Amino Acids

, Volume 47, Issue 9, pp 1805–1815 | Cite as

Use of the guanidination reaction for determining reactive lysine, bioavailable lysine and gut endogenous lysine

  • Shane M. RutherfurdEmail author
Review Article
Part of the following topical collections:
  1. Homoarginine, Arginine and Relatives


Determining the bioavailability of lysine in foods and feedstuffs is important since lysine is often the first limiting indispensable amino acid in diets for intensively farmed livestock (pigs and poultry) and also in many cereal-based diets consumed by humans. When foods or feedstuffs are heat processed, lysine can undergo Maillard reactions to produce nutritionally unavailable products. The guanidination reaction, the reaction of O-methylisourea with the side chain amino group of lysine that produces homoarginine, has been used to determine the unmodified lysine (reactive lysine) in processed foods and feedstuffs and also true ileal digestible reactive lysine (bioavailable lysine). The advantages of the guanidination method in comparison with other reactive lysine methods such as the fluorodinitrobenzene, trinitrobenzenesulphonic acid and dye-binding methods are that it is very specific for reactive lysine and also that the method is relatively straightforward to conduct. The specificity of the guanidination reaction for the lysine side chain amino group is particularly important, since ileal digesta will contain N-terminal groups in the form of free amino acids and peptides. The main disadvantage is that complete conversion of lysine to homoarginine is required, yet it is not straightforward to test for complete guanidination in processed foods and feedstuffs. Another disadvantage is that the guanidination reaction conditions may vary for different food types and sometimes within the same food type. Consequently, food-specific guanidination reaction conditions may be required and more work is needed to optimise the reaction conditions across different foods and feedstuffs.


Homoarginine Lysine Guanidination Reactive Bioavailable 


Conflict of interest

The author declares that there is no conflict of interest.


