Skip to main content
SpringerLink
Log in

Intracellular alpha-keto acid quantification by fluorescence-HPLC

  • Review Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Procedures for the analysis of free α-keto acids in human fluids (i.e. plasma, cerebrospinal fluid, urine, etc.) as well as for studying the dynamic free α-keto acid pools in differentiated tissues and organ cells have been the subject of growing clinical interest in the study of metabolic regulatory and pathophysiological phenomena. Due to the high instability and polarity of the α-keto acids being examined, the development of a quantitative and reproducible analysis of metabolically relevant intracellular α-keto acids still presents a substantial methodological challenge. The aim of small sample size, rapid, non-damaging and “metabolism-neutral” cell isolation, careful sample preparation and stability, as well as reproducible analytics technology is not often achieved. Only few of the methods described can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular α-keto acid analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Al Sawaf HA, Al Ghamdi MA, Al Bekairi AM, Tawfik AF, Abu-Jayyab AR (1993) Changes in free amino acids in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes after treatment with diazepam. Int J Immunopharmacol 15:455–462

    PubMed  CAS  Google Scholar 

  • Alexander RT, Grinstein S (2006) Na+/H+ exchangers and the regulation of volume. Acta Physiol (Oxf) 187:159–167

    CAS  Google Scholar 

  • Algermissen B, Nündel M, Riedel E (1989) Analytik von Aminosäuren mit Fluoreszenz-HPLC. GIT Fachzeitschrift für das Laboratorium 33:783–790

    CAS  Google Scholar 

  • Andrews FJ, Griffiths RD (2002) Glutamine: essential for immune nutrition in the critically ill. Br J Nutr 87(Suppl 1):S3–S8

    PubMed  CAS  Google Scholar 

  • Aussel C, Cynober L, Lioret N, Coudray-Lucas C, Vaubourdolle M, Saizy R, Giboudeau J (1986) Plasma branched-chain keto acids in burn patients. Am J Clin Nutr 44:825–831

    PubMed  CAS  Google Scholar 

  • Aussel C, Cynober L, Giboudeau J (1987) Gas chromatography of branched-chain keto acids analysed as their silylated oxime derivatives. J Chromatogr 423:270–277

    PubMed  CAS  Google Scholar 

  • Baron DN, Ahmed SA (1969) Intracellular concentrations of water and of the principal electrolytes determined by analysis of isolated human leucocytes. Clin Sci 37:205–219

    PubMed  CAS  Google Scholar 

  • Baumgartner RN (1993) Body composition in elderly persons: a critical review of needs and methods. Prog Food Nutr Sci 17:223–260

    PubMed  CAS  Google Scholar 

  • Baumgartner RN, Stauber PM, McHugh D, Wayne S, Garry PJ, Heymsfield SB (1993) Body composition in the elderly using multicompartmental methods. Basic Life Sci 60:251–254

    PubMed  CAS  Google Scholar 

  • Bender DA (1985) Amino acid metabolism, 2nd edn. Chichester, New York

    Google Scholar 

  • Bennet WM, O’Keefe SJ, Haymond MW (1993) Comparison of precursor pools with leucine, alpha-ketoisocaproate, and phenylalanine tracers used to measure splanchnic protein synthesis in man. Metabolism 42:691–695

    PubMed  CAS  Google Scholar 

  • Best L (1997) Glucose and alpha-ketoisocaproate induce transient inward currents in rat pancreatic beta cells. Diabetologia 40:1–6

    PubMed  CAS  Google Scholar 

  • Biolo G, Toigo G, Ciocchi B, Situlin R, Iscra F, Gullo A, Guarnieri G (1997) Metabolic response to injury and sepsis: changes in protein metabolism. Nutrition 13:52–57

    Google Scholar 

  • Böyum A (1983) Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium. Scand J Immunol 17:429–436

    PubMed  Google Scholar 

  • Böyum A (1984) Separation of lymphocytes, granulocytes, and monocytes from human blood using iodinated density gradient media. Methods Enzymol 108:88–102

    PubMed  Google Scholar 

  • Brodelius P (1984) High-performance liquid chromatographic analysis of analogous amino and oxo acids for the determination of amino acid oxidase and transaminase activities. Acta Chem Scand B 38:219–223

