Determination of HEL (Hexanoyl-lysine adduct): a novel biomarker for omega-6 PUFA oxidation.

Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies.

Oxidative stress as a biomarker of respiratory disturbance in patients with severe motor and intellectual disabilities.

Oxidative stress plays an important role in aging and various diseases such as cancer, cardiovascular diseases, diabetes mellitus and bronchial asthma. However, little is known about a potential role of oxidative stress in the pathogenesis of severe motor and intellectual disabilities (SMID) in terms of respiratory disturbance, which is the most common complication. In the present study, we examined the urinary levels of oxidative stress markers, 8-hydroxy-2'-deoxyguanosine (8-OHdG), hexanoyl-lysine adduct (HEL) and acrolein-lysine adduct (ACR) in patients with SMID. The mean level of urinary 8-OHdG in SMID patients was significantly higher than that in normal controls (18.8 +/- 9.0 ng/mg Cre and 10.5 +/- 2.9 ng/mg Cre, respectively) (p < 0.01). There was no significant difference of the mean level of urinary HEL between patients with SMID and normal controls (81.9 +/- 40.3 pmol/mg Cre and 69.2 + /-37.7 pmol/mg Cre, respectively), while the mean level of ACR in patients with SMID was higher than that of normal controls (220.5 +/- 118.6 nmol/mg Cre and 144.9 +/- 62.0 nmol/mg Cre, respectively) (p < 0.05). In addition, the level of 8-OHdG was strongly correlated with the severity of respiratory disturbance evaluated as the respiratory disturbance score (RDS) (Spearman r = 0.73, n = 14, p < 0.01). In contrast, there was no correlation between the levels of these oxidative stress markers and age or medication of antiepileptic drugs. These results suggest that urinary 8-OHdG is a potentially useful biomarker for evaluating the severity of respiratory failure in patients with SMID.

Functional Cyp2e1 is required for substantial in vivo formation of 2,5-hexanedione from n-hexane in the mouse.

Neurotoxicity of n-hexane is mediated by its metabolite 2,5-hexanedione (2,5-HD). Cytochrome P4502E1 (CYP2E1) has been suggested but not shown to be involved in the formation of the metabolite. An objective of the current study was to assess the essentiality of CYP2E1 for in vivo 2,5-HD formation from n-hexane. This was accomplished by comparing urinary levels of the gamma-diketone in n-hexane-treated mice in which the Cyp2e1 gene has been deleted (Cyp2e1-/-) with that in n-hexane-treated wild-type (Cyp2e1+/+) mice. 2,5-HD was detectable not as the free compound but as further metabolites, at levels that were comparable in both strains of mice, following a daily 200 mg/kg i.p. dose of the alkane for 10 days. Continued daily n-hexane treatment resulted in increased urinary levels of 2,5-HD metabolites in Cyp2e1+/+ but not in Cyp2e1-/- mice. Only in Cyp2e1+/+ mice and only on day 21 of n-hexane treatment was a trace level of unchanged 2,5-HD detected. 3-Hexanol was the only other n-hexane metabolite detected in the mice but its concentration was higher in Cyp2e1-/- than in Cyp2e1+/+ mice. In n-hexane-treated rats, in contrast to mice, multiple metabolites of the alkane, including unchanged 2,5-HD, were detected. The results indicate that substantial in vivo formation of 2,5-HD from n-hexane in the mouse requires CYP2E1, and suggest that further detoxification of the metabolite may be very efficient in this species.

Determination of free and glucuronated hexane metabolites without prior hydrolysis by liquid- and gas-chromatography coupled with mass spectrometry.

