Evaluation of the first 5 years of a glutaric aciduria type I neonatal screening programme in Asturias.

INTRODUCTION: . Neonatal screening of glutaric aciduria type 1 (GA-1) has brought radical changes in the course and outcomes of this disease. This study analyses the outcomes of the first 5 years (2015-2019) of the AGA1 neonatal screening programme in our autonomous community. MATERIAL: . We conducted an observational, descriptive and retrospective study. All neonates born between January 1, 2015 and December 31, 2019 that participated in the neonatal screening programme were included in the study. The glutarylcarnitine (C5DC) concentration in dry blood spot samples was measured by means of tandem mass spectrometry applying a cut-off point of 0.25 µmol/L. RESULTS: . A total of 30 120 newborns underwent screening. We found differences in the C5DC concentration based on gestational age, type of feeding and hours of life at sample collection. These differences were not relevant for screening purposes. There were no differences between neonates with weights smaller and greater than 1500 g. Screening identified 2 affected patients and there were 3 false positives. There were no false negatives. The diagnosis was confirmed by genetic testing. Patients have been in treatment since diagnosis and have not developed encephalopathic crises in the first 4 years of life. CONCLUSIONS: . Screening allowed early diagnosis of two cases of GA-1 in the first 5 years since its introduction in our autonomous community. Although there were differences in C5DC levels based on gestational age, type of feeding and hours of life at blood extraction, they were not relevant for screening.

Tricarboxylic acid cycle related-metabolites and risk of atrial fibrillation and heart failure.

BACKGROUND: Tricarboxylic acid (TCA) cycle deregulation may predispose to cardiovascular diseases, but the role of TCA cycle-related metabolites in the development of atrial fibrillation (AF) and heart failure (HF) remains unexplored. This study sought to investigate the association of TCA cycle-related metabolites with risk of AF and HF. METHODS: We used two nested case-control studies within the PREDIMED study. During a mean follow-up for about 10 years, 512 AF and 334 HF incident cases matched by age (±5 years), sex and recruitment center to 616 controls and 433 controls, respectively, were included in this study. Baseline plasma levels of citrate, aconitate, isocitrate, succinate, malate and d/l-2-hydroxyglutarate were measured with liquid chromatography-tandem mass spectrometry. Multivariable conditional logistic regression models were fitted to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for metabolites and the risk of AF or HF. Potential confounders included smoking, family history of premature coronary heart disease, physical activity, alcohol intake, body mass index, intervention groups, dyslipidemia, hypertension, type 2 diabetes and medication use. RESULTS: Comparing extreme quartiles of metabolites, elevated levels of succinate, malate, citrate and d/l-2-hydroxyglutarate were associated with a higher risk of AF [ORQ4 vs. Q1 (95% CI): 1.80 (1.21-2.67), 2.13 (1.45-3.13), 1.87 (1.25-2.81) and 1.95 (1.31-2.90), respectively]. One SD increase in aconitate was directly associated with AF risk [OR (95% CI): 1.16 (1.01-1.34)]. The corresponding ORs (95% CI) for HF comparing extreme quartiles of malate, aconitate, isocitrate and d/l-2-hydroxyglutarate were 2.15 (1.29-3.56), 2.16 (1.25-3.72), 2.63 (1.56-4.44) and 1.82 (1.10-3.04), respectively. These associations were confirmed in an internal validation, except for aconitate and AF. CONCLUSION: These findings underscore the potential role of the TCA cycle in the pathogenesis of cardiac outcomes.

COVID-19 triggered encephalopathic crisis in a patient with glutaric aciduria type 1.