    PubMed  CAS  Google Scholar 

  • Canepa A, Perfumo F, Carrea A, Sanguineti A, Piccardo MT, Gusmano R (1989) Measurement of free amino acids in polymorphonuclear leukocytes by high-performance liquid chromatography. J Chromatogr 491:200–208

    PubMed  CAS  Google Scholar 

  • Canepa A, Filho JC, Gutierrez A, Carrea A, Forsberg AM, Nilsson E, Verrina E, Perfumo F, Bergstrom J (2002) Free amino acids in plasma, red blood cells, polymorphonuclear leukocytes, and muscle in normal and uraemic children. Nephrol Dial Transplant 17:413–421

    PubMed  CAS  Google Scholar 

  • Carrea A, Canepa A, Perfumo F, Ancarani P, Verrina E, Gusmano R (1993) Proteolytic activity and free amino acid concentrations in polymorphonuclear leukocytes. Ann Clin Biochem 30:54–64

    Google Scholar 

  • Chen JG, Kempson SA (1995) Osmoregulation of neutral amino acid transport. Proc Soc Exp Biol Med 210:1–6

    PubMed  CAS  Google Scholar 

  • Delano MJ, Moldawer LL (2006) The origins of cachexia in acute and chronic inflammatory diseases. Nutr Clin Pract 21:68–81

    PubMed  Google Scholar 

  • Early RJ, James R, Thompson R (1984) Branched-chain alpha-ketoacid analysis in biologic fluids. J Chromatogr 310:1–10

    PubMed  CAS  Google Scholar 

  • Engel JM, Mühling J, Weiss S, Kärcher B, Löhr T, Menges T, Little S, Hempelmann G (2006) Relationship of taurine and other amino acids in plasma and in neutrophils of septic trauma patients. Amino Acids 30:87–94

    PubMed  CAS  Google Scholar 

  • Engelhardt H (1986) Praxis der Hochdruck-Flüssigkeits-Chromatographie. Springer, Heidelberg. 1. Auflage

    Google Scholar 

  • Farshidfar G (1990) Vergleichende Untersuchungen zur Bestimmung von alpha-Ketosäuren mittels GLC und HPLC. Diplomarbeit im Fach Biochemie an der Freien Universität Berlin

  • Fauth U, Heinrichs W, Puente-Gonzalez I, Halmagyi M (1990) Maximale Umsatzraten an Enzymen der Glykolyse und des Zitratzyklus von separierten Granulozyten in der postoperativen Phase. [Maximal turnover rates of glycolysis enzymes and of the citrate cycle of separated granulocytes in the postoperative period]. Infusionstherapie 17:178–183

    PubMed  CAS  Google Scholar 

  • Fauth U, Schlechtriemen T, Heinrichs W, Puente-Gonzalez I, Halmagyi M (1993) The measurement of enzyme activities in the resting human polymorphonuclear leukocyte––critical estimate of a method. Eur J Clin Chem Clin Biochem 31:5–16

    PubMed  CAS  Google Scholar 

  • Fernandes AA, Kalhan SC, Njoroge FG, Matousek GS (1986) Quantitation of branched-chain alpha-keto acids as their N-methylquinoxalone derivatives: comparison of O- and N-alkylation versus -silylation. Biomed Environ Mass Spectrom 13:569–581

    PubMed  CAS  Google Scholar 

  • Folin O, Denis W (1912) Tyrosine in proteins as determined by a new calorimetric method. J Biol Chem 12:245–251

    CAS  Google Scholar 

  • Frexes-Steed M, Warner ML, Bulus N, Flakoll P, Abumrad NN (1990) Role of insulin and branched-chain amino acids in regulating protein metabolism during fasting. Am J Physiol 258:E907–E917

    PubMed  CAS  Google Scholar 

  • Frexes-Steed M, Lacy DB, Collins J, Abumrad NN (1992) Role of leucine and other amino acids in regulating protein metabolism in vivo. Am J Physiol 262:E925–E935

    PubMed  CAS  Google Scholar 

  • Frigerio A, Martelli P (1973) Separation of chinoxalinole-derivatives using gas chromatography. J Chromatogr 81:139–150

