Simultaneously quantifying hundreds of acylcarnitines in multiple biological matrices within ten minutes using ultrahigh-performance liquid-chromatography and tandem mass spectrometry

Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances. Here, we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). This enabled simultaneous quantification of 1136 acylcarnitines (C0–C26) within 10-min with good sensitivity (limit of detection < 0.7 fmol), linearity (correlation coefficient > 0.992), accuracy (relative error < 20%), precision (coefficient of variation (CV), CV < 15%), stability (CV < 15%), and inter-technician consistency (CV < 20%, n = 6). We also established a quantitative structure-retention relationship (goodness of fit > 0.998) for predicting retention time (tR) of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters (tR, ion-pairs, and collision energy). Furthermore, we quantified 514 acylcarnitines in human plasma and urine, mouse kidney, liver, heart, lung, and muscle. This provides a rapid method for quantifying acylcarnitines in multiple biological matrices.

Predictive value of presepsin and acylcarnitines for severity and biliary drainage in acute cholangitis

BACKGROUND Bacteremia,which is a major cause of mortality in patients with acute cholangitis,induces hyperactive immune response and mitochondrial dysfunction.Presepsin is responsible for pathogen recognition by innate immunity.Acylcarnitines are established mitochondrial biomarkers.AIM To clarify the early predictive value of presepsin and acylcarnitines as biomarkers of severity of acute cholangitis and the need for biliary drainage.METHODS Of 280 patients with acute cholangitis were included and the severity was stratified according to the Tokyo Guidelines 2018.Blood presepsin and plasma acylcarnitines were tested at enrollment by chemiluminescent enzyme immunoassay and ultra-high-performance liquid chromatography-mass spectrometry,respectively.RESULTS The concentrations of presepsin,procalcitonin,short-and medium-chain acylcarnitines increased,while long-chain acylcarnitines decreased with the severity of acute cholangitis.The areas under the receiver operating characteristic curves(AUC)of presepsin for diagnosing moderate/severe and severe cholangitis(0.823 and 0.801,respectively)were greater than those of conventional markers.The combination of presepsin,direct bilirubin,alanine aminotransferase,temperature,and butyryl-L-carnitine showed good predictive ability for biliary drainage(AUC:0.723).Presepsin,procalcitonin,acetyl-L-carnitine,hydroxydodecenoyl-Lcarnitine,and temperature were independent predictors of bloodstream infection.After adjusting for severity classification,acetyl-L-carnitine was the only acylcarnitine independently associated with 28-d mortality(hazard ratio 14.396;P<0.001)(AUC:0.880).Presepsin concentration showed positive correlation with direct bilirubin or acetyl-L-carnitine.CONCLUSION Presepsin could serve as a specific biomarker to predict the severity of acute cholangitis and need for biliary drainage.Acetyl-L-carnitine is a potential prognostic factor for patients with acute cholangitis.Innate immune response was associated with mitochondrial metabolic dysfunction in acute cholangitis.

Acylcarnitine: Useful biomarker for early diagnosis of hepatocellular carcinoma in non-steatohepatitis patients

