A semi-quantitative method for analysis of HNE-MA and ONE-MA metabolites in the urine of oxidatively stressed rats was previously reported [20
]. While this method allowed for the simultaneous analysis of multiple LPO-MA conjugates, the data was not quantitative. Appropriate internal standards for each of our analytes of interest are necessary in order to perform absolute quantitation. We first synthesized MAd3
following the method of Slatter et al. [25
conjugates of HNE, DHN, HNA, ONE, ONO, and ONA, were then prepared as described by Kuiper et al. [20
]. Quantitation of endogenous LPO-MA conjugates was subsequently achieved by isotope-dilution LC-MS/MS using SRM. We now demonstrate, for the first time, the quantitative determination of ONO-MA in addition to HNE-MA and DHN-MA in vivo
at low mg/g creatinine levels.
In smokers and nonsmokers, we found the urinary levels of ONO-MA to be in the range 0.05-2.26 mg/g creatinine (1.7-177 nM). HNE-MA was present in the range of 0.17-12.19 mg/g creatinine (7.4-225 nM) and DHN-MA at levels of 0.22-17.90 mg/g creatinine (6.6-316 nM). Low LPO-MA conjugate levels in a subgroup of the smokers resulted in the lack of statistical difference between the smoker and nonsmoker groups prior to smoking cessation. Alary et al. [22
] also assessed DHN-MA in humans and found production of 5 μg/24 h in seven healthy human volunteers, which corresponds to 2.7 ng/ml (8.4 nM). These levels are comparable to the low levels of DHN-MA in our study. In a study of the urinary excretion of LPO-MA conjugates in rats, Mally et al. [26
] measured 113.8 ± 36.8 pmol/mg creatinine for HNE-MA (36 μg/g creatinine) and 1.19 ± 0.33 nmol/mg creatinine for DHN-MA (382 μg/g creatinine). Rathahao et al. [21
] and Guéraud et al. [23
] found urinary production of DHN-MA in rats to be in the range of 45-230 ng/24 h (equivalent to 8.8-45 nM, assuming a urine production of 16 ml/24 h), with the higher concentrations appearing in BrCCl3
stressed animals. Alary et al. [22
] also analyzed rat urine and found DHN-MA production of 10 ng/24 h or 0.8 ng/ml (2.5 nM). Urinary levels of DHN-MA in lean and Zucker obese rats (1.1 μM and 2.9 μM, resp.), reported by Orioli et al. [24
], however, are much higher than the other reported values. With the exception of the levels reported by Orioli et al. [24
], urinary levels of HNE-MA and DHN-MA in rats seem to be lower than or at the low end of the range of human levels determined in Alary’s study [22
] and in our present study. Since the urinary levels of LPO-MA conjugates in rats tend to fall within the same range as the levels we found in human samples, our quantitation method should be applicable to animal investigations as well.
Like HNE and ONE, F2α
-isoprostanes are formed from lipid hydroperoxides via radical-mediated pathways. F2α
-isoprostanes are generally considered to be the most reliable markers of in vivo
oxidative stress [30
]. Smoking cessation has been shown to result in significant decreases of urinary F2α
-isoprostane levels after one or two weeks [32
]. The study conducted by Chehne et al. [33
] demonstrated that the decrease of urinary F2α
-isoprostane levels upon smoking cessation was similar between patients having clinically manifested atherosclerosis with or without hypercholesterolemia and/or hypertension, indicating that cigarette smoke is a major contributor to in vivo
oxidative stress compared to other risk factors of atherosclerosis. Similar to urinary F2α
-isoprostane levels, our study of apparently healthy participants showed significant decreases in the levels of MA conjugates of HNE and DHN in the urine upon smoking cessation, reflecting a similar pathway of formation via lipid hydroperoxides.
The LPO metabolites of our study differ from the F2α
-isoprostanes in that they are also products of phase I and phase II metabolism. Thus, the levels of MA conjugates of HNE, DHN, and ONO reflect both formation of HNE and ONE and their subsequent metabolism. Expression levels of GSTs may therefore co-determine urinary levels of HNE-MA, DHN-MA, and ONO-MA. GSTP1, a GST isoenzyme involved in HNE conjugation [34
], was induced in lung tissue of smokers whereas other GSTs, GSTA2 and GSTM1, showed no difference in expression levels between smokers and nonsmokers [36
]. On the other hand, genetic polymorphism of GST may affect gene expression if the mutation is located in the promoter region. Qian et al. [37
] studied single nucleotide polymorphisms (SNPs) of GSTA4, another GST that accepts HNE as a substrate [38
]. Qian et al. [37
] found that the presence of genotypes TA and AA at locus-1718 of GSTA4 was associated with a 37 % significantly decreased risk of lung cancer compared to the TT genotype. The authors suggested that the TA and AA genotypes, with the SNP in the promoter region, may have increased GSTA4 expression and thus greater capacity to detoxify HNE as compared to the TT genotype. Dwivedi et al. [39
] determined that GSTA4 null mice have higher levels of hepatic HNE after CCl4
treatment than wild-type mice, indicating reduced HNE conjugation in GSTA4 null mice. In the study by Qian et al. [40
], the TT genotype had a prevalence of 77 % in lung cancer patients (n
= 500) and 68 % in cancer-free control subjects (n
= 517). The common occurrence of the TT genotype, presumably having reduced GSTA4 expression and reduced capacity to conjugate HNE, may explain the low urinary levels of LPO-MA (< 7 mg/g creatinine) we found in 9 out of 23 smokers.
The low LPO-MA excretion in nine smokers may also be due to smoking-induced phase I metabolism, resulting in enhanced conversion of HNE and ONE into DHN which is not a GST substrate. Aldo-keto reductase 1B10 (AKR1B10) is known to reduce HNE to DHN and to reduce ONE to ONO [41
]. Its up-regulation in smokers, shown by Fukumoto et al. [42
] and Nagaraj et al. [43
], would direct the metabolism of HNE to DHN, resulting in decreased formation of GST-mediated metabolites and MA conjugates ().
We developed a method for the accurate quantitation of ONO-MA, HNE-MA, and DHN-MA in human urine by isotope-dilution LC-MS/MS. We also detected HNA-MA, HNAL-MA, and ONA-MA. The significance of the in vivo detection of ONO-MA and ONA-MA is that these conjugates represent HNE/ONE branching in the breakdown of lipid hydroperoxides as shown in , suggesting that ONO may contribute to the deleterious effects previously ascribed to HNE. Our findings also show that LPO-MA conjugates are elevated in urine obtained from smokers and decrease significantly following smoking cessation, demonstrating the utility of these metabolites as markers of in vivo oxidative stress.