OBJECTIVES: The impact of coronavirus disease-19 (COVID-19) on metabolic outcome in patients with inborn errors of metabolism has rarely been discussed. Herein, we report a case with an acute encephalopathic crisis at the course of COVID-19 disease as the first sign of glutaric aciduria type 1 (GA-1). CASE PRESENTATION: A 9-month-old patient was admitted with encephalopathy and acute loss of acquired motor skills during the course of COVID-19 disease. She had lethargy, hypotonia, and choreoathetoid movements. In terms of COVID-19 encephalopathy, the reverse transcription-polymerase chain reaction assay test for COVID-19 was negative in cerebral spinal fluid. Brain imaging showed frontotemporal atrophy, bilateral subcortical and periventricular white matter, basal ganglia, and thalamic involvement. Elevated glutarylcarnitine in plasma and urinary excretion of glutaric and 3-OH-glutaric acids was noted. A homozygote mutation in the glutaryl-CoA dehydrogenase gene led to the diagnosis of GA-1. CONCLUSIONS: With this report, neurological damage associated with COVID-19 has been reported in GA-1 patients for the first time in literature.

Liver mitochondrial DNA damage and genetic variability of Cytochrome b - a key component of the respirasome - drive the severity of fatty liver disease.

BACKGROUND AND AIMS: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis - NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). METHODS: We included 252 liver specimens of NAFLD patients - in whom histological disease ranged from mild to severe - which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal - a product of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage - were measured. RESULTS: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103  bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. CONCLUSION: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects.

Using arterial-venous analysis to characterize cancer metabolic consumption in patients.

Understanding tumor metabolism holds the promise of new insights into cancer biology, diagnosis and treatment. To assess human cancer metabolism, here we report a method to collect intra-operative samples of blood from an artery directly upstream and a vein directly downstream of a brain tumor, as well as samples from dorsal pedal veins of the same patients. After performing targeted metabolomic analysis, we characterize the metabolites consumed and produced by gliomas in vivo by comparing the arterial supply and venous drainage. N-acetylornithine, D-glucose, putrescine, and L-acetylcarnitine are consumed in relatively large amounts by gliomas. Conversely, L-glutamine, agmatine, and uridine 5-monophosphate are produced in relatively large amounts by gliomas. Further we verify that D-2-hydroxyglutarate (D-2HG) is high in venous plasma from patients with isocitrate dehydrogenases1 (IDH1) mutations. Through these paired comparisons, we can exclude the interpatient variation that is present in plasma samples usually taken from the cubital vein.

Clinical pharmacokinetics and pharmacodynamics of ivosidenib, an oral, targeted inhibitor of mutant IDH1, in patients with advanced solid tumors.

Background Mutant isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) enzymes produce the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (AG-120) is a targeted mutant IDH1 inhibitor under evaluation in a phase 1 dose escalation and expansion study of IDH1-mutant advanced solid tumors including cholangiocarcinoma, chondrosarcoma, and glioma. We explored the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of ivosidenib in these populations. Methods Ivosidenib was administered orally once (QD) or twice (BID) daily in continuous 28-day cycles; 168 patients received ≥1 dose within the range 100 mg BID to 1200 mg QD. PK and PD were assessed using validated liquid chromatography-tandem mass spectrometry assays. Results Ivosidenib demonstrated good oral exposure after single and multiple doses, was rapidly absorbed, and had a long terminal half-life (mean 40-102 h after single dose). Exposure increased less than dose proportionally. Steady state was reached by day 15, with moderate accumulation across all tumors (1.5- to 1.7-fold for area-under-the-curve at 500 mg QD). None of the intrinsic and extrinsic factors assessed affected ivosidenib exposure, including patient/disease characteristics and concomitant administration of weak CYP3A4 inhibitors/inducers. After multiple doses in patients with cholangiocarcinoma or chondrosarcoma, plasma 2-HG was reduced by up to 98%, to levels seen in healthy subjects. Exposure-response relationships for safety and efficacy outcomes were flat across the doses tested. Conclusions Ivosidenib demonstrated good oral exposure and a long half-life. Robust, persistent plasma 2-HG inhibition was observed in IDH1-mutant cholangiocarcinoma and chondrosarcoma. Ivosidenib 500 mg QD is an appropriate dose irrespective of various intrinsic and extrinsic factors. Trial RegistrationClinicalTrials.gov (NCT02073994).

Metabolitic profiling of amino acids in paraquat-induced acute kidney injury.

