Clinical significance of small molecule metabolites in the blood of patients with different types of liver injury.

To understand the characteristic of changes of serum metabolites between healthy people and patients with hepatitis B virus (HBV) infection at different stages of disease, and to provide reference metabolomics information for clinical diagnosis of liver disease patients. 255 patients with different stages of HBV infection were selected. 3 mL blood was collected from each patient in the morning to detect differences in serum lysophosphatidylcholine, acetyl-L-carnitine, oleic acid amide, and glycocholic acid concentrations by UFLC-IT-TOF/MS. The diagnostic values of four metabolic substances were evaluated by receiver operating characteristic (ROC) curve. The results showed that the optimal cut-off value of oleic acid amide concentration of the liver cirrhosis and HCC groups was 23.6 mg/L, with a diagnostic sensitivity of 88.9% and specificity of 70.6%. The diagnostic efficacies of the three substances were similar in the hepatitis and HCC groups, with an optimal cut-off value of 2.04 mg/L, and a diagnostic sensitivity and specificity of 100% and 47.2%, respectively. The optimal cut-off value of lecithin of the HBV-carrier and HCC groups was 132.85 mg/L, with a diagnostic sensitivity and specificity of 88.9% and 66.7%, respectively. The optimal cut-off value of oleic acid amide of the healthy and HCC groups was 129.03 mg/L, with a diagnostic sensitivity and specificity of 88.4% and 83.3%, respectively. Lysophosphatidylcholine, acetyl-L-carnitine, and oleic acid amide were potential metabolic markers of HCC. Among them, lysophosphatidylcholine was low in the blood of HCC patients, and its diagnostic efficacy was better than that of acetyl-L-carnitine and oleic acid amide, providing reference metabolomics information in clinical diagnosis and future research.

Association of gut-related metabolites with outcome in acute heart failure.

BACKGROUND: Trimethylamine N-oxide (TMAO), a gut-related metabolite, is associated with heart failure (HF) outcomes. However, TMAO is the final product of a complex metabolic pathway (ie, choline/carnitine) that has never been entirely investigated in HF. The present study investigates a panel of metabolites involved in the TMAO-choline/carnitine metabolic pathway for their associations with outcome in acute HF patients. METHODS: In total, 806 plasma samples from acute HF patients were analyzed for TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, γ-butyrobetaine, crotonobetaine, trimethylamine, betaine aldehyde, choline, and betaine using a developed liquid chromatography-tandem mass spectrometry method. Associations with outcome of all-cause mortality (death) and a composite of all-cause mortality and/or rehospitalization caused by HF (death/HF) at 30 days and 1 year were investigated. RESULTS: TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, and γ-butyrobetaine were associated with death and death/HF at 30 days (short term; hazard ratio 1.30-1.49, P≤ .021) and at 1 year (long term; hazard ratio 1.15-1.25, P≤ .026) when adjusted for cardiac risk factors. L-carnitine and acetyl-L-carnitine were superior for short-term outcomes whereas TMAO was the superior metabolite for association with long-term outcomes. Furthermore, acetyl-L-carnitine and L-carnitine were superior for in-hospital mortality and improved risk stratification when combined with current clinical risk scores (ie, Acute Decompensated HEart Failure National REgistry, Organized Program To Initiate Lifesaving Treatment In Hospitalized Patients With Heart Failure, and Get With The Guidelines-Heart Failure; odds ratio (OR) ≥ 1.52, P≤ .020). CONCLUSIONS: Carnitine-related metabolites show associations with adverse outcomes in acute HF, in particular L-carnitine and acetyl-L-carnitine for short-term outcomes, and TMAO for long-term outcomes. Further studies are warranted to investigate the role and implications of carnitine metabolites including intervention in the pathogenesis of HF.

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.

The Type 2 Diabetes Susceptibility PROX1 Gene Variants Are Associated with Postprandial Plasma Metabolites Profile in Non-Diabetic Men.

