Our previous study suggested that the Mono-ADP ribosylhydrolase 2 (MACROD2) rs6110695 A>G polymorphism is significantly associated with white blood cell (WBC) count in the Korean population. The present study aimed to evaluate the clinical relevance of the MACROD2 rs6110695 A>G polymorphism for predicting WBC count by utilizing plasma metabolites and a single-nucleotide polymorphism (SNP). Two groups were characterized by MACROD2 rs6110695 A>G SNP genotypes among 139 healthy subjects based on the genetic information provided in our previous work: rs6110695 AA genotype group (n = 129) and rs6110695 AG genotype group (n = 10). Plasma global metabolic profiling was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). To estimate the predictive abilities of WBC count models using the rs6110695 genotype and/or significant differential metabolites, multiple linear regression analysis and receiver operating characteristic (ROC) curve analysis were conducted. The AG genotype had greater WBC-to-apolipoprotein (apo) A-I ratios; counts of WBCs, lymphocytes, monocytes, and granulocytes; monocyte-to-lymphocyte ratio (MLR); and monocyte-to-platelet ratio (MPR) than the AA genotype. In terms of metabolic profile, indoleacetic acid, and butyrylcarnitine levels were considerably distinct between the two groups, and these metabolites were considered to be meaningful prognostic variables for the rs6110695 genotype. Finally, ROC curve analysis demonstrated that the model containing the rs6110695 genotype and the two main metabolites was reliable. The present study revealed that individuals carrying the rs6110695 AG genotype with high plasma indoleacrylic acid and butyrylcarnitine levels might have elevated WBC counts. The rs6110695 genotype and the concentrations of indoleacrylic acid and butyrylcarnitine could contribute to reducing the risk of chronic diseases in the future.

OBJECTIVE: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4).
METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children\'s Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types.
RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations.
CONCLUSIONS: ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.
目的: 探讨丁酰基肉碱（C4）代谢异常新生儿的基因型和生化表型特征。方法: 收集2018年1月至2023年6月在浙江大学医学院附属儿童医院经串联质谱法筛查单纯C4增高的120例新生儿初筛和召回复查的C4、C4/C3检测数据，并换算为C4增高倍数。采用液相捕获技术靶向捕获酰基辅酶A脱氢酶8（ACAD8）和短链酰基辅酶A脱氢酶（ACADS）基因的外显子及邻近50 bp区域，通过高通量测序和生物信息学分析获取基因变异信息，参考美国医学遗传学与基因组学学会分类标准进行致病性评估。采用威尔科克森秩和检验分析不同基因型新生儿C4增高倍数的差异。结果: 共检出32种ACAD8基因变异型，其中7种变异型未见报道；检出41种ACADS基因变异型，其中17种变异型未见报道。ACAD8双等位基因变异39例，ACAD8单等位基因变异3例，ACADS双等位基因变异34例，ACADS单等位基因变异36例，ACAD8和ACADS双基因变异5例。ACAD8双等位基因变异组、ACADS双等位基因变异组C4增高倍数初筛值和召回值均高于ACADS单等位基因变异组（均P<0.01），且ACAD8双等位基因变异组C4增高倍数初筛值和召回值较ACADS双等位基因变异组更高（均P<0.01）。所有携带ACAD8或ACADS双等位基因变异新生儿的初筛C4增高倍数均大于1.5；而仅有25%（9/36）携带ACADS单等位基因变异的新生儿初筛C4增高倍数大于1.5。结论: ACAD8和（或）ACADS基因变异是浙江地区新生儿C4增高的主要遗传学原因，其变异型具有高度异质性。双等位基因变异者C4水平高于单等位基因变异者。常规串联质谱法新生儿筛查结果为单纯的C4增高时，其“筛查切值”可以适当提升。.

