was collected from 10 patients and 13 control subjects. The concentration of protein-conjugated acrolein in saliva and plasma was measured by either Western blotting or enzyme-linked immunosorbent assay., Results: The concentration of protein-conjugated acrolein, especially albumin-conjugated acrolein, was greatly increased in saliva from patients with primary Sjögren's syndrome (p<0.001). The concentration of protein-conjugated acrolein was inversely correlated with the flow rate of saliva., Conclusion: The results indicate that the concentration of protein-conjugated acrolein, a marker of cell or tissue damage, in saliva is well correlated with seriousness of primary Sjögren's syndrome., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background and Purpose: We recently found that increases in plasma levels of protein-conjugated acrolein and polyamine oxidases, enzymes that produce acrolein, are good markers for stroke. The aim of this study was to determine whether the level of protein-conjugated acrolein is increased and levels of spermine and spermidine, the substrates of acrolein production, are decreased at the locus of infarction., Methods: A unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. The level of protein-conjugated acrolein at the locus of infarction and in plasma was measured by Western blotting and enzyme-linked immunosorbent assay, respectively. The levels of polyamines at the locus of infarction and in plasma were measured by high-performance liquid chromatography., Results: The level of protein-conjugated acrolein was greatly increased, and levels of spermine and spermidine were decreased at the locus of infarction at 24 hours after the induction of stroke. The size of infarction was significantly decreased by N-acetylcysteine, a scavenger of acrolein. It was also found that the increases in the protein-conjugated acrolein, polyamines, and polyamine oxidases in plasma were observed after the induction of stroke., Conclusions: The results indicate that the induction of infarction is well correlated with the increase in protein-conjugated acrolein at the locus of infarction and in plasma.

We found previously that increased levels of polyamine oxidase (PAO) [acetylpolyamine oxidase (AcPAO) plus spermine oxidase (SMO)], and acrolein (CH(2)CHCHO) are good markers of stroke. We then investigated whether silent brain infarction (SBI) can be detected by measuring acrolein, PAO, or other biomarkers. Several biomarkers were measured in the plasma of 53 normal subjects and 44 subjects with SBI. It was found that the levels of protein-conjugated acrolein (PC-Acro), interleukin-6 (IL-6) and C-reactive protein (CRP) were significantly higher in SBI than in normal subjects. PAO was slightly higher in SBI than in normal subjects. Since the probability of SBI was increased with age, values were analyzed including age as a factor. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age using a receiver operating characteristic curve, SBI was indicated with 89% sensitivity and 91% specificity. The results indicate that measurement of PC-Acro together with IL-6 and CRP makes it possible to identify SBI with high sensitivity and specificity.

The pharmacokinetics and metabolism of 2'-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague-Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent. BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels. In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h. BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid. In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.

Background: The molecular mechanisms of cerebral vasospasm following aneurysmal subarachnoid haemorrhage (aSAH) remain unclear. Acrolein, a reactive metabolite produced in many models of mechanical and ischemic injury, has been shown to cause vasospasm in coronary artery and aorta models. These traits suggest it may play a role in post-aSAH cerebral vasospasm. This pilot study was designed as a preliminary investigation to determine if acrolein levels could be used as a clinical tool to predict the presence of vasospasm., Methods: Eleven patients with aSAH and Hunt and Hess admission grades of III-V were prospectively enrolled. Patients were stratified according to the presence or absence of vasospasm, defined as a delayed ischaemic neurological deficit in which all other possible causes have been excluded. Soluble acrolein levels were determined at two times points: early (day 1-3 post-SAH) and late (day 8-12 post-SAH) and the change in acrolein levels over this period was computed using a Mann-Whitney test., Results: The change in acrolein levels over this period between the vasospasm and non-vasospasm group trended toward but did not achieve statistical significance (means: 5.68 versus -5.54; medians: 5.27 versus -3.99; range: -8.067 to 22.904 versus -13.83 to 5.199 p=0.13). Five out of six vasospasm patients showed an increase in acrolein levels over the vasospasm period. Three out of four non-vasospasm patients showed a decrease over the vasospasm period., Conclusions: The results of this pilot study suggest that acrolein levels increase in patients undergoing vasospasm during the vasospasm window. This suggests that acrolein may play a role in the pathways leading up to or following vasospasm. There is a need for larger more definitive studies.