The prospero homeobox 1 (PROX1) gene may show pleiotropic effects on metabolism. We evaluated postprandial metabolic alterations dependently on the rs340874 genotypes, and 28 non-diabetic men were divided into two groups: high-risk (HR)-genotype (CC-genotype carriers, n = 12, 35.3 ± 9.5 years old) and low-risk (LR)-genotype (allele T carriers, n = 16, 36.3 ± 7.0 years old). Subjects participated in two meal-challenge-tests with high-carbohydrate (HC, carbohydrates 89%) and normo-carbohydrate (NC, carbohydrates 45%) meal intake. Fasting and 30, 60, 120, and 180 min after meal intake plasma samples were fingerprinted by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In HR-genotype men, the area under the curve (AUC) of acetylcarnitine levels was higher after the HC-meal [+92%, variable importance in the projection (VIP) = 2.88] and the NC-meal (+55%, VIP = 2.00) intake. After the NC-meal, the HR-risk genotype carriers presented lower AUCs of oxidized fatty acids (-81-66%, VIP = 1.43-3.16) and higher linoleic acid (+80%, VIP = 2.29), while after the HC-meal, they presented lower AUCs of ornithine (-45%, VIP = 1.83), sphingosine (-48%, VIP = 2.78), linoleamide (-45%, VIP = 1.51), and several lysophospholipids (-40-56%, VIP = 1.72-2.16). Moreover, lower AUC (-59%, VIP = 2.43) of taurocholate after the HC-meal and higher (+70%, VIP = 1.42) glycodeoxycholate levels after the NC-meal were observed. Our results revealed differences in postprandial metabolites from inflammatory and oxidative stress pathways, bile acids signaling, and lipid metabolism in PROX1 HR-genotype men. Further investigations of diet-genes interactions by which PROX1 may promote T2DM development are needed.

Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Analysis of serum metabolite profiles in syphilis patients by untargeted metabolomics.

BACKGROUND: Global metabolomics analysis can provide substantial information on energy metabolism, physiology, possible diagnostic biomarkers and intervention strategies for pathogens. OBJECTIVE: To gain a better understanding of the mechanisms of syphilis and analysis of serum metabolite profiles in syphilis patients. METHODS: We conducted an untargeted metabolomics analysis of serum from 20 syphilis patients and 20 healthy controls. RESULTS: A total of 2890 molecular features were extracted from each sample, and the peak intensity of each feature was obtained. Distinct differential metabolites were identified by principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis. Furthermore, five metabolites were identified as significantly different by Student's t-test, including trimethylamine N-oxide, l-arginine, lysoPC(18:0), betaine and acetylcarnitine. KEGG analysis showed that these differential metabolites were in various pathways, including Chagas disease, fatty acid biosynthesis, primary bile acid biosynthesis, Salmonella infection, ABC transporters, glycerophospholipid metabolism and choline metabolism. Among them, trimethylamine N-oxide was 3.922 times in patients with syphilis than healthy controls. CONCLUSION: Trimethylamine N-oxide may be used as an indicator to distinguish between syphilis patients and healthy controls. The changes in these metabolites suggest that Treponema pallidum affects the normal metabolic activity of host cells, providing some clues for elucidating the pathogenesis of T. pallidum.

Deep Vein Thrombosis Exhibits Characteristic Serum and Vein Wall Metabolic Phenotypes in the Inferior Vena Cava Ligation Mouse Model.

OBJECTIVES: Deep vein thrombosis (DVT) is a major health problem, responsible for significant morbidity and mortality. The identification of a simple and effective diagnostic biomarker of DVT remains a challenge. Metabolomics have recently emerged as a new powerful scientific tool to characterise metabolic phenotypes of complex diseases and investigate small molecules in biofluids. The aim of the study was to identify the blood and vein wall metabolomic signature of DVT in a murine experimental model. METHODS: An established inferior vena cava ligation mouse model of DVT (n=10) was used and compared with sham surgery controls (n=10). Comprehensive untargeted metabolic profiling of serum and vein wall extracts was undertaken using liquid chromatography coupled mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Multivariate and univariate statistical analysis demonstrated a differential metabolic profile when comparing DVT mice and control animals. Serum from DVT mice was characterised by differential concentrations of adenosine (decreased in DVT mice 9.6 fold), adenine (decreased 10.6 fold), and tricyclic acid cycle (TCA) intermediates, including citrate, succinate, and fumarate (1.5, 2.3, and 2.8 fold decreases, respectively). l-carnitine was found to be of greater abundance in the serum of DVT animals (67.0 fold change). A number of lipid moiety classes, including sphingomyelins, phosphatidylcholines, and triglycerides, were differentially abundant. Several metabolites were found in vein wall, including acetylcarnitine (increased in DVT mice 1.9 fold), adenosine (increased 2.2 fold), and ceramide (increased 2.7 fold). Correlation analysis illustrated the biochemical relationships between assigned metabolites, with the discriminatory molecules being highly correlated with each other, in both serum and vein wall. CONCLUSIONS: The present findings demonstrate that metabolic dysregulations in DVT centre on energy metabolism, sphingolipid, and adenosine metabolism, representing a DVT specific metabolite signature in a murine experimental model.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

Acetylcarnitine Is a Candidate Diagnostic and Prognostic Biomarker of Hepatocellular Carcinoma.