Acylcarnitines are formed when an acyl group is transferred from coenzyme A to a molecule of L-carnitine. In organic acidemias, and in fatty acid oxidation disorders, specific acylcarnitine species accumulate in a pattern that is characteristic for each disease. For this reason, acylcarnitine analysis is widely used for screening and diagnosis of inherited disorders of metabolism. The most common method for acylcarnitine analysis uses flow injection tandem mass spectrometry. Flow injection analysis allows for high throughput, however, does not provide separation of isomeric and isobaric compounds. Among the acylcarnitine species which can be affected by the presence of isomeric/isobaric compounds, C4-carnitine and C5DC-carnitine are probably the ones encountered most often. The method presented here is performed on urine and utilizes butanolic HCL to derivatize acylcarnitines, ultra-performance liquid chromatography to resolve C4- and C5-DC isomers and isobars, and quantitation of these species using multiple-reaction monitoring (MRM).

Current standard of care imaging, cytology, or cystic fluid analysis cannot reliably differentiate malignant from benign pancreatic cystic neoplasms. This study sought to determine if the metabolic profile of cystic fluid could distinguish benign and malignant lesions, as well as mucinous and non-mucinous lesions.
Metabolic profiling by untargeted mass spectrometry and quantitative nuclear magnetic resonance was performed in 24 pancreatic cyst fluid from surgically resected samples with pathological diagnoses and clinicopathological correlation.
(Iso)-butyrylcarnitine distinguished malignant from benign pancreatic cysts, with a diagnostic accuracy of 89%. (Iso)-butyrylcarnitine was 28-fold more abundant in malignant cyst fluid compared with benign cyst fluid (P=.048). Furthermore, 5-oxoproline (P=.01) differentiated mucinous from non-mucinous cysts with a diagnostic accuracy of 90%, better than glucose (82% accuracy), a previously described metabolite that distinguishes mucinous from non-mucinous cysts. Combined analysis of glucose and 5-oxoproline did not improve the diagnostic accuracy. In comparison, standard of care cyst fluid carcinoembryonic antigen (CEA) and cytology had a diagnostic accuracy of 40% and 60% respectively for mucinous cysts. (Iso)-butyrylcarnitine and 5-oxoproline correlated with cyst fluid CEA levels (P<.0001 and P<.05 respectively). For diagnosing malignant pancreatic cysts, the diagnostic accuracies of cyst size > 3 cm, ≥ 1 high-risk features, cyst fluid CEA, and cytology are 38%, 75%, 80%, and 75%, respectively.
(Iso)-butyrylcarnitine has potential clinical application for accurately distinguishing malignant from benign pancreatic cysts, and 5-oxoproline for distinguishing mucinous from non-mucinous cysts.

To identify endotypes of osteoarthritis (OA) by a metabolomics analysis.
Study participants included hip/knee OA patients and controls. Fasting plasma samples were metabolomically profiled. Common factor analysis and K-means clustering were applied to the metabolomics data to identify the endotypes of OA patients. Logistic regression was utilized to identify the most significant metabolites contributing to the endotypes. Clinical and epidemiological factors were examined in relation to the identified OA endotypes.
Six hundred and fifteen primary OA patients and 237 controls were included. Among the 186 metabolites measured, 162 passed the quality control analysis. The 615 OA patients were classified in three clusters (A, 66; B, 200; and C, 349). Patients in cluster A had a significantly higher concentration of butyrylcarnitine (C4) than other clusters and controls (all P < 0.0002). Elevated C4 is thought to be related to muscle weakness and wasting. Patients in cluster B had a significantly lower arginine concentration than other clusters and controls (all P < 7.98 × 10-11). Cluster C patients had a significantly lower concentration of lysophosphatidylcholine (with palmitic acid), which is a pro-inflammatory bioactive compound, than other clusters and controls (P < 3.79 × 10-6). Further, cluster A had a higher BMI and prevalence of diabetes than other clusters (all P ≤ 0.0009), and also a higher prevalence of coronary heart disease than cluster C (P = 0.04). Cluster B had a higher prevalence of coronary heart disease than cluster C (P = 0.003) whereas cluster C had a higher prevalence of osteoporosis (P = 0.009).
Our data suggest three possible clinically actionable endotypes in primary OA: muscle weakness, arginine deficit and low inflammatory OA.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive disorder of β-oxidation caused by pathogenic variants in the ACADS gene. Analyte testing for SCADD in blood and urine, including newborn screening (NBS) using tandem mass spectrometry (MS/MS) on dried blood spots (DBSs), is complicated by the presence of two relatively common ACADS variants (c.625G>A and c.511C>T). Individuals homozygous for these variants or compound heterozygous do not have clinical disease but do have reduced short-chain acyl-CoA dehydrogenase (SCAD) activity, resulting in elevated blood and urine metabolites. As part of a larger study of the potential role of exome sequencing in NBS in California, we reviewed ACADS sequence and MS/MS data from DBSs from a cohort of 74 patients identified to have SCADD. Of this cohort, approximately 60% had one or more of the common variants and did not have the two rare variants, and thus would need no further testing. Retrospective analysis of ethylmalonic acid, glutaric acid, 2-hydroxyglutaric acid, 3-hydroxyglutaric acid, and methylsuccinic acid demonstrated that second-tier testing applied before the release of the newborn screening result could reduce referrals by over 50% and improve the positive predictive value for SCADD to above 75%.