Background: Acrolein is a very reactive aldehyde present in cigarette smoke and endogenously generated by pathways such as lipid peroxidation and threonine metabolism by phagocytes. Acrolein has been shown to affect uptake of cholesterol by HDL. We hypothesized that acrolein could also have deleterious effects on paraoxonase 1 (PON-1) activity. We also determined whether free serum acrolein levels are higher in renal failure, and assessed whether they decrease after hemodialysis (HD) and whether this change correlates with increases in PON-1 activity., Methods: We incubated human HDL with 0-10 mmol/l acrolein for 2 h and measured PON-1 activity and structural changes. Acrolein was also measured in 40 end stage renal disease (ESRD) patients (before and after a hemodialysis session), and 40 control subjects., Results: We found that acrolein inhibits PON-1 activity in HDL in a time and concentration dependent fashion. Inhibition occurred at 40% at 0.5 mmol/l and was cancelled by cysteine but not by aminoguanidine or carnosine. We confirm that free serum acrolein levels are higher in chronic renal failure patients and demonstrate that they are partially removed by HD. Decrease in acrolein levels after dialysis correlate with increases in PON-1 activity (r=0.32, p 0.01)., Conclusion: Acrolein inactivates paraoxonase 1 in HDL, a process that is inhibited by N-acetylcysteine. We confirm that acrolein levels are higher in ESRD and show for the first time, data supporting that acrolein is partially removed by hemodialysis. Decrease in acrolein levels after dialysis correlates with increase in PON-1 activity. This could offer new insights to explain low PON-1 activities in smokers and renal failure subjects as well as pointing at thiol-conserving reducing compounds such as N-acetylcysteine, as putative therapeutic palliatives.

The levels of polyamines (putrescine, spermidine and spermine) and polyamine oxidase in plasma of patients with chronic renal failure were determined. The level of putrescine was increased but the level of spermine was decreased in the plasma of these patients. The patients also had increased plasma polyamine oxidase activity leading to increased degradation of spermine. As acrolein was a major toxic compound produced from spermine by polyamine oxidase, the levels of free and protein-conjugated acrolein in plasma were also measured. Acrolein levels were enhanced in plasma of patients with chronic renal failure. The accumulated acrolein found as protein conjugates was equivalent to 170 microM, which was about 5-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by spermine oxidase in plasma. An increase in putrescine, spermine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. After patients with chronic renal failure had undergone hemodialysis, their levels of plasma polyamines, spermine oxidase and acrolein returned towards normal. It is likely that acrolein produced from spermine accumulates in the blood due to decreased excretion into urine and may function as a uremic "toxin".

Background and Purpose: We found previously that plasma levels of acrolein (CH2=CHCHO) and spermine oxidase (SMO) were well correlated with the degree of severity of chronic renal failure. The aim of this study was to test whether the levels of these 2 markers and of acetylpolyamine oxidase (AcPAO) were increased in the plasma of stroke patients., Methods: The activity of AcPAO and SMO and the level of protein-conjugated acrolein in plasma of the stroke patients and normal subjects were measured by high-performance liquid chromatography and ELISA, respectively. Focal infarcts were estimated by MRI or computed tomography (CT)., Results: The levels of AcPAO, SMO, and acrolein were significantly increased in the plasma of stroke patients. The size of stroke was nearly parallel with the multiplied value of acrolein and total polyamine oxidase (AcPAO plus SMO). After the onset of stroke, an increase in AcPAO first occurred, followed by increased levels of SMO and finally acrolein. In 1 case, an increase in AcPAO and SMO preceded focal damage as detected by MRI or CT. Furthermore, stroke was confirmed by MRI in a number of mildly symptomatic patients (11 cases) who had increased levels of total polyamine oxidase and acrolein. Among apparently normal subjects (8 cases) who had high values of acroleinxtotal polyamine oxidase, stroke was found in 4 cases by MRI., Conclusions: The results indicate that increased levels of AcPAO, SMO, and acrolein are good markers of stroke.

Cyclophosphamide (CY) is an alkylating agent used for the treatment of various types of cancer and is also used as a potent immunosuppressant. Acrolein, a metabolite of CY is cytotoxic and has the ability to covalently bind with proteins in vitro to form acrolein-protein adducts. These protein adducts are considered to be putative markers of oxidative stress and cause damage to protein in aging, atherosclerosis and diabetes. We have, for the first time, detected acrolein-lysine adducts in plasma low-density lipoprotein (LDL) and in the aorta of CY-treated animals by agarose gel electrophoresis, immunoblot and immunohistochemical methods. The extent of lipid peroxidation caused by the metabolite acrolein in plasma LDL was also measured quantitatively by using high-performance liquid chromatography. These results confirm the role of acrolein-lysine adducts in the development of atherosclerosis or atherogenesis.