  1. Allen LC, Viswanatha T (1970) Reaction of amino acids with guanidinating agents. Can J Biochem 48:1189–1191CrossRefPubMedGoogle Scholar
  2. Almeida FN, Htoo JK, Thomson J, Stein HH (2014) Effects of heat treatment on the apparent and standardized ileal digestibility of amino acids in canola meal fed to growing pigs. Anim Feed Sci Technol 187:44–52CrossRefGoogle Scholar
  3. Angkanaporn K, Ravindran V, Mollah Y, Bryden WL (1997) Secretion of homoarginine into the gut of chickens. Vet Res Comm 21:161–167CrossRefGoogle Scholar
  4. Awati A, Rutherfurd SM, Kies AK, Veyry A, Moughan PJ (2009) Endogenous lysine in ileal digesta in the growing rat determined using different methods. J Sci Food Agric 89:2200–2206CrossRefGoogle Scholar
  5. Batterham ES, Murison RD, Lewis CE (1979) Availability of lysine in protein concentrates as determined by the slope-ratio assay with growing pigs and rats and by chemical techniques. Brit J Nutr 41:383–391CrossRefPubMedGoogle Scholar
  6. Beardsley RL, Reilly JP (2002) Optimization of guanidination procedures for MALDI mass mapping. Anal Chem 74:1884–1890CrossRefPubMedGoogle Scholar
  7. Booth VH (1971) Problems in the determination of FDNB-available lysine. J Sci Food Agric 22:658–666CrossRefPubMedGoogle Scholar
  8. Bunjapamai S, Mahoney RR, Fagerson IS (1982) Determination of D-amino acids in some processed foods and effect of racemization on in vitro digestibility of casein. J Food Sci 47:1229–1234CrossRefGoogle Scholar
  9. Butts CA, Moughan PJ, Smith WC (1991) Endogenous amino acid flow at the terminal ileum of the rat determined under conditions of peptide alimentation. J Sci Food Agric 55:175–187CrossRefGoogle Scholar
  10. Butts CA, Moughan PJ, Smith WC (1992) Protein nitrogen, peptide nitrogen and free amino acid nitrogen in endogenous digesta nitrogen at the terminal ileum of the rat. J Sci Food Agric 59:291–298CrossRefGoogle Scholar
  11. Caine WR, Sauer WC, Huang GS, Diebold G, Schollenberger M, Mosenthin R (2008) Influence of guanidination on apparent ileal digestibility of amino acids in pigs fed diets with soybean meal, rapeseed meal or peas as a protein source. Livest Sci 116:300–308CrossRefGoogle Scholar
  12. Carpenter KJ (1960) The estimation of available lysine in animal-protein foods. Biochem J 77:604–610PubMedCentralCrossRefPubMedGoogle Scholar
  13. Catrein I, Herrmann R, Bosserhoff A, Ruppert T (2005) Experimental proof for a signal peptidase 1 like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type 1 SPase. FEBS J 272:2892–2900CrossRefPubMedGoogle Scholar
  14. Chervenka CH, Wilcox PE (1956) Chemical derivatives of chymotrypsinogen. II Reaction with O-methylisourea. J Biol Chem 222:635–647PubMedGoogle Scholar
  15. de Lange CFM, Sauer WC, Souffrant WB (1989) The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum. J Anim Sci 67:755–762PubMedGoogle Scholar
  16. de Vrese M, Middendorf K, Hagemeister H (1994) Prevention of amino acid racemization during guanidination—a prerequisite for measurement of protein digestibility by homoarginine labelling. Z Ernahrungswiss 33:310–312CrossRefPubMedGoogle Scholar
  17. Desrosiers T, Savoie L, Bergeron G, Parent G (1989) Estimation of lysine damage in heated whey proteins by furosine determinations in conjunction with the digestion cell technique. J Agric Food Chem 37:1385–1391CrossRefGoogle Scholar
  18. Eklund M, Caine WR, Sauer WC, Huang GS, Diebold G, Schollenberger M, Mesenthin R (2013) Guanidination procedures increases standardised ileal digestibilities of nitrogen and amino acids in rapeseed meal, soybean meal and peas fed to growing pigs. Anim Prod Sci 53:265–273Google Scholar
  19. Erbersdobler HF, Hupe A (1991) Determination of lysine damage and calculation of lysine bio-availability in several processed foods. Z Ernährungswiss 30:46–49CrossRefPubMedGoogle Scholar
  20. Finot PA, Viani R, Bricout J, Mauron J (1969) Detection and identification of pyridosine, a second lysine derivative obtained upon acid hydrolysis of heated milk. Experientia 25:134–135CrossRefPubMedGoogle Scholar