The identification of serum biomarkers to improve the diagnosis and prognosis of hepatocellular carcinoma has been elusive to date. In this study, we took a mass spectroscopic approach to characterize metabolic features of the liver in hepatocellular carcinoma patients to discover more sensitive and specific biomarkers for diagnosis and progression. Global metabolic profiling of 50 pairs of matched liver tissue samples from hepatocellular carcinoma patients was performed. A series of 62 metabolites were found to be altered significantly in liver tumors; however, levels of acetylcarnitine correlated most strongly with tumor grade and could discriminate between hepatocellular carcinoma tumors and matched normal tissues. Post hoc analysis to evaluate serum diagnosis and progression potential further confirmed the diagnostic capability of serum acetylcarnitine. Finally, an external validation in an independent batch of 58 serum samples (18 hepatocellular carcinoma patients, 20 liver cirrhosis patients, and 20 healthy individuals) verified that serum acetylcarnitine was a meaningful biomarker reflecting hepatocellular carcinoma diagnosis and progression. These findings present a strong new candidate biomarker for hepatocellular carcinoma with potentially significant diagnostic and prognostic capabilities. Cancer Res; 76(10); 2912-20. ©2016 AACR.

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism.

Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured acylcarnitine profiles at several time points and calculated net transorgan acylcarnitine fluxes. Fasting plasma acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle acylcarnitine flux was observed. We conclude that liver has a key role in acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different acylcarnitine species.

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines.

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.

Serum vitamin B12 levels during the first trimester of pregnancy correlate with newborn screening markers of vitamin B12 deficiency.

INTRODUCTION: Low maternal vitamin B12 status is a risk factor for various adverse pregnancy outcomes. Although vitamin B12 deficiency is not a primary target of newborn screening (NBS) programs, measurements of propionylcarnitine (C3) and its ratios with acetylcarnitine (C3/C2) and palmitoylcarnitine (C3/C16) may incidentally identify vitamin B12-deficient newborns. The objective of this study was to measure vitamin B12 levels in women during the first trimester of pregnancy, evaluate predictors of these concentrations, and study their relationship with newborn screening results. DESIGN: Vitamin B12 concentrations were evaluated in 204 women during the first trimester of pregnancy and possible confounding factors were analyzed. After giving birth, data of their newborns (189) were collected (sex, gestational age, birthweight) and the acylcarnitine profile obtained by tandem mass spectrometry during NBS was analyzed. To assess the effects of the variables on vitamin B12 serum concentrations and newborn screening markers, stepwise multiple linear regression models were used. RESULTS: The mean serum concentration of vitamin B12 was 370.8 pmol/L (502.4 pg/mL) (SD 142.81). Vitamin B12 concentrations were significantly lower in smokers (p=0.027), and in women with low meat consumption (p=0.040). There was a significant inverse correlation between mothers'’ vitamin B12 concentrations and their children’'s C3 (r=-0.24; p=0.001), C3/C2 (r=-0.23; p=0.002) and C3/C16 levels (r=-0.20; p=0.006). CONCLUSIONS: Newborn screening markers (C3, C3/C2, and C3/C16) present an inverse correlation with maternal vitamin B12 status in the first trimester of pregnancy. Regarding factors that may influence maternal serum vitamin B12 levels during the first trimester, smoking seems to have a negative effect, and meat consumption a positive effect.

Proton NMR-based metabolite analyses of archived serial paired serum and urine samples from myeloma patients at different stages of disease activity identifies acetylcarnitine as a novel marker of active disease.

BACKGROUND: Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an 'ideal' fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm. METHODS: We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial. FINDINGS: Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies. CONCLUSIONS: These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.

Effect of short- and long-term treatment with valproate on carnitine homeostasis in humans.

