First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC–ESI–MS/MS
Introduction
Type 2 diabetes is the most common form of diabetes, and its prevalence is dramatically increasing in both developed and developing countries [1], [2]. As an endocrine metabolic disease, diabetes would lead to the metabolic disorder of several kinds of endogenetic metabolites [3]. Therefore, early identification of individuals at risk is particularly important for delaying or preventing the onset of type 2 diabetes and decreasing the burden of the condition worldwide [4], [5].
Recent studies have shown that the amino acid metabolism plays a potential key role in the pathogenesis of diabetes, and amino acid profiling is very helpful in diabetic risk assessment [3], [6]. Higher concentrations of branched chain amino acids (Leu, Ile, and Val) and aromatic amino acids (Trp, Tyr and Phe) have emerged as predictors of the future development of diabetes [7], [8], [9], [10]. In previous studies, we recently identified the N-acetyl leucine (Ac-Leu) metabolite to be a potential biomarker candidate related to diabetes. Metabolic signature of N-acetyl leucine (Ac-Leu) strongly predicts diabetes development in the mice hair [11]. These results proved that amino acids play essential roles in the energy metabolism as a cluster metabolite.
Urine and serum samples have been extensively investigated for the amino acid assay in biological specimens [3], [8], [12], [13]. However, the inherent problems of serum and urine specimens, such as the fluctuation in its composition during the day and the hygienic practice during its collection and handling, prompted us to search for other types of noninvasive samples. Recently, the value of hair as a noninvasive biosample has been recognized in the biomedical field [14], [15]. In contrast, the human hair is relatively clean and the sampling is easy and noninvasively collected and easily stored. Hair analysis provides one important means for determining the individual past history of long-term chemical exposures, because many substances have been detected in the hair [16], [17], [18], [19], [20], [21]. Many studies concerning hair analysis have dealt with drugs of abuse, such as heroin, cocaine and amphetamines [22]. In recent years, however, interest in hair analysis has gradually shifted to other drug species, e.g., doping agents and therapeutic drugs [23], [24]. Hair is a biological sample that is not considered to be important in the field of clinical testing, except in limited cases such as the detection of drug abuse [25], [26], [27]. Furthermore, hair samples contain long-term histories of diseases and medicines used for treatment as compared to other biological samples; it is a notable biological sample in that it compensates for the limitations of blood or urine samples. However, for analyzing hair samples, an appropriate extraction method is required for sample preparation and the method is usually time-consuming. Also, the concentrations of the target compounds obtained from the hair extracts are expected to be lower compared to those obtained from blood or urine samples; therefore, highly sensitive detection methods are required for hair analyses. According to recent reports, human hair may be used to obtain physiologic information, and may serve as the noninvasive biosample for the diagnosis of chronic disease. Basic compounds are efficiently incorporated into hair. Certain kinds of endogenous biogenic amino acids have been detected in human hair [28], [29], [30]. However, a method for the determination of Ac-Leu and Ac-Ile in human hair has not been reported.
Section snippets
Materials and reagents
The Ac-Leu and Ac-Ile were from Watanabe Chemical Industries, Co. Ac-Nle (Watanabe Chemical Industries) was used as the internal standard (IS). Formic acid (FA), hydrochloric acid (HCl), trifluoroacetic acid (TFA), sodium dodecylsulfate (SDS), methanol (CH3OH), and acetonitrile (CH3CN) were of special reagent grade (Wako Pure Chemicals). All other chemicals were of analytical reagent grade and were used without further purification. Deionized water and distilled water (H2O) were used throughout
Optimization of separation and detection conditions
The Ac-Ile, Ac-Leu and Ac-Nle are hydrophilic and strong basic compounds (Fig. 1). Therefore, the simultaneous separation of acetyl-amino acids by reversed-phase chromatography using an ODS column is very difficult due to adsorption on the resins. For the separation, an anti-pressurized column packed with small porous resins, Acquity UPLC BEH C18 (100 × 2.1 mm i.d., 1.7 μm), was used for the rapid separation of Ac-Ile, Ac-Leu and Ac-Nle by UPLC. For the detection of Ac-Ile, Ac-Leu and Ac-Nle, the
Conclusion
We developed a novel, sensitive, and simple LC–MS/MS method for the simultaneous determination of Ac-Ile and Ac-Leu in human hair. It takes around 12 h to extract the components from a hair sample using the traditional protocol. In this study, micropulverized extraction methods were used and the samples were efficiently extracted in 30 min. This study was useful for detecting the Ac-Ile and Ac-Leu from the hairs of DP and HV. This determination of Ac-Ile and Ac-Leu in the human hair by
Acknowledgments
The present research was supported by a Grant-in-Aid for Young Scientists (A) (KAKENHI, No. 26713021), and Challenging Exploratory Research (KAKENHI, No. 25560058) from the Japan Society for the Promotion of Science, and the National Natural Science Foundation of China (81360487).
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