  21. Finot PA, Bujard E, Mottu F, Mauron J (1977) Availability of the true Schiff’s bases of lysine. Chemical evaluation of the Schiff’s base between lysine and lactose in milk. In: Friedman M (ed) Protein crosslinking-B: nutritional and medical consequences. Plenum, New York, pp 343–366CrossRefGoogle Scholar
  22. Fontaine J, Zimmer U, Moughan PJ, Rutherfurd SM (2007) Effect of heat damage in an autoclave on the reactive lysine contents of soy products and corn distillers dried grains with solubles. Use of the results to check on lysine damage in common qualities of these ingredients. J Agric Food Chem 55:10737–10743CrossRefPubMedGoogle Scholar
  23. Friesen MJ, Kairie E, Hyachoti CM (2006) Ileal amino acid digestibility and reactive lysine content in peas (Pisum sativum) fed to growing pigs. Anim Feed Sci Technol 129:210–223CrossRefGoogle Scholar
  24. Fuller MF (2004) The encyclopaedia of farm animal nutrition. CAB Int, Wallingford, pp 448–451CrossRefGoogle Scholar
  25. Henle T, Walter H, Klostermeyer H (1991) Evaluation of the extent of the early Maillard reaction in milk products by direct measurement of the Amadori-product lactuloselysine. Z Lebensm -Forsch A 193:119–122CrossRefGoogle Scholar
  26. Hodgkinson SM, Moughan PJ (2007) An effect of dietary protein content on endogenous ileal lysine flow in the growing rat. J Sci Food Agric 87:233–238CrossRefGoogle Scholar
  27. Hodgkinson SM, Moughan PJ, Reynolds GW, James KA (2000) The effect of dietary peptide concentration on endogenous ileal amino acid loss in the growing pig. Br J Nutr 83:421–430PubMedGoogle Scholar
  28. Hodgkinson SM, Souffrant WB, Moughan PJ (2003) Comparison of the enzyme-hydrolyzed casein, guanidination, and isotope dilution methods for determining ileal endogenous protein flow in the growing rat and pig. J Anim Sci 81:2525–2534PubMedGoogle Scholar
  29. Hurrell RF, Carpenter KJ (1974) Mechanisms of heat damage in proteins 4. The reactive lysine content of heat-damaged material as measured in different ways. Br J Nutr 32:589–604CrossRefPubMedGoogle Scholar
  30. Hurrell RF, Carpenter KJ (1981) The estimation of available lysine in foodstuffs after Maillard reactions. Prog Food Nutr Sci 5:159–176PubMedGoogle Scholar
  31. Imbeah M, Angkanaporn K, Ravindran V, Bryden WL (1996) Investigations on the guanidination of lysine in proteins. J Sci Food Agric 72:213–218CrossRefGoogle Scholar
  32. Klee WA, Richards FM (1957) The reaction of O-methylisourea with bovine pancreatic ribonuclease. J Biol Chem 229:489–504PubMedGoogle Scholar
  33. Krause R, Knoll K, Henle T (2003) Studies on the formation of furosine and pyridosine during acid hydrolysis of different Amadori products of lysine. Eur Food Res Technol 216:277–283Google Scholar
  34. Maga JA (1981) Measurement of available lysine using the guanidination reaction. J Food Sci 46:132–134CrossRefGoogle Scholar
  35. Mao L-C, Lee K-H, Erbersbobler HF (1993) Effects of heat treatment on lysine in soya protein. J Sci Food Agric 62:307–309CrossRefGoogle Scholar
  36. Marty BJ, Chavez ER, de Lange CF (1994) Recovery of amino acids at the distal ileum for determining apparent and true ileal amino acid digestibilities in growing pigs fed various heat-processed full-fat soybean products. J Anim Sci 72:2029–2037PubMedGoogle Scholar
  37. Mauron J, Mottu F, Bujard E, Egli RH (1955) The availability of lysine, methionine and tryptophan in condensed milk and milk powder. In vitro digestion studies. Arch Biochem Biophys 59:433–451CrossRefPubMedGoogle Scholar
  38. Moehn S, Bertolo RFP, Pencharz PB, Ball RO (2005) Development of the indicator amino acid oxidation technique to determine the availability of amino acids from dietary protein in pigs. J Nutr 135:2866–2870PubMedGoogle Scholar
  39. Moughan PJ (2003) AA digestibility and availability in food and feedstuffs. In: Ball RO (ed) Digestive physiology in pigs. Proc. 9th Intl. Symp. Univ. Alberta, Alberta, Canada, pp 199–221Google Scholar