    CAS  Google Scholar 

  • Fuchs M (1990) Entwicklung einer vollautomatischen quantitativen Aminosäurebestimmung mittels hochauflösender Liquidchromatographie nach Fluoreszenzderivatisierung. Diplomarbeit im Fach Biochemie an der Freien Universität Berlin

  • Fuchs M (1998) Untersuchungen zur Regulation der Homöostase im Aminosäure und alpha-Ketosäurestoffwechsel von Hepatocyten im Perfusionsmodell. Inaugural dissertation im Fach Biochemie an der Freien Universität Berlin

  • Fuchs M, Riedel E (1994) Quantitative fluoreszenz-HPLC, Analytik von Aminosäuren und Polyaminen. GIT Verlag, Darmstadt

    Google Scholar 

  • Fuchs M, Gerlach J, Unger J, Encke J, Smith M, Neuhaus P, Nündel M, Riedel E (1994) Alpha-keto acid metabolism by hepatocytes cultured in a hybrid liver support bioreactor. Int J Artif Organs 17:554–558

    PubMed  CAS  Google Scholar 

  • Funchal C, Tramontina F, Dos Santos AQ, de Souza DF, Goncalves CA, Pessoa-Pureur R, Wajner M (2007) Effect of the branched-chain alpha-keto acids accumulating in maple syrup urine disease on S100B release from glial cells. J Neurol Sci 260(1–2):87–94. May 11, Epub ahead of print

    PubMed  CAS  Google Scholar 

  • Fürst P (1998) Old and new substrates in clinical nutrition. J Nutr 128:789–796

    PubMed  Google Scholar 

  • Fürst P, Stehle P, Graser TA (1987) Fortschritte in der Aminosäurenanalytik unter besonderer Berücksichtigung der Ermittlung intrazellulärer Aminosäurenmuster. Infusionsther Klin Ernähr 14:137–146

    PubMed  Google Scholar 

  • Fürst P, Alvestrand A, Bergstrom J (1992) Branched-chain amino acids and branched-chain ketoacids in uremia. Contrib Nephrol 98:44–58

    PubMed  Google Scholar 

  • Fürst P, Pogan K, Hummel M, Herzog B, Stehle P (1997) Design of parenteral synthetic dipeptides for clinical nutrition: in vitro and in vivo utilization. Ann Nutr Metab 41:10–21

    PubMed  Google Scholar 

  • Gerlach JC, Fuchs M, Smith MD, Bornemann R, Encke J, Neuhaus P, Riedel E (1996) Is a clinical application of hybrid liver support systems limited by an initial disorder in cellular amino acid and alpha-keto acid metabolism, rather than by later gradual loss of primary hepatocyte function? Transplantation 62:224–228

    PubMed  CAS  Google Scholar 

  • Grimble RF (2001) Nutritional modulation of immune function. Proc Nutr Soc 60:389–397

    PubMed  CAS  Google Scholar 

  • Grimble RF, Grimble GK (1998) Immunonutrition: role of sulfur amino acids, related amino acids, and polyamines. Nutrition 14:605–610

    PubMed  CAS  Google Scholar 

  • Hammarqvist F, Wernerman J, von der Decken A, Vinnars E (1990) Alanyl-glutamine counteracts the depletion of free glutamine and the postoperative decline in protein synthesis in skeletal muscle. Ann Surg 212(5):637–644

    PubMed  CAS  Google Scholar 

  • Hammarqvist F, Ejesson B, Wernerman J (2001) Stress hormones initiate prolonged changes in the muscle amino acid pattern. Clin Physiol 21:44–50

    PubMed  CAS  Google Scholar 

  • Hanke MT, Koessler KK (1920) Studies on proteinogenous amines. IV. The quantitative colorimetric method for estimating imidazole derivatives. J Biol Chem 43:527–542

    CAS  Google Scholar 

  • Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y (1985) Determination of alpha-keto acids in serum and urine by high-performance liquid chromatography with fluorescence detection. J Chromatogr 344:33–39

    PubMed  CAS  Google Scholar 

  • Hasselgren PO (2000) Catabolic response to stress and injury: implications for regulation. World J Surg 24:1452–1459

    PubMed  CAS  Google Scholar 

  • Hasselgren PO, Fischer JE (1998) Sepsis: stimulation of energy-dependent protein breakdown resulting in protein loss in skeletal muscle. World J Surg 22:203–208

