Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry

Background: Amino acidopathies are a class of inborn errors of metabolism (IEM) that can be diagnosed by analysis of amino acids (AA) in plasma. Current strategies for AA analysis include cation exchange HPLC with post-column ninhydrin derivatization, GC-MS, and LC-MS/MS-related methods. Major drawbacks of the current methods are time-consuming procedures, derivative problems, problems with retention, and MS-sensitivity. The use of hydrophilic interaction liquid chromatography (HILIC) columns is an ideal separation mode for hydrophilic compounds like AA. Here we report a HILIC-method for analysis of 36 underivatized AA in plasma to detect defects in AA metabolism that overcomes the major drawbacks of other methods. Methods: A rapid, sensitive, and specific method was developed for the analysis of AA in plasma without derivatization using HILIC coupled with tandem mass-spectrometry (Xevo TQ, Waters). Results: Excellent separation of 36 AA (24 quantitative/12 qualitative) in plasma was achieved on an Acquity BEH Amide column (2.1×100 mm, 1.7 μm) in a single MS run of 18 min. Plasma of patients with a known IEM in AA metabolism was analyzed and all patients were correctly identified. Conclusion: The reported method analyzes 36 AA in plasma within 18 min and provides baseline separation of isomeric AA such as leucine and isoleucine. No separation was obtained for isoleucine and allo-isoleucine. The method is applicable to study defects in AA metabolism in plasma

Determination of low isotopic enrichment of L-[1-C-13]valine by gas chromatography combustion isotope ratio mass spectrometry: a robust method for measuring protein fractional synthetic rates in vivo

A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-C-13]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC separation of valine from the other branched-chain amino acids, leucine and isoleucine, is easily obtained. A good lineair relationship was observed between the increment of the C-13/C-12 isotope ratio in CO, gas derived from the combustion-of derivatized valine and the tracer mole ratio of L-[1-C-13]valine to unlabelled valine. The limit of quantitation was at an L-[1-C-13]valine tracer mole ratio of 0.0002. The method was used to measure the isotopic enrichment of L-[1-C-13]valine-in standard mixtures and in skeletal muscle of six growing piglets infused with L-[1-C-13]valine (2 mg kg(-1) h(-1) for 6 h). After infusion of L-[1-C-13]valine the mean tracer mole ratio in plasma of L-[1-C-13]valine at the isotopic steady state was 0.0740 +/- 0.0056 (GC/MS, mean +/- SEM) and the mean tracer mole ratio of valine in muscle protein fraction at 6 h was 0.000236 +/- 0.000038 (GC/C/IRMS). The resulting mean protein fractional synthetic rate in piglet skeletal muscle was 0.052 +/- 0.007% h(-1), which is in good agreement with literature data obtained with alternative, more elaborate techniques. By this method protein fractional synthetic rates Can be measured at low isotopic enrichment levels using L-[1-C-13]valine, the MCM derivative and on-line GC/C/IRMS. (C) 1998 John Whey & Sons, Ltd

A new approach for fast metabolic diagnostics in CMAMMA

Background: The presence of increased urinary concentrations of both methylmalonic acid (MMA) and malonic acid (MA) is assumed to differentiate combined malonic and methylmalonic aciduria (CMAMMA), due to mutations in the ACSF3 gene, from other causes of methylmalonic aciduria (classic MMAemia). Detection of MA in urine, however, is challenging since excretion of MA can be easily missed. The objective of the study was to develop a method for quantification of MA in plasma to allow differentiation between CMAMMA and classic MMAemia. Methods: Compound heterozygosity for mutations in the ACSF3 gene was detected in two female siblings using diagnostic exome sequencing. Urine (MMA and MA) was analyzed with GC/MS, while plasma was analyzed with UPLC-MS/MS. MA/MMA ratios were calculated. Results: Both patients had a severe psychiatric presentation (at the age of 6 years and 5.5 years, respectively) after a viral infection. MA excretion in the patients was only just above the highest control value in several samples. MA concentrations in plasma from the two patients were clearly above the highest value observed in control subjects. However, MA concentrations in plasma from patients with classic MMAemia were also elevated. Additional, calculation of MA/MMA ratio in plasma allowed to fully differentiate between CMAMMA and classic MMAemia. Conclusions: Calculating the MA/MMA ratio in plasma allows differentiation between CMAMMA and classic MMAemia. The full clinical spectrum of CMAMMA remains to be delineated