BACKGROUND: The herbicide paraquat (1, 1'-dimethyl-4, 4'-bipyridylium dichloride; PQ) is a poison well-known to cause delayed mortality due to acute kidney injuries (AKI). This study examines the changes in serum amino acids (AAs) metabolite profiles as surrogate markers of renal cell metabolism and function after paraquat poisoning. METHODS: To identify the metabolic profiling of free serum AAs and its metabolites, serum from 40 paraquat-poisoned patients with or without AKI is collected. LC-MS/GC-MS is performed to analyze AA molecules. A Cox proportional hazard model was used to assess for incidence of AKI. Receiver operating characteristic (ROC) curve is applied to evaluate AKI occurrence and prognosis. RESULTS: A total of 102 serum AAs and its metabolites were identified. Compared with non-AKI patients, 37 varied significantly in AKI patients. The univariate Cox proportional hazard model analysis revealed that the estimated PQ amount, plasma PQ concentration, urine PQ concentration, APACHE, SOFA scores and 16 amino acids correlated with the incidence of AKI. Further analyses revealed that 3-methylglutarylcarnitine, 1-methylimidazoleacetate, and urea showed higher cumulative hazard ratios for the occurrence of AKI during follow-up (P < 0.05). The area under the curve (AUC) of 3-methylglutarylcarnitine, 1-methylimidazoleacetate and urea were 0.917, 0.857, 0.872, respectively. CONCLUSION: 3-methylglutarylcarnitine, 1-methylimidazoleacetate and urea were associated with AKI in patients with paraquat intoxication.

Dyskinesia in a Child: A Concern for a Rare Neuro-Metabolic Disorder.

A 3-year child is discussed who presented with dyskinesia, large head size, developmental delay, and recurrent infections necessitating multiple hospital admissions. The diagnosis was not made at initial presentation or even after multiple hospital admissions. An organic acidemia was suspected, based on raised ammonia and lactate levels and metabolic acidosis and the diagnosis of glutaric aciduria Type 1 was established by finding markedly elevated levels of glutaric acid and its specific metabolites on urine organic acids analysis by gas chromatography-mass spectrometry, in the setting of specific clinical features. Further supporting evidence was provided by CT scan brain showing subdural hygroma along left cerebral hemisphere causing gyral flattening and widening of sylvian fissure.

Isocitrate dehydrogenase 1 and 2 mutations, 2-hydroxyglutarate levels, and response to standard chemotherapy for patients with newly diagnosed acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) cells harboring mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) produce the oncometabolite 2-hydroxyglutarate (2HG). This study prospectively evaluated the 2HG levels, IDH1/2 mutational status, and outcomes of patients receiving standard chemotherapy for newly diagnosed AML. METHODS: Serial samples of serum, urine, and bone marrow aspirates were collected from patients newly diagnosed with AML, and 2HG levels were measured with mass spectrometry. Patients with baseline serum 2HG levels greater than 1000 ng/mL or marrow pellet 2HG levels greater than 1000 ng/2 × 106 cells, which suggested the presence of an IDH1/2 mutation, underwent serial testing. IDH1/2 mutations and estimated variant allele frequencies were identified. AML characteristics were compared with the Wilcoxon test and Fisher's exact test. Disease-free survival and overall survival (OS) were evaluated with log-rank tests and Cox regression. RESULTS: Two hundred and two patients were treated for AML; 51 harbored IDH1/2 mutations. IDH1/2-mutated patients had significantly higher 2HG levels in serum, urine, bone marrow aspirates, and aspirate cell pellets than wild-type patients. A serum 2HG level greater than 534.5 ng/mL was 98.8% specific for the presence of an IDH1/2 mutation. Patients with IDH1/2-mutated AML treated with 7+3-based induction had a 2-year event-free survival (EFS) rate of 44% and a 2-year OS rate of 57%. There was no difference in complete remission rates, EFS, or OS between IDH1/2-mutated and wild-type patients. Decreased serum 2HG levels on day 14 as a proportion of the baseline were significantly associated with improvements in EFS (P = .047) and OS (P = .019) in a multivariate analysis. CONCLUSIONS: Among patients with IDH1/2-mutated AML, 2HG levels are highly specific for the mutational status at diagnosis, and they have prognostic relevance in patients receiving standard chemotherapy.