    PubMed  CAS  Google Scholar 

  • Hayashi T, Sugiura T, Terada H, Kawai S, Ohno T (1976) High-speed liquid chromatographic determination of phenylpyruvic acid. J Chromatogr 118:403–408

    PubMed  CAS  Google Scholar 

  • Hayashi T, Tsuchiya H, Todoriki H, Naruse H (1982) High-performance liquid chromatographic determination of alpha-keto acids in human urine and plasma. Anal Biochem 122:173–179

    PubMed  CAS  Google Scholar 

  • Hayashi T, Tsuchiya H, Naruse H (1983) High-performance liquid chromatographic determination of alpha-keto acids in plasma with fluorometric detection. J Chromatogr 273:245–252

    PubMed  CAS  Google Scholar 

  • Henschen A, Hupf KP, Lottspeich F (1989) High-performance-liquid-chromatography in biochemistry. VCH Verlagsgesellschaft, Weinheim, 1. Auflage

  • Hockenhull DJ, Floodgate GD (1952) o-Phenylenediamine and 1:2-diamino-4-nitrobenzene as reagents for a-keto acids. Biochem J 52:38–40

    PubMed  CAS  Google Scholar 

  • Holecek M (2002) Relation between glutamine, branched-chain amino acids, and protein metabolism. Nutrition 18:130–133

    PubMed  CAS  Google Scholar 

  • Holecek M, Sprongl L, Tichy M, Pecka M (1998) Leucine metabolism in rat liver after a bolus injection of endotoxin. Metabolism 47:681–685

    PubMed  CAS  Google Scholar 

  • Horber FF, Kahl J, Lecavalier L, Krom B, Haymond MW (1989) Determination of leucine and alpha-ketoisocaproic acid concentrations and specific activity in plasma and leucine specific activities in proteins using high-performance liquid chromatography. J Chromatogr 495:81–94

    PubMed  CAS  Google Scholar 

  • Horvath CG, Preiss BA, Lipsky SR (1967) Fast liquid chromatography: an investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers. Anal Chem 39:1422–1428

    PubMed  CAS  Google Scholar 

  • Huber JFK (1969) High efficiency, high speed liquid chromatography in columns. J Chromatogr Sci 7:85–90

    CAS  Google Scholar 

  • Johnson C, Metcoff J (1986) Relation of protein synthesis to plasma and cell amino acids in neonates. Pediatr Res 20:140–146

    PubMed  CAS  Google Scholar 

  • Kallio H, Linko RR (1972) Gas-liquid chromatographic analysis of 2,4-dinitrophenylhydrazones of carbonyl compounds. J Chromatogr 65:355–360

    CAS  Google Scholar 

  • Kallio H, Linko RR (1973) Gas-liquid chromatographic analysis of 2,4-dinitrophenylhydrazones of keto acid methyl esters. J Chromatogr 76:229–232

    PubMed  CAS  Google Scholar 

  • Kapeller-Adler R (1933) Über eine neue Methode zur quantitativen Histidinbestimmung und über deren Anwendbarkeit zur Untersuchung von biologischen Flüssigkeiten, insbesondere von Gravidenharnen. Biochem Z 264:131–141

    CAS  Google Scholar 

  • Katrukha SP, Kukes VG (1986) Derivatization of endogenous and exogenous compounds in plasma for high-performance liquid chromatographic analysis. J Chromatogr 365:105–110

    PubMed  CAS  Google Scholar 

  • Kiba N, Muto M, Furusawa M (1989) High-performance liquid chromatographic determination of branched-chain alpha-keto acids in serum using immobilized leucine dehydrogenase as post-column reactor. J Chromatogr 497:236–242

    PubMed  CAS  Google Scholar 

  • Kieber DJ, Mopper K (1983) RP-HPLC analysis of alpha-ketoacid quinoxalinol. J Chromatogr 281:135–144

    CAS  Google Scholar 

  • Kielducka A, Paradies G, Papa S (1981) A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats. J Bioenerg Biomembr 13:123–132

    PubMed  CAS  Google Scholar 

  • Kirkland J (1971) High speed liquid-partition chromatography with chemically bonded organic stationary phases. J Chromatogr Sci 9:206–214