OBJECTIVE: The two primary pathophysiological characteristics of patients with type 2 diabetes mellitus (T2DM) are insulin resistance (IR) and beta cell dysfunction. It has been proposed that the development of IR is secondary to the accumulation of triacylglycerols and fatty acids in the muscle and liver, which is in turn thought to be secondary to an enzymatic defect in mitochondrial beta-oxidation. The purpose of the present study was to analyze the molecules of intermediary metabolism to determine if an alteration in mitochondrial function exists in T2DM patients and, if so, to determine whether this alteration is caused by excess nutrients or an enzymatic defect.
METHODS: Seventy-seven subjects were recruited and divided into four groups (21 T2DM patients, 17 non-diabetic overweight/obese subjects, 20 offspring of T2DM patients, and 19 healthy subjects). Anthropometric parameters were determined by air plethysmography, and biochemical and metabolic parameters were measured, including 31 acylcarnitines (ACs) and 13 amino acids quantified by MS/MS and 67 organic acids measured by GC/MS.
RESULTS: Patients with T2DM showed elevation of short-chain ACs (C2, C4), a glycogenic amino acid (valine), a glycogenic and ketogenic amino acid (tyrosine), and a ketogenic amino acid (leucine) as well as altered excretion of dicarboxylic acids. T2DM offspring with abnormal glucose tolerance test GTT showed increased levels of C16. Subjects in the obese group who were dysglycemic also showed altered urinary excretion of dicarboxylic acids and lower levels of a long-chain AC (C14:2).
CONCLUSIONS: These results suggest that mitochondrial beta-oxidation is altered in T2DM patients and that the alteration is most likely caused by nutrient overload through a different pathway from that observed in obese subjects.

Measurement of carnitine and acylcarnitines in plasma is important in diagnosis of fatty acid β-oxidation disorders and organic acidemia. The usual method uses flow injection tandem mass spectrometry (FIA-MS/MS), which has limitations. A rapid and more accurate method was developed to be used for high-risk screening and diagnosis. Carnitine and acylcarnitines were separated by hydrophilic interaction liquid chromatography (HILIC) without derivatization and detected with a QTRAP MS/MS System. Total analysis time was 9.0min. The imprecision of within- and between-run were less than 6% and 17%, respectively. Recoveries were in the range of 85-110% at three concentrations. Some acylcarnitine isomers could be separated, such as dicarboxylic and hydroxyl acylcarnitines. The method could also separate interferent to avoid false positive results. 216 normal samples and 116 patient samples were detected with the validated method, and 49 patients were identified with fatty acid oxidation disorders or organic acidemias.

In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.