Since n-hexane metabolites are excreted as glucuronide conjugates, most conventional analytical procedures require preliminary hydrolysis, yielding to the 'total' 2,5-hexanedione (2,5-HD), but also giving rise to a number of artifacts. The whole pattern of n-hexane metabolites, both conjugated and unconjugated, as well as different methods of sample pretreatment have been evaluated by hyphenated techniques (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS)). Aliquots of urine from rats exposed to n-hexane underwent enzymatic or acid hydrolysis or both; whereas one aliquot was applied to LC-MS, dichloromethane extracts were analyzed by GC-MS. In untreated urine, four glucuronides (-G) were identified and characterized by LC-MS: 2-hexanol-G, 5-hydroxy-2-hexanone-G, 4,5-dyhydroxy-2-hexanone-G, and 2,5-hexanediol-G. 'Free' 2,5-HD was detectable in non-hydrolyzed samples by both GC- and LC-MS. Whereas enzymatic hydrolysis did not increase the amount of 2,5-HD, acid hydrolysis led to increase 2,5-HD in variable amount and produced gamma-valerolactone as a result of a complete transformation of 4,5-dihydroxy-2-hexanone-G and the partial conversion from 5-hydroxy-2-hexanone-G. Further experiments showed that both 5-hydroxy-2-hexanone-G and 4,5-dihydroxy-2-hexanone-G, isolated by solid-phase extraction and hydrolyzed, yield comparable amount of 2,5-HD and gamma-valerolactone. In samples treated by acid hydrolysis, GC-MS only does not allow to understand the true source of 'total' 2,5-HD, which may be produced not only from 4,5-dihydroxy-2-hexanone-G but also from the more abundant 5-hydroxy-2-hexanone-G, which thus represents the main source of analytical artifacts. 'Free' 2,5-HD seems to be both suitable from an analytical point of view and meaningful for biological monitoring purposes, provided that conjugate metabolites are rapidly removed from the body leading to a negligible neurotoxic risk.

Hydrolysis, absorption and metabolism of di(2-ethylhexyl) terephthalate in the rat.

1. The hydrolysis of di(2-ethylhexyl) terephthalate (DEHT) and di(2-ethylhexyl) phthalate (DEHP) were studied using rat gut homogenate fractions in vitro. Both isomers were hydrolysed by the intestinal fraction; however, DEHP was hydrolysed to 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) in about equal proportions, whereas DEHT was hydrolysed to 2-EH and terephthalic acid (TPA). The half-lives for disappearance of the diesters were determined to be 12.6 min for DEHP and 53.3 min for DEHT. 2. The absorption and metabolism of DEHT were studied by administering [hexyl-2-14C]DEHT (in corn oil) by oral gavage at a dose level of 100 mg/kg to 10 adult male Sprague-Dawley rats. Urine, faeces and expired air were collected for 144 h and analysed for the presence of radioactivity, and faeces and urine were analysed for unlabelled metabolites. 3. Radioactivity was eliminated in faeces (56.5 +/- 12.1% of dose) primarily as unchanged DEHT, small amounts of MEHT and polar metabolites; excreted in urine (31.9 +/- 10.9% of dose) principally as MEHT and metabolic products of 2-EH; and expired as 14CO2 (3.6 +/- 0.9% of dose). Less than 2% of the administered radioactivity was found in the carcass. Small amounts of 14C were found in the tissues with the highest amounts found in liver and fat. 4. Metabolites identified in urine included terephthalic acid (equivalent to 51% of dose), oxidized metabolites of 2-EH and MEHT, and glucuronic and sulphuric acid conjugates (equivalent to about 10% of dose).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers.

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.

Changes of n-hexane metabolites in urine of rats exposed to various concentrations of n-hexane and to its mixture with toluene or MEK.

It is well known that n-hexane produces peripheral neuropathy, and 2,5-hexanedione, one of the metabolites of n-hexane, is thought to be the main causative agent. Recently, the metabolites of n-hexane in urine have been measured by gas chromatography, and 2,5-hexanedione was proved to be useful for the biological monitoring of n-hexane exposure. In the present experiment, we intended to clarify the change of n-hexane metabolites in the urine of rats exposed to various concentrations of n-hexane and to its mixture with toluene of MEK. In the first experiment, five separate groups of five rats each were exposed to 100, 500, 1000, or 3000 ppm of n-hexane, or fresh air respectively in an exposure chamber for 8 h a day. Urinary samples were gathered during exposure, 16, 24, and 40 h after exposure. Half of each sample was analyzed by gas chromatography after hydrolysis with acid and enzymes, and the other half was analyzed without hydrolysis. 2,5-Dimethylfuran, MBK, 2-hexanol, 2,5-hexanedione, and gamma-valerolactone could be identified as n-hexane metabolites in the urine. The main metabolites were 2-hexanol and 2,5-hexanedione. 2-Hexanol was mostly excreted during exposure, while most of the 2,5-hexanedione was excreted after the end of exposure. The amount of metabolites in the urine correlatively increased with the concentration of n-hexane from 100 to 1000 ppm, but the amount of metabolites scarcely increased when the concentration of n-hexane increased from 1000 to 3000 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity and metabolism of methyl n-butyl ketone.

MEK (2-butanone) when combined with MBK (2-hexanone) markedly enhanced MBK neurotoxicity. MBK in rat plasma after exposure to MBK/MEK increased with time. Metabolites of MBK identified in blood and urine of rats and guinea pigs were 2-hexanol and 2,5-hexanedione.