    CAS  Google Scholar 

  • Kirkland J (2000) Ultrafast reversed-phase high-performance liquid chromatographic separations: an overview. J Chromatogr Sci 38:535–544

    PubMed  CAS  Google Scholar 

  • Kjeldahl J (1883) Neue Methode zur Bestimmung des Stickstoffs in organischen Körpern. Z Anal Chem 22:366–382

    Article  Google Scholar 

  • Koessler KK, Hanke MT (1919) Studies on proteinogenous amines. II. A microchemical colorimetric method for estimating imidazole derivatives. J Biol Chem 39:497–519

    CAS  Google Scholar 

  • Koike K, Koike M (1984) Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography. Anal Biochem 141:481–487

    PubMed  CAS  Google Scholar 

  • Lang F, Busch GL, Völkl H (1998) The diversity of volume regulatory mechanisms. Cell Physiol Biochem 8:1–45

    PubMed  CAS  Google Scholar 

  • Law RO (1991) Amino acids as volume-regulatory osmolytes in mammalian cells. Comp Biochem Physiol A 99:263–277

    PubMed  CAS  Google Scholar 

  • Learn DB, Fried VA, Thomas EL (1990) Taurine and hypotaurine content of human leukocytes. J Leukoc Biol 48:174–182

    PubMed  CAS  Google Scholar 

  • Livesey G, Edwards TE (1985) Quantification of branched chain a-keto-acids as quinoxalinols: importance of excluding oxygen during derivatisation. J Chromatogr B Biomed Sci Appl 337:98–102

    CAS  Google Scholar 

  • Mårtensson J (1986) The effect of fasting on leukocyte and plasma glutathione and sulfur amino acid concentrations. Metabolism 35:118–121

    PubMed  Google Scholar 

  • Mårtensson J, Larsson J, Nordstrom H (1987) Amino acid metabolism during the anabolic phase of severely burned patients: with special reference to sulphur amino acids. Eur J Clin Invest 17:130–135

    PubMed  Google Scholar 

  • Martin AJP, Synge RLM (1941) A new form of chromatogramm employing two liquid phases. Biochem J 35:1358–1368

    PubMed  CAS  Google Scholar 

  • Matsukawa T, Hasegawa H, Shinohara Y, Hashimoto T (2001) Gas chromatographic-mass spectrometric determination of alpha-ketoisocaproic acid and [2H7]alpha-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine. J Chromatogr B Biomed Sci Appl 751:213–220

    PubMed  CAS  Google Scholar 

  • Matsuo Y, Yagi M, Walser M (1993) Arteriovenous differences and tissue concentrations of branched-chain ketoacids. J Lab Clin Med 121:779–784

    PubMed  CAS  Google Scholar 

  • Metcoff J (1986) Intracellular amino acid levels as predictors of protein synthesis. J Am Coll Nutr 5:107–120

    PubMed  CAS  Google Scholar 

  • Metcoff J, Fürst P, Scharer K, Distler G, Weber R, Mangold J, Graser TA, Pfaff G, Schönberg D (1989) Energy production, intracellular amino acid pools, and protein synthesis in chronic renal disease. J Am Coll Nutr 8:271–284

    PubMed  CAS  Google Scholar 

  • Meyer V (1986) Praxis der HPLC. Verlag Diesterweg-Salle-Sauerländer, Frankfurt am Main

    Google Scholar 

  • Milley JR, Sweeley JC (1993) High-performance liquid chromatographic measurement of leucine and alpha-ketoisocaproate in whole blood: application to fetal protein metabolism. J Chromatogr 613:23–33

    PubMed  CAS  Google Scholar 

  • Moore S, Stein WH (1948a) Chromatography of amino acids on starch columns. Solvent mixtures for the fractionation of protein hydrolysates. J Biol Chem 178:53–77

    Google Scholar 

  • Moore S, Stein WH (1948b) Photometric ninhydrin method for use in the chromatography with starch. J Biol Chem 176:367–388

    PubMed  CAS  Google Scholar 

  • Moore S, Stein WH (1952) Chromatography of amino acids on sulfonated polystyrene resins. J Biol Chem 192:663–681

