Screening for fatty acid beta oxidation disorders: Acylglycine analysis by electron impact ionization gas chromatography–mass spectrometry

Urinary acylglycine analysis by chemical ionization (CI) GC–MS has been utilized for more than a decade to screen of fatty acid oxidation disorders. We have developed an alternative GC–MS method involving tert.-butyldimethylsilyl derivatization and standard electron impact ionization. Using six stable isotope labeled internal standards, this method allows the biochemical diagnosis of glutaric aciduria type II and medium chain acyl-CoA dehydrogenase deficiency, and could contribute to the diagnosis of other FAO disorders when used in combination with other biochemical investigations on blood and urine. This method can be conveniently applied to GC–MS system routinely used for organic acid analysis.     

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers^1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer^3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)^1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis⁹. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity^10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids¹². This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites¹³.Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.     

Electrospray ionisation mass spectrometry: principles and clinical applications

This mini-review provides a general understanding of electrospray ionisation mass spectrometry (ESI-MS) which has become an increasingly important technique in the clinical laboratory for structural study or quantitative measurement of metabolites in a complex biological sample. The first part of the review explains the electrospray ionisation process, design of mass spectrometers with separation capability, characteristics of the mass spectrum, and practical considerations in quantitative analysis. The second part then focuses on some clinical applications. The capability of ESI-tandem-MS in measuring bio-molecules sharing similar molecular structures makes it particularly useful in screening for inborn errors of amino acid, fatty acid, purine, pyrimidine metabolism and diagnosis of galactosaemia and peroxisomal disorders. Electrospray ionisation is also efficient in generating cluster ions for structural elucidation of macromolecules. This has fostered a new and improved approach (vs electrophoresis) for identification and quantification of haemoglobin variants. With the understanding of glycohaemoglobin structure, an IFCC reference method for glycohaemoglobin assay has been established using ESI-MS. It represents a significant advancement for the standardisation of HbA1c in diabetic monitoring. With its other applications such as in therapeutic drug monitoring, ESI-MS will continue to exert an important influence in the future development and organisation of the clinical laboratory service.     

ESI-MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders

Acylcarnitines in urine from 45 patients with organic acidemias and fatty acid oxidation disorders were evaluated using ESI-MS/MS. The urinary acylcarnitine profiles in organic acidemias, SCAD deficiency and MCAD deficiency were compatible with blood acylcarnitine profiles, and abnormalities in urinary acylcarnitine profiles in these conditions were enhanced following carnitine loading. Urinary acylcarnitine profiles were not helpful for characterization of long-chain fatty acid disorders, but a combination of urine and blood acylcarnitine analysis was useful for differential diagnosis of carnitine deficit.     

Prenatal diagnosis for organic acid disorders using two mass spectrometric methods, gas chromatography mass spectrometry and tandem mass spectrometry

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.     

Diagnosis of inborn errors of metabolism using filter paper urine,urease treatment,isotope dilution and gas chromatography–mass spectrometry

This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography–mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC–MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.     

Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPR^dn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.     

A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid: diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency

Inherited disorders of fatty acid oxidation are a group of acute life-threatening but treatable disorders, clinically complicated by severe hypoketotic hypoglycemia precipitated by prolonged fasting. Among them, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is by far the most frequent disorder. Here we report a modified method for quantitative acylcarnitine profiling by electrospray ionisation-tandem mass spectrometry (ESI-MS-MS) in human skin fibroblasts using unlabelled palmitic acid as substrate. The reliability of this method was tested in cultured skin fibroblasts from previously diagnosed patients with specific carnitine cycle and fatty acid beta-oxidation defects. Furthermore, acylcarnitine profiling was investigated in fibroblasts and dried blood spots from patients with different variants of MCAD deficiency. ESI-MS-MS-based investigation of cultured skin fibroblasts from patients with disorders of fatty acid oxidation revealed a pathognomonic acylcarnitine profiling. In addition, this method delineated different variants of MCAD deficiency, i.e. mild and classical. The octanoylcarnitine (C8)-to-decanoylcarnitine (C10) and C8-to-acetylcarnitine (C2) ratios were the most specific markers to differentiate mild and classical forms of MCAD deficiency in fibroblasts. Similar results were obtained by quantitative acylcarnitine profiling in dried blood spots. In conclusion, this novel technique is a powerful tool for the investigation of fatty acid oxidation disorders under standardized conditions in fibroblasts.     