Acrolein, an alpha,beta-unsaturated aldehyde, is a ubiquitous environmental toxic pollutant. Because of potential human exposure, there is a need for a sensitive, reliable, and specific method to monitor acrolein exposure. Acrolein is a potent electrophile and reacts with proteins mainly through Michael addition reaction, leading to acrolein-protein adducts (APA). The present study aimed to develop a competitive enzyme-linked immunosorbent assay (ELISA) method for the quantitation of APA in biological samples. Antibody to acrolein-keyhole limpet hemocyanin adduct was raised in rabbits, and the specificity of the antibody was determined by ELISA using acrolein-albumin adduct (AAA) or native albumin. A dose-dependent response was observed with AAA, but no immunoreactivity with native albumin. Further, lack of cross-reactivity of anti-acrolein antibody with formaldehyde-, malondialdehyde-, or 4-hydroxynonenal-albumin adducts indicates its specificity for acrolein. For the competitive ELISA, 1:16,000 diluted antisera was used with varying concentrations of AAA, which provided a linear detection range between 250 and 10,000 pg. To test the efficacy of the method for possible use as a biomarker of acrolein exposure, SD rats were orally administered 1 or 7 doses of 9.2 mg/kg/d acrolein. APA levels, quantitated in the serum, showed significantly greater formation (32% and 58% after 1 and 7 doses, respectively) in acrolein-treated rats as compared to the controls. Western blot analyses of APA in the sera from acrolein-treated rats showed APA bands (especially 29, 31, and 100 kD) with greater intensity in comparison to controls, further supporting our ELISA results. These results suggest that quantitation of APA has potential to be used as biomarker of acrolein exposure and eventually for molecular dosimetry and risk assessment.

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.

Accumulating evidence indicates that protein modification by acrolein is one of the major hallmarks of atherosclerosis. The purpose of the present study was to evaluate the serum acrolein-modified protein adduct (Acr) level in end-stage renal disease (ESRD), and to elucidate the efficacy of vitamin E-bonded hemodialyzer in reducing Acr in a crossover trial. A significant increase in Acr was found in ESRD patients compared with healthy controls (p <.001). In ESRD, the Acr level of those patients with type 2 diabetes mellitus (DM) was significantly higher compared with the non-DM group (p <.05). Forty-one ESRD patients who exhibited Acr levels higher than the mean value in ESRD were treated by vitamin E-bonded hemodialyzer for 6 months. After 6 months of treatment, Acr levels were decreased to those found in healthy individuals (p <.001). When hemodialyzers were switched back from vitamin E bonded to the original regular ones, Acr levels increased to nearly their initial levels after 3 months (p <.001), compared with the 6 month time point. These results suggest the potential of Acr as an oxidative stress marker in ESRD, and that vitamin E-bonded hemodialyzer treatment is a reasonable approach to reduce oxidative stress in ESRD.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

A rapid, sensitive and specific high-performance liquid chromatographic method for the quantification of acrolein (1), one of the toxic metabolites of oxazaphosphorine alkylating agents (cyclophosphamide and ifosfamide) was developed. Condensation of acrolein with Luminarin 3 afforded a fluorescent derivative that could be specifically detected and quantified. Chromatographic conditions involved a C18 RP column Uptisphere and a gradient elution system to optimize resolution and time analysis. The method showed high sensitivity with a limit of detection of 100 pmol/ml and a limit of quantification of 300 pmol/ml. This technique is particularly suitable for pharmacokinetic studies on plasma of oxazaphosphorine-receiving patients.

Protein adducts are used as markers of chemical exposure. Determination of the clearance rate of these adducts from the blood circulation will provide the time frame for their measurement. Radioactive albumin was prepared biosynthetically by repeated intraperitoneal injections of L-[4,5-3H]lysine to a rat. After an affinity purification, an aliquot of this native [3H-lysine]albumin was adducted with 5 mM acrolein. Both the native albumin (A-treated group) and the albumin-acrolein adduct (AAA-treated group) were intravenously injected to separate groups of rats, and the clearance of radioactivity from the plasma was measured as a function of time. At the end of the experiment (33 h after the injection), radioactivity in the whole plasma, and in homogenates of liver, kidney and spleen and their trichloroacetic acid(TCA)-soluble and -insoluble fractions in both A- and AAA-treated groups, was measured. The results, at the initial 11 h after the injection, showed that the radioactivity was cleared from the circulating plasma more rapidly in the AAA-treated group (32% of the injected radioactivity remained) than the A-treated group (52%). At 33 h after the injection, 22% of the injected radioactivity remained in the plasma in the AAA-treated group as compared to 32% in the A-treated group. The whole homogenates of liver and kidney and their corresponding TCA-soluble fractions showed higher radioactivity in the AAA-treated group as compared to the A-treated group. However, the TCA-insoluble fractions from livers and kidneys of the AAA-treated group showed lower radioactivity as compared to the A-treated group. These results indicated that the albumin-acrolein adduct was removed more rapidly from the circulation than the native albumin, and degraded more rapidly by the liver and kidney. There was no preferential removal or degradation of the adducted albumin by the spleen.