    Google Scholar 

  • Mühling J, Fuchs M, Dehne MG, Sablotzki A, Menges T, Weber B, Hempelmann G (1999) Quantitative determination of free intracellular amino acids in single human polymorphonuclear leucocytes. Recent developments in sample preparation and high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 728:157–166

    PubMed  Google Scholar 

  • Mühling J, Fuchs M, Campos ME, Gonter J, Engel JM, Sablotzki A, Menges T, Weiss S, Dehne MG, Krüll M, Hempelmann G (2003) Quantitative determination of free intracellular alpha-keto acids in neutrophils. J Chromatogr B Analyt Technol Biomed Life Sci 789:383–392

    PubMed  Google Scholar 

  • Mühling J, Burchert D, Langefeld TW, Matejec R, Harbach H, Engel J, Wolff M, Welters ID, Fuchs M, Menges T, Krüll M, Hempelmann G (2006a) Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and alpha-keto acid homeostasis or immunocompetence. Amino Acids 33(3):511–524. Oct 31, Epub ahead of print

    PubMed  Google Scholar 

  • Mühling J, Engel J, Halabi M, Müller M, Fuchs M, Krüll M, Harbach H, Langefeld TW, Wolff M, Matejec R, Welters ID, Menges T, Hempelmann G (2006b) Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and alpha-keto acid profiles or host defense capability. Amino Acids 31:11–26

    PubMed  Google Scholar 

  • Mühling J, Paddenberg R, Hempelmann G, Kummer W (2006c) Hypobaric hypoxia affects endogenous levels of alpha-keto acids in murine heart ventricles. Biochem Biophys Res Commun 342:935–939

    PubMed  Google Scholar 

  • Mühling J, Nickolaus KA, Matejec R, Langefeld TW, Harbach H, Engel J, Wolff M, Weismüller K, Fuchs M, Welters ID, Krüll M, Heidt MC, Hempelmann G (2007) Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and alpha-keto acid homeostasis? Amino Acids (Mar 2, Epub ahead of print)

  • Nakahara T, Ishida J, Yamaguchi M, Nakamura M (1990) Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection. Anal Biochem 190:309–313

    PubMed  CAS  Google Scholar 

  • Nissen SL, Van Huysen C, Haymond MW (1981) Measurement of plasma alpha-ketoisocaproate concentrations and specific radioactivity by high-performance liquid chromatography. Anal Biochem 110:389–392

    PubMed  CAS  Google Scholar 

  • Nissen SL, Van Huysen C, Haymond MW (1982) Measurement of branched chain amino acids and branched chain alpha-ketoacids in plasma by high-performance liquid chromatography. J Chromatogr 232:170–175

    PubMed  CAS  Google Scholar 

  • Pailla K, Blonde-Cynober F, Aussel C, De Bandt JP, Cynober L (2000) Branched-chain keto-acids and pyruvate in blood: measurement by HPLC with fluorimetric detection and changes in older subjects. Clin Chem 46:848–853

    PubMed  CAS  Google Scholar 

  • Pereira C, Murphy KD, Herndon DN (2005a) Altering metabolism. J Burn Care Rehabil 26:194–199

    PubMed  Google Scholar 

  • Pereira C, Murphy K, Jeschke M, Herndon DN (2005b) Post burn muscle wasting and the effects of treatments. Int J Biochem Cell Biol 37:1948–1961

    PubMed  CAS  Google Scholar 

  • Qureshi GA (1987) High-performance liquid chromatographic methods with fluorescence detection for the determination of branched-chain amino acids and their alpha-keto analogues in plasma samples of healthy subjects and uraemic patients. J Chromatogr 400:91–99

    PubMed  CAS  Google Scholar 

  • Radeck W, Beck K, Staib W (1988) Simple method for rapid quantification of branched-chain 2-oxo acids in physiological fluids as quinoxalinol derivatives by high-performance liquid chromatography. J Chromatogr 432:297–301

    PubMed  CAS  Google Scholar 

  • Reid CL (2004) Nutritional requirements of surgical and critically-ill patients: do we really know what they need? Proc Nutr Soc 63:467–472

    PubMed  Google Scholar 

  • Riedel E, Hampl H, Nündel M, Busche D, Fuchs M (1992a) Severity of anaemia influences pattern of amino acids and alpha-keto acids in haemodialysis patients. Contrib Nephrol 98:98–104