Plasma 2-hydroxyglutarate, a promising prognostic biomarker candidate for skeletal muscle injury in Fischer 344 rats.

Previously, we have demonstrated the potential of plasma 2-hydroxyglutarate (2HG) as an easily detectable biomarker for skeletal muscle injury in rats. Here, we examined whether plasma 2HG was superior to conventional skeletal muscle damage biomarkers, including aspartate aminotransferase (AST), creatine kinase (CK), and skeletal muscle-type CK isoenzyme (CK-MM) levels, in rats. Skeletal muscle injury was induced in 4- or 9-week-old male Fischer 344 rats by cerivastatin (CER) or tetramethyl-p-phenylenediamine (TMPD) administration. Plasma 2HG levels were measured on days 4, 8, and 11 (CER group) and at 6 and 24 hr post-administration (TMPD group). Plasma AST, CK, and CK-MM activities and histopathological changes in the rectus femoris muscle were evaluated at the study endpoints. In the CER group, AST, CK, and CK-MM increased in 4- and 9-week-old rats, whereas increases in CK (4- and 9-week-old rats) and CK-MM (4-week-old rats) were not obvious in the TMPD group. In both 4- and 9-week-old rats, plasma 2HG increased on day 8 and at 24 hr post-administration in the CER and TMPD groups, respectively. Histopathological analysis revealed myofiber vacuolation and necrosis in both groups. The histopathological damage to the rectus femoris muscle was more severe in the CER than in the TMPD group. Increased plasma 2HG was associated with CER- and TMPD-induced skeletal muscle injuries in rats and was not affected by age differences or repeated blood collection. The results suggest that plasma 2HG is superior to CK and CK-MM as a biomarker for mild skeletal muscle injury.

Quantitation of plasma and urine 3-hydroxyglutaric acid, after separation from 2-hydroxyglutaric acid and other compounds of similar ion transition, by liquid chromatography-tandem mass spectrometry for the confirmation of glutaric aciduria type 1.

BACKGROUND: Glutaric aciduria type 1, a deficiency of glutaryl-CoA dehydrogenase, causes an accumulation of neurotoxic metabolites glutaric acid and 3-hydroxyglutaric acid (3-HGA). Testing of these analytes is routinely done by GC-MS but seldom account for interference from isomers or compounds with similar ion transitions. We developed a liquid chromatography tandem mass spectrometry method that accurately measures 3-HGA in urine and plasma specimens, while utilizing similar reagents and instrumentation used for the routine performance of amino acid and acylcarnitine analysis in determining the diagnosis of several metabolic disorders. METHOD: Plasma and urine samples were added aliquots of the deuterated 3-HGA internal standard and acetonitrile. The protein-free supernatant was brought to dryness, and the residue derivatized using 3 M HCL in 1-butanol with heating. The dried derivative was then reconstituted in 50% methanol-water solution and aliquot transferred to an HPLC vial for analysis by LC-MS/MS. Separation was performed using a C8 HPLC column under flow gradient conditions of 0.2% formic acid in water and methanol, respectively. Ionization was by ESI and detection of selected precursor-product ion transitions by multiple reaction monitoring (MRM) in positive mode. RESULTS: The butyl-ester derivative of 3-HGA eluted at 7.82 min while 2-hydroxyglutaric acid (2-HGA) eluted at 8.21 min. This was equivalent to a separation factor of 1.05 and a resolution of 1.03, respectively. The 3-HGA calibration curve was linear over the range 6.20-319 ng mL-1 (r2 = 0.9996), and the reportable range determined by the linearity was found to be 1.54-384 ng mL-1. The calculated limits of detection and quantitation were 0.348 and 1.56 ng mL-1, respectively. Intra- and Inter-assay %CVs for quality control plasma and urine samples ranged from 2 to 18%, with recoveries of 66-115%. The method correlated to the gold standard GC-MS method for both serum (r2 ≥ 0.996) and urine analysis (r2 ≥ 0.949). The concentration of 3-HGA in normal, non-GA1 individuals was ≤25.2 ng mL-1 (in plasma) and ≤ 35.0 µmol mmol-1 of creatinine (in urine). CONCLUSIONS: This LC-MS/MS method accurately quantified plasma and urine 3-HGA concentration after successful resolution from 2-HGA and other compounds with similar ion transitions. This method is suitable for confirmatory testing of 3-HGA, as a follow-up to an abnormal newborn screen test result, with concern for GA type 1.

Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations.

Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)1-8. Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants9,10. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML11. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies.

Separation and determination of the enantiomers of lactic acid and 2-hydroxyglutaric acid by chiral derivatization combined with gas chromatography and mass spectrometry.

Lactic acid and 2-hydroxyglutaric acid are chiral metabolites that have two distinct d- and l-enantiomers with distinct biochemical properties. Perturbations of a single enantiomeric form have been found to be closely related to certain diseases. Therefore, the ability to differentiate the d and l enantiomers is important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2-hydroxyglutaric acid enantiomers by chiral derivatization (with l-menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of above-mentioned enantiomers exhibited linear calibration curves with a correlation coefficient (R2 ) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17-102.30% with inter- and intraday precisions (relative standard deviations) in the range of 4.23-17.26%. The limits of detection for d-lactic acid, l-lactic acid, d-2-hydroxyglutaric acid, and l-2-hydroxyglutaric acid were 0.13, 0.11, 1.12, and 1.16 µM, respectively. This method was successfully applied to analyze mouse plasma. The d-lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t-test) than those of normal mice, suggesting that d-lactic acid may serve as an indicator for type 2 diabetes mellitus.

Isocitrate dehydrogenase 2 mutations correlate with leukemic transformation and are predicted by 2-hydroxyglutarate in myelodysplastic syndromes.

PURPOSE: The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by the presence of somatically mutated hematopoietic stem cells (HSCs) that increase the risk of progression to secondary acute myeloid leukemia (sAML). Mutations in isocitrate dehydrogenase (IDHmut) are thought to correlate with the increased production of the oncogenic protein 2-hydroxyglutarate (2-HG) in AML. The aim of this study was to examine whether serum 2-HG has utility as a prognostic biomarker, and whether elevated 2-HG levels are predictive of IDH mutations in patients with MDS. METHODS: Genetic profiling was utilized to determine the genetic composition of a large cohort of MDS patients, including the presence or absence of IDH1 or IDH2 mutations (n = 281). Serum 2-HG levels were detected by liquid chromatography-tandem mass spectrometry. RESULTS: In the current study of MDS patients, elevated serum 2-HG levels were predictive of inferior overall- and leukemia-free survival irrespective of IPSS risk grouping. Higher serum 2-HG levels predicted the presence of IDH mutations. IDH2mut patients had a higher risk of leukemic transformation. The co-occurrence of DNMT3A or SRSF2 mutations was found to be increased in IDH2mut patients. IDH2 mutations were associated with significantly worse OS and LFS amongst patients with low-risk MDS by IPSS grouping. CONCLUSIONS: The noted predictive value of serum 2-HG levels and IDH2 mutations on OS and LFS support the use of biomarkers and/or underlying cytogenetics in novel prognostic scoring systems for MDS.

Validation of a routine gas chromatography mass spectrometry method for 2-hydroxyglutarate quantification in human serum as a screening tool for detection of idh mutations.

High circulating levels of 2-hydroxyglutarate (2HG) have been reported in patients with determinate isocitrate dehydrogenase (IDH) mutated tumors. Recent studies indicate that in malignancies such as acute myeloid leukemia (AML), measurements of 2HG in serum provide useful diagnostic and prognostic information and improve patient selection and monitoring of IDH-targeted treatments. In the current study, we validated a sensitive and specific gas chromatography mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HG in routine clinical laboratories. Extraction was liquid-liquid with ethyl acetate, and derivatization was reduced to 3 min of microwave irradiation. The analytical method was linear over a wide dynamic range, presenting acceptable intraday and day-to-day precision and accuracy. The limit of quantification was 10 ng/mL, process efficiency ranged between 38% and 49%, and recovery of added 2HG was 99-105%. 2HG was found to be stable in serum for up to 48 h at both 4 °C and at ambient temperature, and after three freeze-thaw cycles. Microwave derivatizated extracts in the autosampler were found to be stable for up to 120 h. In summary, the present method is useful for quantification of 2HG serum levels in patients with IDH mutated malignancies in clinical laboratories.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies.