AIMS: The aim of this study was to identify the mechanisms of hypocarnitinemia in patients treated with valproate. METHODS: Plasma concentrations and urinary excretion of carnitine, acetylcarnitine, propionylcarnitine, valproylcarnitine, and butyrobetaine were determined in a patient starting valproate treatment and in 10 patients on long-term valproate treatment. Transport of carnitine and valproylcarnitine by the proximal tubular carnitine transporter OCTN2 was assessed in vitro. RESULTS: In the patient starting valproate, the plasma carnitine and acetylcarnitine levels dropped for 1-3 weeks and had recovered after 3-5 weeks, whereas the plasma levels of propionyl and valproylcarnitine increased steadily over 5 weeks. The renal excretion and excretion fractions (EFs) of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine decreased substantially after starting valproate. Compared with controls, patients on long-term valproate treatment had similar plasma levels of carnitine, acetylcarnitine, and propionylcarnitine, whereas valproylcarnitine was found only in patients. Urinary excretion and renal clearance of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine were decreased in valproate-treated compared with that in control patients, reaching statistical significance for carnitine. The EFs of carnitine, acetylcarnitine, and propionylcarnitine were <5% of the filtered load in controls and were lower in valproate-treated patients. In contrast, the EF for valproylcarnitine approached 100%, resulting from a low affinity of valproylcarnitine for the carnitine transporter OCTN2 and competition with concomitantly filtered carnitine. CONCLUSIONS: The initial drop in plasma carnitine levels of valproate-treated patients is most likely due to impaired carnitine biosynthesis, whereas the recovery of the plasma carnitine levels is explainable by an increased renal expression of OCTN2. Renally excreted valproylcarnitine does not affect renal handling of carnitine in vivo.

Plasma metabolomic profiles reflective of glucose homeostasis in non-diabetic and type 2 diabetic obese African-American women.

Insulin resistance progressing to type 2 diabetes mellitus (T2DM) is marked by a broad perturbation of macronutrient intermediary metabolism. Understanding the biochemical networks that underlie metabolic homeostasis and how they associate with insulin action will help unravel diabetes etiology and should foster discovery of new biomarkers of disease risk and severity. We examined differences in plasma concentrations of >350 metabolites in fasted obese T2DM vs. obese non-diabetic African-American women, and utilized principal components analysis to identify 158 metabolite components that strongly correlated with fasting HbA1c over a broad range of the latter (r = -0.631; p<0.0001). In addition to many unidentified small molecules, specific metabolites that were increased significantly in T2DM subjects included certain amino acids and their derivatives (i.e., leucine, 2-ketoisocaproate, valine, cystine, histidine), 2-hydroxybutanoate, long-chain fatty acids, and carbohydrate derivatives. Leucine and valine concentrations rose with increasing HbA1c, and significantly correlated with plasma acetylcarnitine concentrations. It is hypothesized that this reflects a close link between abnormalities in glucose homeostasis, amino acid catabolism, and efficiency of fuel combustion in the tricarboxylic acid (TCA) cycle. It is speculated that a mechanism for potential TCA cycle inefficiency concurrent with insulin resistance is "anaplerotic stress" emanating from reduced amino acid-derived carbon flux to TCA cycle intermediates, which if coupled to perturbation in cataplerosis would lead to net reduction in TCA cycle capacity relative to fuel delivery.

Hidrólisis de acilcarnitinas durante el análisis en sangre y plasma por espectrometría de masas en tandem / Hydrolysis of acylcarnitines during measurement in blood and plasma by tandem mass spectrometry

La determinación de acilcarnitinas en sangre es una herramienta importante para el diagnóstico de algunas enfermedades hereditarias, así como deficiencias metabólicas secundarias. Bajo las condiciones acídicas y la alta temperatura utilizadas durante el proceso de derivatización, es posible que pueda ocurrir algún grado de hidrólisis de acilcarnitinas, lo cual puede potencialmente interferir con las determinaciones de la carnitina libre. El objetivo del presente estudio fue investigar la hidrólisis de las acilcarnitinas (de cadena corta, media y larga) durante el proceso de derivatización y analizar su efecto sobre la determinación de carnitina libre. El porcentaje de hidrólisis fue de 27% para acilcarnitinas de cadena corta, 17% para acilcarnitinas de cadena media y 5% para acilcarnitinas de cadena larga. Estos resultados pueden ocasionar un incremento en los niveles de carnitina libre de las muestras analizadas.

The measurement of acylcarnitines in blood is an important tool for diagnosis of some inherited metabolic diseases and secondary metabolic deficiencies. Under the acidic conditions and the high temperature used for the derivatisation process, it is feasible that some degree of hydrolysis of acylcarnitines to free carnitine may occur and therefore potentially interfere with free carnitine measurements. The objective of the present study was to investigate the hydrolysis of acylcarnitines (short-chain-, medium-chain, and long chain acylcarnitines) during derivatisation process and to analyse its effect on free carnitine measurement. The average percentage of hydrolysis was 27% for short-chain acylcarnitines, 17% for medium-chain acylcarnitines, and 5% for long-chain acylcarnitines. These results can increase the free carnitine levels in the analysed samples.