    PubMed  CAS  Google Scholar 

  • Riedel E, Hampl H, Nündel M, Farshidfar G (1992b) Essential branched-chain amino acids and alpha-ketoanalogues in haemodialysis patients. Nephrol Dial Transplant 7:117–120

    PubMed  CAS  Google Scholar 

  • Riedel E, Nündel M, Hampl H (1996) alpha-Ketoglutarate application in hemodialysis patients improves amino acid metabolism. Nephron 74:261–265

    Article  PubMed  CAS  Google Scholar 

  • Riedel E, Nündel M, Wendel G, Hampl H (2000) Amino acid and alpha-keto acid metabolism depends on oxygen availability in chronic hemodialysis patients. Clin Nephrol 53:S56-S60

    PubMed  CAS  Google Scholar 

  • Rocchiccioli F, Leroux JP, Cartier P (1981) Quantitation of 2-ketoacids in biological fluids by gas chromatography chemical ionization mass spectrometry of O-trimethylsilyl-quinoxalinol derivatives. Biomed Mass Spectrom 8:160–164

    PubMed  CAS  Google Scholar 

  • Roth E, Karner J (1987) Intrazelluläre Aminosäurekonzentrationen bei verschiedenen Krankheitszuständen. Infusionsther Klin Ernähr 14:147–150

    PubMed  CAS  Google Scholar 

  • Roth E, Karner J, Roth-Merten A, Winkler S, Valentini L, Schaupp K (1991) Effect of alpha-ketoglutarate infusions on organ balances of glutamine and glutamate in anaesthetized dogs in the catabolic state. Clin Sci (Lond) 80:625–631

    CAS  Google Scholar 

  • Sakaguchi S (1925) Über eine neue Farbenreaktion von Protein und Arginin. J Biochem 5:25–31

    CAS  Google Scholar 

  • Schäfer G, Schauder P (1988) Assessment of effects of amino acids and branched chain keto acids on leucine oxidation in human lymphocytes. Scand J Clin Lab Invest 48:531–536

    PubMed  Google Scholar 

  • Shestopalov AI, Kristal BS (2007) Branched chain keto-acids exert biphasic effects on alpha-ketoglutarate-stimulated respiration in intact rat liver mitochondria. Neurochem Res 32:947–951

    PubMed  CAS  Google Scholar 

  • Smeaton TC, Owens JA, Robinson JS (1989) Micro-method for measurement of branched-chain keto acid concentrations in plasma from sheep and man. J Chromatogr 487:434–439

    PubMed  CAS  Google Scholar 

  • Spackman DH, Stein WH, Moore S (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30:1190–1206

    CAS  Google Scholar 

  • Stein WH, Moore S (1948) Chromatography on starch columns. Separation of phenylalanine, leucine, isoleucine, methionine, tyrosine and valine. J Biol Chem 176:337–366

    PubMed  CAS  Google Scholar 

  • Strange K (2004) Cellular volume homeostasis. Adv Physiol Educ 28:155–159

    PubMed  Google Scholar 

  • Synge RLM (1944) Analysis of a partial hydrolysate of gramidicin by partition chromatography with starch. Biochem J 38:285–294

    PubMed  CAS  Google Scholar 

  • Teigland M, Klungsayr L (1983a) Accumulation of alpha-ketoisocapronat from leucine in homogenates of tissue from rainbow and rat. Comp Biochem Physiol A 75:703–705

    CAS  Google Scholar 

  • Teigland M, Klungsayr L (1983b) Accumulation of alpha-ketoisocaproate from leucine in homogenates of tissues from rainbow trout (Salmo gairdnerii) and rat. An improved method for determination of branched chain keto acids. Comp Biochem Physiol B 75:703–705

    PubMed  CAS  Google Scholar 

  • Trachtman H (1991) Cell volume regulation: a review of cerebral adaptive mechanisms and implications for clinical treatment of osmolal disturbances. I. Pediatr Nephrol 5:743–750

    PubMed  CAS  Google Scholar 

  • Tsuchiya H, Hashizume I, Tokunaga T, Tatsumi M, Takagi N, Hayashi T (1983) High-performance liquid chromatography of alpha-keto acids in human saliva. Arch Oral Biol 28:989–992

