Automated GC–MS analysis of free amino acids in biological fluids

Kaspar, Hannelore; Dettmer, Katja; Gronwald, Wolfram; Oefner, Peter J.

doi:10.1016/j.jchromb.2008.06.018

Cited by 169 publications

(130 citation statements)

References 44 publications

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“…Levels of urinary phosphate and urinary metabolites were assessed in knockout and control mice with the use of established analytic procedures. 15 Bone studies were performed in the mice at 45 and 51 weeks of age. For details of the studies in knockout mice, see the Methods section in the Supplementary Appendix.…”

Section: Ehhadh Knockout Micementioning

confidence: 99%

Mistargeting of Peroxisomal EHHADH and Inherited Renal Fanconi's Syndrome

et al. 2014

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Background: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. Methods: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance (1H-NMR) spectroscopy. Results: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. 1H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. Conclusions: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.)

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Section: Ehhadh Knockout Micementioning

confidence: 99%

Mistargeting of Peroxisomal EHHADH and Inherited Renal Fanconi's Syndrome

et al. 2014

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“…Interest in sarcosine as a marker molecule remains topical, as shown by a recent modification of sarcosine assay by GC/MS in urine. 8 We present measurements in plasma of prostate cancer patients characterised by PSA, tumour stage and Gleason score using GC/MS for sarcosine methodology 9,10 to elaborate further on the suitability of sarcosine as a marker for prostate cancer.…”

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confidence: 99%

Plasma sarcosine does not distinguish early and advanced stages of prostate cancer

et al. 2012

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The identification of prostate cancer remains problematic; no single, simple procedure exists for a reliable diagnosis. Prostate specific antigen (PSA), while non-invasive and easily measurable in serum, is not specific owing to false-positives from benign prostatic hyperplasia (BPH), inflammatory conditions and prostatic trauma. Additional diagnostic information must therefore be obtained by transrectal ultrasonography (TRUS) and digital rectal examination (DRE) to assess prostate size and morphology. Final confirmation of a malignancy requires histopathological analysis of several biopsies. A remaining hurdle in the identification process is the existence of indolent organ-confined disease and aggressive metastatic prostate cancer that can only be distinguished by additional imaging or nuclear medicine procedures.In a new approach to this diagnostic dilemma, it has been argued that screening for changes in metabolite expression resulting from gene silencing and gene activation could be used to identify a specific biological marker that increases in the transformation process. Therefore, a paper elaborating on metabolite expression in clinical samples of benign, localised and metastatic prostate cancer 1 was enthusiastically received and debated in editorials. [2][3][4][5] The proposal that sarcosine (N-methylglycine) (a metabolite of choline found in urine) may be characteristic for prostate cancer progression has since been examined in other laboratories. 1 These investigations found sarcosine levels in urine 6 and serum 7 to be constant, irrespective of sample pathology. From our own and published preliminary measurements, it is nevertheless clear that sarcosine-alanine ratios in prostate tumour patients are often elevated above controls and are spread over a wider range. 1,7,17 A relationship between sarcosine and disease therefore cannot as yet be totally ruled out. Interest in sarcosine as a marker molecule remains topical, as shown by a recent modification of sarcosine assay by GC/MS in urine. 8 We present measurements in plasma of prostate cancer patients characterised by PSA, tumour stage and Gleason score using GC/MS for sarcosine methodology 9,10 to elaborate further on the suitability of sarcosine as a marker for prostate cancer. MethodsPatient blood was collected by venipuncture and centrifuged, and clear supernatants were stored at -20°C. The AxSYM total PSA was determined by a MEIA microparticle enzyme immune assay (Abbott) and expressed as ng/ml. Ethical approval was granted by the University of Stellenbosch Health Research Ethics Committee (N09/11/330).Sarcosine and alanine were quantified in plasma by gaschromatograph mass spectrometry. Briefly, amino acids were extracted from 100 µl of acidified plasma by solid phase extraction onto a cation exchange column before being washed, eluted and derivatised to their corresponding alkyl chloroformates using a commercial kit (EZ:faast, Phenomenex, Torrance CA, USA). After derivatisation, the amino acids were extracted into solvent and 2 µl injected in...

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“…SH, OH, NH and COOH) with nonpolar trimethylsilane groups (TMS). According to some studies, by replacing acidic protons with nonpolar TMS groups, polarity of an amino acid is reduced and its vola" lity is increased [11][12][13][14][15][16][17] which is an advantage in gas chromatographic analysis.…”

Section: Resultsmentioning

confidence: 99%

Method for determination of amino acid content in protein products for medical use

Macovescu¹,

Chelaru²,

Gurau³

2017

LFJ

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ABSTRACT. The paper presents a method for determina$ on of amino acid content in collagen biomaterials for medical use produced in the Collagen Department of INCDTP -Division ICPI and its valida$ on. The method has three stages: the fi rst consists in hydrolysing the collagen biomaterial sample down to amino acids; the second step refers to deriva$ za$ on of amino acids with N, O-bis (trimethylsilyl) trifl uoroacetamide (BSTFA) which is a silyla$ on reagent; deriva$ zed amino acids are detected using a mass spectrometer a+ er gas chromatography. The method was validated to establish performance parameters and to check compliance with the intended purpose. KEY WORDS: collagen, amino acids, biomaterials METODĂ PENTRU DETERMINAREA CONŢINUTULUI DE AMINOACIZI DIN PRODUSELE PROTEICE PENTRU UZ MEDICALREZUMAT. Se prezintă o metodă de determinare a conţinutului de aminoacizi din biomaterialele colagenice pentru uz medical produse în Departamentul Colagen al INCDTP -Sucursala ICPI şi validarea ei. Metoda are trei etape: prima constă în hidroliza probei de biomaterial colagenic până la stadiul de aminoacizi; a doua etapă se referă la deriva$ zarea aminoacizilor cu N, O-bis (trime$ lsilil) trifl uoracetamidă (BSTFA) care este un reac$ v de sililare; aminoacizii deriva$ zaţi sunt detectaţi pe un spectrometru de masă după gaz cromatografi e. Metoda a fost validată pentru a se stabili parametrii de performanţă şi pentru verifi carea conformării cu scopul propus. CUVINTE CHEIE: colagen, aminoacizi, biomateriale PROCÉDÉ DE DÉTERMINATION DE LA TENEUR EN ACIDES AMINÉS DANS DES PRODUITS PROTÉIQUES À USAGE MÉDICALRÉSUMÉ. On présente une méthode de détermina$ on de la teneur en acides aminés de biomatériaux de collagène à usage médical fabriqués dans le Département Collagène de INCDTP-ICPI et sa valida$ on. La méthode comporte trois étapes : la première consiste en l'hydrolyse de l'échan$ llon de biomatériau de collagène au stade des acides aminés ; la deuxième étape se rapporte à la dériva$ sa$ on d'acides aminés avec du N, O-bis (triméthylsilyl) trifl uoroacétamide (BSTFA) qui est un réac$ f de silyla$ on ; les acides aminés dérivés sont détectés sur un spectromètre de masse après chromatographie en phase gazeuse. La méthode a été validée pour établir les paramètres de performance et vérifi er la conformité avec l'objec$ f prévu.

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Automated GC–MS analysis of free amino acids in biological fluids

Cited by 169 publications

References 44 publications

Mistargeting of Peroxisomal EHHADH and Inherited Renal Fanconi's Syndrome

Mistargeting of Peroxisomal EHHADH and Inherited Renal Fanconi's Syndrome

Plasma sarcosine does not distinguish early and advanced stages of prostate cancer

Method for determination of amino acid content in protein products for medical use

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