The toxicokinetic profile of cinnamaldehyde (CNMA) was investigated in Fischer 344 rats. CNMA was found to be unstable in blood. After iv administration, a large fraction of CNMA was immediately oxidized to cinnamic acid. The biological half-life of CNMA after iv administration was found to be 1.7 hr. After administration by gavage of CNMA at 250 or 500 mg/kg body weight using corn oil as vehicle, the maximum blood concentrations of CNMA were in the order of 1 microgram/ml. These low blood concentrations were maintained over a 24-hr period after a dose of 500 mg/kg, which is relatively long considering the short (1.7 hr) biological half-life of CNMA. The estimated oral bioavailability of CNMA was less than 20% for both the 250 and 500 mg/kg doses. No CNMA was present in blood at any time in rats dosed with 50 mg CNMA/kg body weight. Only a small amount of the administered CNMA was excreted in rat urine as free cinnamic acid or beta-glucuronide-conjugated cinnamic acid. The majority of CNMA administered orally was excreted in urine as hippuric acid within 24 hr. The maximum excretion rate occurred at 8 hr after gavage. Hippuric acid recovered in 50-hr urine samples was found to be directly proportional to the oral dose of CNMA.

A rapid and sensitive high performance liquid chromatographic (HPLC) method is described for the quantitation of cinnamaldehyde (CNMA) in rat blood at concentrations of 0.1-100 micrograms/mL. One of the metabolites of CNMA, cinnamic acid, can also be quantified simultaneously. CNMA is unstable in rat blood, probably because of rapid oxidation to cinnamic acid by enzymatic catalysis and nonenzymatic Schiff base formation with free amine groups of blood proteins. The disappearance of CNMA from rat blood follows first-order reaction kinetics with a half-life of 9 min at room temperature. The current analysis method involves the addition of an agent that will prevent CNMA degradation by denaturing protein and competitively blocking nucleophilic addition reactions, resulting in the nearly complete recovery of CNMA from blood. Recovery of cinnamic acid was approximately 80% at concentrations of 1-10 micrograms/mL.

The subacute inhalation toxicity of acrolein was examined in 4 groups of 20 hamsters, 12 rats and 4 rabbits each, exposed repeatedly to acrolein vapour at concentrations of 0, 0.4, 1.4 and 4.9 ppm (6 h/day, 5 days/week) for a 13-week period. The most important effects found at the highest level included mortality in rats, ocular and nasal irritation, growth depression and histopathological changes of the respiratory tract in each of the animal species exposed. The aberrations in the airways consisted of destruction and hyper- and metaplasia of the lining epithelium accompanied by inflammatory alterations. Rats appeared to be the most susceptible of the species examined and showed treatment-related abnormalities even at 0.4 ppm, whereas this exposure level was found to be a no-toxic effect level in both hamsters and rabbits.

The effect of methoxyflurane anesthesia on allyl alcohol-induced hepatotoxicity and the metabolism of allyl alcohol was studied in male rats. Hepatotoxicity was assessed by the measurement of serum alanine aminotransferase activity and histopathological examination. Allyl alcohol-induced hepatotoxicity was enhanced when allyl alcohol (32 mg/kg) was administered 4 hr before or up to 8 days after a single 10-min exposure to methoxyflurane vapors. The possibility that methoxyflurane increases alcohol dehydrogenase-dependent oxidation of allyl alcohol to acrolein, the proposed toxic metabolite, was evaluated by measuring the rate of acrolein formation in the presence of allyl alcohol and liver cytosol. The effect of methoxyflurane on alcohol dehydrogenase activity in liver cytosol was also assessed by measuring the rate of NAD+ utilization in the presence of ethyl alcohol or allyl alcohol. Alcohol dehydrogenase activity and rate of acrolein formation were elevated in methoxyflurane-pretreated rats. The results suggest that a modest increase in alcohol dehydrogenase activity and rate of acrolein formation markedly enhances allyl alcohol-induced hepatotoxicity.