    PubMed  CAS  Google Scholar 

  • Tsuchiya H, Sato M, Yamamoto K, Yamauchi M, Tani H, Namikawa I, Takagi N (1990) High-performance liquid chromatographic analysis of alpha-keto acids produced from amino acid metabolism in oral bacteroides. J Appl Bacteriol 69:125–133

    PubMed  CAS  Google Scholar 

  • Van Slyke DD (1911) The analysis of proteins by determination of the chemical group characteristics of the different amino-acids. J Biol Chem 10:15–55

    Google Scholar 

  • Van Slyke DD (1912) The quantitative determination of aliphatic amino groups. J Biol Chem 12:275–284

    Google Scholar 

  • Verbalis JG (2006) Whole-body volume regulation and escape from antidiuresis. Am J Med 119:S21–S29

    PubMed  Google Scholar 

  • Walser M, Swain LM, Alexander V (1987) Measurement of branched-chain ketoacids in plasma by high-performance liquid chromatography. Anal Biochem 164:287–291

    PubMed  CAS  Google Scholar 

  • Walser M, Jarskog FL, Hill SB (1989) Branched-chain-ketoacid metabolism in patients with chronic renal failure. Am J Clin Nutr 50:807–813

    PubMed  CAS  Google Scholar 

  • Wang ZJ, Zaitsu K, Ohkura Y (1988) High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent. J Chromatogr 430:223–231

    PubMed  CAS  Google Scholar 

  • Wang X, Jurkovitz C, Price SR (1997) Branched-chain amino acid catabolism in uremia: dual regulation of branched-chain alpha-ketoacid dehydrogenase by extracellular pH and glucocorticoids. Miner Electrolyte Metab 23:206–209

    PubMed  CAS  Google Scholar 

  • Willems HL, de Kort TF, Trijbels FJ, Monnens LA, Veerkamp JH (1978) Determination of pyruvate oxidation rate and citric acid cycle activity in intact human leukocytes and fibroblasts. Clin Chem 24:200–203

    PubMed  CAS  Google Scholar 

  • Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L (2000) Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest 80:617–653

    PubMed  CAS  Google Scholar 

  • Woolf LI, Hasinoff C, Perry A (1982) Estimation of branched-chain alpha-keto acids in blood by gas chromatography. J Chromatogr 231:237–245

    PubMed  CAS  Google Scholar 

  • Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 208:2819–2830

    PubMed  CAS  Google Scholar 

  • Young E, Bradley RF (1967) Cerebral edema with irreversible coma in severe diabetic ketoacidosis. N Engl J Med 276:665–669

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dr. Ing. Herbert Knauer GmbH, Berlin, Germany

    M. Fuchs & M. Campos

  2. Clinics of Anaesthesiology, Intensive Care Medicine and Pain Therapy, University Hospital Giessen and Marburg GmbH, Campus Giessen, Justus-Liebig-University Giessen, Rudolf-Buchheim-Strasse 7, 35385, Giessen, Germany

    J. Engel, R. Matejec, M. Henrich, H. Harbach, M. Wolff, K. Weismüller, T. Menges, G. Hempelmann & J. Mühling

  3. Department of Cardiovascular Surgery, University Hospital Giessen and Marburg, Justus-Liebig-University Giessen, Giessen, Germany

    M. C. Heidt

  4. University Department of Anaesthesia, Royal Liverpool University Hospital, Liverpool, UK

    I. D. Welters

  5. Department of Internal Medicine/Infectious Diseases, Charité, Medical School of Humboldt University, Berlin, Germany

    M. Krüll

Authors
  1. M. Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Engel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Campos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Matejec
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Henrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. H. Harbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Wolff
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. K. Weismüller
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. T. Menges
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. C. Heidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. I. D. Welters
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. M. Krüll
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. G. Hempelmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. J. Mühling
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Mühling.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fuchs, M., Engel, J., Campos, M. et al. Intracellular alpha-keto acid quantification by fluorescence-HPLC. Amino Acids 36, 1–11 (2009). https://doi.org/10.1007/s00726-008-0033-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-008-0033-x

Keywords

Search

Navigation