BACKGROUND Early diagnosis of hepatocellular carcinoma(HCC)is necessary to improve the prognosis of patients.However,the currently available tumor biomarkers are insufficient for the early detection of HCC.Acylcarnitine is essential in fatty acid metabolic pathways.A recent study reported that a high level of acylcarnitine may serve as a useful biomarker for the early diagnosis of HCC in steatohepatitis(SH)patients.In contrast,another study reported that the level of acetylcarnitine(AC2)-one of the acylcarnitine species-in non-SH patients with HCC was decreased vs that reported in those without HCC.AIM To investigate the usefulness of acylcarnitine as a biomarker for the early diagnosis of HCC in non-SH patients.METHODS Thirty-three non-SH patients(14 with HCC and 19 without HCC)were enrolled in this study.Blood samples were obtained from patients at the time of admission.The levels of acylcarnitine and AC2 in the serum were determined through tandem mass spectrometry.The levels of vascular endothelial growth factor(VEGF)and VEGF receptor 2(VEGFR-2)were determined by enzymelinked immunosorbent assay.Univariate and multivariate analyses were used to determine early diagnostic factors of HCC.RESULTS The level of acylcarnitine was significantly lower in non-SH patients with HCC vs those without HCC(P<0.05).In contrast,the level of lens culinaris agglutininreactive fraction ofα-fetoprotein(AFP)-AFP-L3%-was significantly higher in non-SH patients with HCC vs those without HCC(P<0.05).However,the levels of total carnitine,free carnitine,AFP,des-γ-carboxy prothrombin,VEGF,and VEGFR-2 were not different between patients with and without HCC.The multivariate analysis showed that a low level of acylcarnitine was the only independent factor for the early diagnosis of HCC.The patients with a low level of AC2 had a significantly higher level of VEGF vs those with a high level of AC2(P<0.05).CONCLUSION The metabolic pathways of fatty acids may differ between SH HCC and non-SH HCC.Further studies are warranted to investigate these differences.

Association of altered serum acylcarnitine levels in early pregnancy and risk of gestational diabetes mellitus

Gestational diabetes mellitus(GDM)is a high-prevalence disease and diagnosed in middle pregnancy.Acylcarnitines are a series of fatty acid esters of carnitine and play important roles in fatty acid and carbohydrate metabolism.However,the role of acylcarnitine on the development of GDM remains unclear.This case-control study involving 214 study participants(107 GDM cases and 107 matched controls)was conducted in a cohort,in China,from 2013 to 2015.The levels of carnitine and 36 acylcarnitines in serum samples collected at the early stage of pregnancy were determined by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry.The associations of the levels of the 37 targeted compounds with GDM risk were investigated by using binary conditional logistic regression models.Alterations in acylcarnitine levels were observed 9–17 weeks before GDM diagnosis.The increases in levels of propionyl-carnitine,malonyl-carnitine,isovaleryl-carnitine,palmitoyl-carnitine and linoleoyl-carnitine were associated with GDM risk with odds ratios(ORs)per standard deviation(SD)increment greater than 1(p<0.05),after adjustment for potential confounding factors(pre-pregnancy body mass index and parity).On the contrary,the increases of decanoyl-carnitine,decenoyl-carnitine,tetradecenoyl-carnitine,tetradecandienoylcarnitine levels were associated with the reduced risk for GDM(ORs per SD<1,p<0.05).To our knowledge,the present study is the largest case-control study to investigate the association between early-pregnancy acylcarnitine levels in serum and GDM risk.The findings add to the evidence for the association between acylcarnitine levels and GDM risk.

Metabolic and biological changes in children with obesity and diabetes

The World Health Organization has stated that obesity in childhood is one of themost serious public health challenges of the 21st century. Overweightness andobesity in early childhood lead to a higher risk of overweightness and obesity inadulthood, thus conferring an increased risk of chronic inflammatory conditions,including type 2 diabetes mellitus, cardiovascular diseases, non-alcoholic fattyliver disease, and some cancers. Therefore, metabolome analysis, targeted atscreening and intervening in childhood obesity, is very important. Recent studieshave indicated that amino acid and lipid metabolism could influence metabolicpathways in children with obesity. For this review, we searched clinical dataaddressing metabolomic profiles and insulin resistance (IR) in children withobesity from inception to February 2021 in Medline, Web of Science, and Scopus.According to our search, branched-chain amino acids (BCAAs), aromatic aminoacids, and acylcarnitines have reportedly been associated with IR as biomarkersfor diabetes in children. BCAAs, tyrosine, and phenylalanine could be predictorsof the future development of diabetes in nondiabetic subjects. In addition, it iswell known that insulin regulates BCAA metabolism, and BCAA is a biomarkerfor IR. To interpret the mechanism behind metabolic changes in obesity, it is veryimportant to understand the pathways and combinations related with amino acid,lipid and glucose metabolism. In this review, we summarize studies on metabolicchanges to understand metabolomics in children with obesity.