Diagnosis and follow-up of inborn errors of amino acid metabolism: Use of proton magnetic resonance of biological fluids

Summary Proton magnetic resonance spectra of biological fluids such as urine, plasma and cerebro-spinal fluid can be used for multi-component analysis of highly concentrated species, thus providing information about the general metabolism of the patient. Hydrogen containing analytes in concentration higher than 10µM are indeed often detectable in biological fluid in 15 minutes by means of an unexpensive 200 MHz spectrometer essentially without sample manipulation. Amino acids, keton bodies, organic acids and other metabolites can be easily estimated by this approach; consequently this technique represents a powerful tool particularly in the diagnosis of inborn errors of amino acid metabolism, when improving the prognosis often depends on a very early diagnosis and on an effective method for monitoring the effects of therapy.In the present paper, several cases of inherited diseases related to amino acid impaired metabolism will be presented to illustrate the importance in the diagnosis. Phenylketonuria, tyrosinemia, cystinuria, ornithinemia, argininosuccinic aciduria, maple syrup urine disease (MSUD), alkaptonuria, lysinuria and other genetic pathologies were in fact unambiguously and rapidly diagnosed by means of the identification in the biological fluids of the relevant accumulating amino acids and/or of their metabolites. The proposed technique is suitable to become, in the future, a useful routine tool for a wide neonatal screening.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

Rapid characterization of the fatty acyl composition of complex lipids by collision-induced dissociation time-of-flight mass spectrometry

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample.     

Human placenta metabolizes fatty acids: implications for fetal fatty acid oxidation disorders and maternal liver diseases

The role of fat metabolism during human pregnancy and in placental growth and function is poorly understood. Mitochondrial fatty acid oxidation disorders in an affected fetus are associated with maternal diseases of pregnancy, including preeclampsia, acute fatty liver of pregnancy, and the hemolysis, elevated liver enzymes, and low platelets syndrome called HELLP. We have investigated the developmental expression and activity of six fatty acid beta-oxidation enzymes at various gestational-age human placentas. Placental specimens exhibited abundant expression of all six enzymes, as assessed by immunohistochemical and immunoblot analyses, with greater staining in syncytiotrophoblasts compared with other placental cell types. beta-Oxidation enzyme activities in placental tissues were higher early in gestation and lower near term. Trophoblast cells in culture oxidized tritium-labeled palmitate and myristate in substantial amounts, indicating that the human placenta utilizes fatty acids as a significant metabolic fuel. Thus human placenta derives energy from fatty acid oxidation, providing a potential explanation for the association of fetal fatty acid oxidation disorders with maternal liver diseases in pregnancy.     

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8–C12) and long-chain (C14–C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K_i of 18.8 μM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.     

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.     

Application of gas chromatography mass spectrometry (GC–MS) in conjunction with multivariate classification for the diagnosis of gastrointestinal diseases

Gastrointestinal diseases such as irritable bowel syndrome, Crohn’s disease (CD) and ulcerative colitis are a growing concern in the developed world. Current techniques for diagnosis are often costly, time consuming, inefficient, of great discomfort to the patient, and offer poor sensitivities and specificities. This paper describes the development and evaluation of a new methodology for the non-invasive diagnosis of such diseases using a combination of gas chromatography mass spectrometry (GC–MS) and chemometrics. Several potential sample matrices were tested: blood, breath, faeces and urine. Faecal samples provided the only statistically significant results, providing discrimination between CD and healthy controls with an overall classification accuracy of 85 % (78 % specificity; 93 % sensitivity). Differentiating CD from other diseases proved more challenging, with overall classification accuracy dropping to 79 % (83 % specificity; 68 % sensitivity). This diagnostic performance compares well with the gold standard technique of colonoscopy, suggesting that GC–MS may have potential as a non-invasive screening tool.     

Lipid metabolism in peroxisomes: enzymology, functions and dysfunctions of the fatty acid alpha- and beta-oxidation systems in humans

Peroxisomes are subcellular organelles present in virtually all eukaryotic cells catalysing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. One of the major functions of peroxisomes concerns their role in lipid metabolism, which includes: (i) fatty acid betaoxidation; (ii) ether phospholipid synthesis; (iii) fatty acid alpha-oxidation; and (iv) isoprenoid biosynthesis. In this paper, we review the current state of knowledge concerning the peroxisomal fatty acid alpha- and beta-oxidation systems with particular emphasis on the enzymes involved and the various disorders of fatty acid oxidation in peroxisomes. We also pay attention to the fact that some of the metabolites that accumulate as the result of a defect in peroxisomal alpha- and/or beta-oxidation are activators of members of the family of nuclear receptors, including peroxisome-proliferator-activated receptor alpha.     

Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic–mass spectrometric (GC–MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC–MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16 246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and α-aminoadipic aciduria/α-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16 246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.     

Triacylglycerol profiling of marine microalgae by mass spectrometry

We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and 1H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.     

A method for the simultaneous determination of 3T3-L1 adipocyte metabolites by liquid chromatography/mass spectrometry using [(13)C]-stable isotopes

A useful method employing liquid chromatography mass spectrometry (LC/MS) and a stable isotope was developed for simultaneous examination of major metabolism in adipocytes, de novo fatty acid synthesis, glycerol output, and glucose uptake with high sensitivity. The addition of thiazolidinediones, potent agonists of peroxisome proliferator-activated receptors-γ, for 10 d increased glucose uptake in a dose-dependent manner. Fatty acid (FA) synthesis increased at low concentrations of thiazolidinediones (TZDs) and decreased at high concentrations. It is important to assess adipocytes from various examples of metabolism, because each example of adipocyte metabolism is directly related to obesity or metabolic syndrome in various ways. The technique makes metabolic examination easier than conventional methods by means of radioisotopes and makes it possible to identify metabolites and to apply them in biomarker screening.