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Circulating oncometabolite D-2-hydroxyglutarate enantiomer is a surrogate marker of isocitrate dehydrogenase-mutated intrahepatic cholangiocarcinomas.

Therapeutic resources are limited for advanced biliary tract cancers and prognosis remains poor. Somatic mutations in isocitrate dehydrogenase (IDH)1/2 gene are found in 5-36% of patients with intrahepatic cholangiocarcinoma (ICC). The mutant forms of IDH1/2 catalyse the non-reversible accumulation of 2-hydroxyglutarate (2HG). Increasing numbers of indirect or direct-targeted therapies are developed to IDH1/2 mutations and could be assisted by a routinely feasible, rapid and inexpensive serum 2HG measurement by liquid chromatography coupled to tandem mass spectrometry. By comparing eight patients with an IDH1/2-mutated ICC to nine patients with wild-type IDH1/2 ICC, we found significantly higher levels of 2HG in patients with IDH1/2 mutations versus the wild-type group (median, 10.9 vs. 0.8 µmol/L, p = 0.0037). D and L-2HG enantiomer levels significantly differed between the two groups with a higher level of D-2HG (p < 0.0001) in patients with IDH1/2 mutations. Accordingly, the D/L ratio was markedly higher in the patients with IDH1/2 mutations compared with the wild-type group (38.0 vs. 0.9 µmol/L, p < 0.0001). D-2HG measurement ensured 100% sensitivity and specificity at a cut-off of 0.6 µmol/L. D-2HG levels were correlated with tumour burden and tumour response to treatment with IDH-targeted therapies or indirect therapies. D-2HG serum level measurement by liquid chromatography coupled to tandem mass spectrometry is a sensitive, specific, precise (a coefficient of variation <10% and an accuracy >95%), fast (9 min run per sample) and inexpensive surrogate marker of IDH1/2 somatic mutation in ICC. Systematic measurement in patients with ICC may facilitate access to, and monitoring of, IDH-driven therapies.

Plasma 2-hydroxyglutarate and hexanoylcarnitine levels are potential biomarkers for skeletal muscle toxicity in male Fischer 344 rats.

To identify new candidate biomarkers for skeletal muscle toxicity, an unbiased metabolomic analysis was performed in rats treated with two distinct myotoxicants, cerivastatin (CER) and tetramethyl-p-phenylenediamine (TMPD). Skeletal muscle toxicity was induced in male Fischer 344 rats by administering CER or TMPD and monitored using established endpoints, such as increased plasma creatine kinase (CK) activity and histopathology, and a metabolomic analysis of skeletal muscle and plasma samples. Plasma CK levels in CER-treated rats were markedly elevated at Day 11; however, those in TMPD-treated rats showed a statistically significant decrease at 24 hr after dosing. Light microscopy revealed that vacuolated or necrotic fibers were evident in all CER-treated rats on Day 11, and slightly vacuolated fibers were observed in TMPD-treated rats at 6 and 24 hr after dosing. Metabolomic analysis of the rectus femoris indicated increases in 2-hydroxyglutarate (2HG) in CER-treated rats and hexanoylcarnitine in CER- and TMPD-treated rats. There were also increases in plasma 2HG in CER-treated rats on Days 8 and 11 and in TMPD-treated rats at 24 hr after dosing and increases in plasma hexanoylcarnitine in CER-treated rats on Day 11 and in TMPD-treated rats at 6 and 24 hr after dosing. These experiments demonstrated the potential of plasma 2HG and hexanoylcarnitine as specific and easily detectable biomarkers for skeletal muscle toxicity in rats and demonstrated the value of metabolomics for biomarker detection and identification in toxicological studies.