A New Approach for Fast Metabolic Diagnostics in CMAMMA

Background: The presence of increased urinary concentrations of both methylmalonic acid (MMA) and malonic acid (MA) is assumed to differentiate combined malonic and methylmalonic aciduria (CMAMMA), due to mutations in the ACSF3 gene, from other causes of methylmalonic aciduria (classic MMAemia). Detection of MA in urine, however, is challenging since excretion of MA can be easily missed. The objective of the study was to develop a method for quantification of MA in plasma to allow differentiation between CMAMMA and classic MMAemia. Methods: Compound heterozygosity for mutations in the ACSF3 gene was detected in two female siblings using diagnostic exome sequencing. Urine (MMA and MA) was analyzed with GC/MS, while plasma was analyzed with UPLC-MS/MS. MA/MMA ratios were calculated. Results: Both patients had a severe psychiatric presentation (at the age of 6 years and 5.5 years, respectively) after a viral infection. MA excretion in the patients was only just above the highest control value in several samples. MA concentrations in plasma from the two patients were clearly above the highest value observed in control subjects. However, MA concentrations in plasma from patients with classic MMAemia were also elevated. Additional, calculation of MA/MMA ratio in plasma allowed to fully differentiate between CMAMMA and classic MMAemia. Conclusions: Calculating the MA/MMA ratio in plasma allows differentiation between CMAMMA and classic MMAemia. The full clinical spectrum of CMAMMA remains to be delineated

Reliable Diagnosis of Carnitine Palmitoyltransferase Type IA Deficiency by Analysis of Plasma Acylcarnitine Profiles

Item does not contain fulltextBACKGROUND: Carnitine palmitoyltransferase IA (CPT-IA) deficiency is an inherited disorder of the carnitine cycle (MIM #255120). Patients affected by this deficiency might be missed easily because of lack of specific and sensitive biochemical markers. In this study, sensitivity and specificity of plasma free carnitine (C0) and long-chain acylcarnitines (lc-ac: C16:0-, C16:1-, C18:0-, C18:1- and C18:2-ac) was evaluated, including the sum of lc-ac ( summation operatorlc-ac) and the molar ratios C0/(C16:0-ac+C18:0-ac) and C0/ summation operatorlc-ac. METHODS: Nine plasma acylcarnitine profiles of 4 CPT-IA deficient patients were compared with profiles of 2,190 subjects suspected of or diagnosed with an inherited disorder of metabolism. Age-dependent reference values were calculated based on the patient population without a definite diagnosis of an inborn error of metabolism (n = 1,600). Sensitivity, specificity, and Receiver Operating Characteristic (ROC) curves were calculated based on samples of the whole patient population. RESULTS: Concentrations of C0 in plasma were normal in all CPT-IA deficient patient samples. ROC analyses showed highest diagnostic values for C18:0-ac, C18:1-ac, and summation operatorlc-ac (AUC 1.000) and lowest for C0 (AUC 0.738). Combining two markers, i.e., a plasma C18:1-ac concentration 587, specificity to diagnose CPT-IA deficiency increased to 99.3% compared with either C18:1-ac (97.4%) or C0/(C16:0-ac+C18:0-ac) (96.9%) alone, all at a sensitivity of 100%. CONCLUSIONS: Combination of a low concentration of C18:1-ac with a high molar ratio of C0/(C16:0-ac+C18:0-ac) ratio in plasma has high diagnostic value for CPT-IA deficiency. Patients with a clinical suspicion of CPT-IA deficiency can be diagnosed with this test combination.7 p

Increased de novo lipogenesis and delayed conversion of large VLDL into intermediate density lipoprotein particles contribute to Hyperlipidemia in glycogen storage disease type 1a

Glycogen storage disease type 1a (GSD-1a) is a metabolic disorder characterized by fasting-induced hypoglycemia, hepatic steatosis, and hyperlipidemia. The mechanisms underlying the lipid abnormalities are largely unknown. To investigate these mechanisms seven GSD-1a patients and four healthy control subjects received an infusion of [1-C-13]acetate to quantify cholesterogenesis and lipogenesis. In a subset of patients, [1-C-13] valine was given to assess lipoprotein metabolism and [2-C-13] glycerol to determine whole body lipolysis. Cholesterogenesis was 274 +/- 112 mg/d in controls and 641 +/- 201 mg/d in GSD-1a patients (p <0.01). Plasma triglyceride-palmitate derived from de novo lipogenesis was 7.1 +/- 9.4 and 86.3 +/- 42.5 mu mol/h in controls and patients, respectively.(p <0.01). Production of VLDL did not show a consistent difference between the groups, but conversion of VLDL into intermediate density lipoproteins was relatively retarded in all patients (0.6 +/- 0.5 pools/d) compared with controls (4.3 +/- 1.8 pools/d). Fractional catabolic rate of intermediate density lipoproteins was lower in patients (0.8 +/- 0.6 pools/d) compared with controls (3.1 +/- 1.5 pools/d). Whole body lipolysis was similar, i.e., 4.5 +/- 1.9 mu mol/kg/min in patients and 3.8 +/- 1.9 mu mol/kg/min in controls. Hyperlipidemia in GSD-1a is associated with strongly increased lipid production and a slower relative conversion of VLDL to LDL