title: First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.
abstract: BACKGROUND
  Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic β-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP.
  
  
  METHODS
  Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS.
  
  
  RESULTS
  Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01).
  
  
  CONCLUSIONS
  The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes.
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan; Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji 133002, Jilin Province, China. Electronic address: junzhe@u-shizuoka-ken.ac.jp.
authors: Jun Zhe
authors: JZ
authors: Min
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors: Yuki
authors: Y
authors: Tomiyasu
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors: Takashi
authors: T
authors: Morotomi
authors: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji 133002, Jilin Province, China.
authors: Ying-Zi
authors: YZ
authors: Jiang
authors: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji 133002, Jilin Province, China.
authors: Gao
authors: G
authors: Li
authors: Western Hospital Pharmacy, Affiliated Hospital of Yanbian University Yanji, 133000, Jilin, Province, China.
authors: Qing
authors: Q
authors: Shi
authors: Fengxian Branch of Shanghai Sixth People's Hospital, Shanghai 201400, China.
authors: Hai-Fu
authors: HF
authors: Yu
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors: Koichi
authors: K
authors: Inoue
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors: Kenichiro
authors: K
authors: Todoroki
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Electronic address: toyooka@u-shizuoka-ken.ac.jp.
authors: Toshimasa
authors: T
authors: Toyo'oka
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006197:Hair / Q000737:chemistry*; D064368:Healthy Volunteers; D006801:Humans; D007532:Isoleucine / Q000032:analysis*; D007930:Leucine / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization; D055815:Young Adult
chemical_list: D007532:Isoleucine; D007930:Leucine
keywords: Diabetes; Human hair; N-Acetyl isoleucine; N-Acetyl leucine; UPLC–ESI–MS/MS