Metabolomics identifies perturbations in human disorders of propionate metabolism.

BACKGROUND: We applied untargeted mass spectrometry-based metabolomics to the diseases methylmalonic acidemia (MMA) and propionic acidemia (PA). METHODS: We used a screening platform that used untargeted, mass-based metabolomics of methanol-extracted plasma to find significantly different molecular features in human plasma samples from MMA and PA patients and from healthy individuals. Capillary reverse phase liquid chromatography (4 microL/min) was interfaced to a TOF mass spectrometer, and data were processed using nonlinear alignment software (XCMS) and an online database (METLIN) to find and identify metabolites differentially regulated in disease. RESULTS: Of the approximately 3500 features measured, propionyl carnitine was easily identified as the best biomarker of disease (P value 1.3 x 10(-18)), demonstrating the proof-of-concept use of untargeted metabolomics in clinical chemistry discovery. Five additional acylcarnitine metabolites showed significant differentiation between plasma from patients and healthy individuals, and gamma-butyrobetaine was highly increased in a subset of patients. Two acylcarnitine metabolites and numerous unidentified species differentiate MMA and PA. Many metabolites that do not appear in any public database, and that remain unidentified, varied significantly between normal, MMA, and PA, underscoring the complex downstream metabolic effects resulting from the defect in a single enzyme. CONCLUSIONS: This proof-of-concept study demonstrates that metabolomics can expand the range of metabolites associated with human disease and shows that this method may be useful for disease diagnosis and patient clinical evaluation.

L-carnitine supplementation in breeding pigeons: impact on zootechnical performance and carnitine metabolism.

In the first experiment (Exp1), three consecutive breeding rounds were performed by two groups of six pigeon couples in order to study the impact of L-carnitine supplementation (80 mg x d(-1)) of parent pigeons on zootechnical performance. Both in the second and third experiments (Exp2, Exp3), one breeding round was performed by two groups of six pigeon couples to reveal the biochemical background of the increase in squab growth, the limitation of body weight decrease in male parent birds and the tendency for an improved cumulative feed efficiency due to L-carnitine supplementation in Exp1. Growth improvement of the squabs with L-carnitine was only seen when the parent pigeons were supplemented, together with a marked rise in the body weight of the parent birds around hatching. Based on the results of the crop milk analysis, growth improvement was probably due to a quantitative impact on crop milk production. The crop milk from the supplemented groups in both Exp2 and Exp3 had increased levels of carnitine. Carnitine, gamma-butyrobetaine and acetylcarnitine were increased in plasma samples of the supplemented parent pigeons. No differences were present in the squabs' plasma for these parameters. In the squabs of Exp3, no changes were seen in the proportional growth or the protein content of the heart, breast muscle and liver, but the breast muscle of the squabs from the supplemented group in Exp3 showed a considerable rise in carnitine and a marked decrease in gamma-butyrobetaine.

The effect of marathon running on carnitine metabolism and on some aspects of muscle mitochondrial activities and antioxidant mechanisms.

Carnitine is an essential co-factor in the catabolism of fats as an energy source. The primary purpose of this study was to investigate the effect of running a marathon on the metabolism of carnitine by endurance-trained athletes, and to evaluate the effect of carnitine administration on the performance of such exercise. The effects of marathon running on mitochondrial enzymes and cellular anti-oxidants were also examined to assess whether the expression of these activities is altered by exercise. Subjects were 10 experienced male marathon runners aged between 19 and 25 years. Running a marathon caused a fall in the plasma content of unesterified carnitine (37%) and an increase in the level of acetylcarnitine present (288%). Loading of the athletes with L-carnitine for 10 days before running a marathon abolished the exercise-induced fall in plasma-free carnitine (P less than 0.05) whilst amplifying the production of acetylcarnitine (P less than 0.05). Carnitine loading of the athletes studied made no detectable improvement in performance of the marathon (P greater than 0.05). Cytochrome oxidase, succinate cytochrome C reductase and superoxide dismutase activities present in skeletal muscle were unaltered by marathon running. However, such exercise caused a large increase in the tissue content of oxidized glutathione (189%) at the expense of reduced glutathione (-18%).