  40. Moughan PJ, Rutherfurd SM (1990) Endogenous flow of total lysine and other amino acids at the distal ileum of the protein- and peptide-fed rat: the chemical labelling of gelatin protein by transformation of lysine to homoarginine. J Sci Food Agric 52:179–192CrossRefGoogle Scholar
  41. Moughan PJ, Rutherfurd SM (1996) A new method for determining digestible reactive lysine in foods. J Agric Food Chem 44:2202–2209CrossRefGoogle Scholar
  42. Moughan PJ, Schuttert G (1991) Composition of nitrogen-containing fractions in digesta from the distal ileum of pigs fed a protein-free diet. J Nutr 121:1570–1574PubMedGoogle Scholar
  43. Moughan PJ, Darragh AJ, Smith WC, Butts CA (1990) Trichloroacetic and perchloric acids as precipitants of protein in endogenous digesta from the rat. J Sci Food Agric 52:13–21CrossRefGoogle Scholar
  44. Moughan PJ, Gall MPJ, Rutherfurd SM (1996) Absorption of lysine and deoxyketosyllysine in an early-Maillard browned casein by the growing pig. J Agric Food Chem 44:1520–1525CrossRefGoogle Scholar
  45. Nyachoti CM, de Lange CF, Schulze H (1997) Estimating endogenous amino acid flows at the terminal ileum and true ileal amino acid digestibilities in feedstuffs for growing pigs using the homoarginine method. J Anim Sci 75:3206–3213PubMedGoogle Scholar
  46. Nyachoti CM, McNeilage-Van de Wiele EM, de Lange CF, Gabert VM (2002) Evaluation of the homoarginine technique for measuring true ileal amino acid digestibilities in pigs fed a barley-canola meal-based diet. J Anim Sci 802:440–448Google Scholar
  47. Pahm AA (2008) Utilization of amino acids in corn distillers dried grains with solubles (DDGS) by pigs and poultry and the use of reactive lysine procedures to evaluate DDGS quality. Ph.D. dissertation, University of IllinoisGoogle Scholar
  48. Pahm AA, Pedersen C, Stein HH (2008) Application of the reactive lysine procedure to estimate lysine digestibility in distillers dried grains with solubles fed to growing pigs. J Agric Food Chem 56:9441–9446CrossRefPubMedGoogle Scholar
  49. Pahm AA, Pedersen C, Simon D, Stein HH (2010) A preliminary study on the length of incubation needed to maximize guanidination of lysine in distiller dried grains with solubles (DDGS) and in pig ileal digesta. Anim Feed Sci Technol 159:68–71CrossRefGoogle Scholar
  50. Ravindran V, Hew LI, Ravindran G, Bryden WL (2004) Endogenous amino acid flow in the avian ileum: quantification using three techniques. Br J Nutr 92:217–223CrossRefPubMedGoogle Scholar
  51. Rehman Z-U (2006) Storage effects on nutritional quality of commonly consumed cereals. Food Chem 92:53–57CrossRefGoogle Scholar
  52. Rutherfurd SM, Gilani GS (2009) Amino Acid Analysis. Curr Protoc Protein Sci 58:11.9.1–11.9.37Google Scholar
  53. Rutherfurd SM, Moughan PJ (1990) Guanidination of lysine in selected dietary proteins. J Agric Food Chem 38:209–211CrossRefGoogle Scholar
  54. Rutherfurd SM, Moughan PJ (1997) Application of a new method for determining digestible reactive lysine to variably heated protein sources. J Agric Food Chem 45:1582–1586CrossRefGoogle Scholar
  55. Rutherfurd SM, Moughan PJ (2007) Development of a novel bioassay for determining the available lysine contents of foods and feedstuffs. Nutr Res Rev 20:3–16CrossRefPubMedGoogle Scholar
  56. Rutherfurd SM, Moughan PJ, Morel PCH (1997a) Assessment of the true ileal digestibility of reactive lysine as a predictor of lysine uptake from the small intestine of the growing pig. J Agric Food Chem 45:4378–4383CrossRefGoogle Scholar
  57. Rutherfurd SM, Moughan PJ, van Osch L (1997b) Digestible reactive lysine in processed feedstuffs-Application of a new bioassay. J Agric Food Chem 45:1189–1194CrossRefGoogle Scholar
  58. Rutherfurd SM, Torbatinejad NM, Moughan PJ (2006) Available (ileal digestible reactive) lysine in selected cereal-based food products. J Agric Food Chem 54:9453–9457CrossRefPubMedGoogle Scholar
  59. Rutherfurd SM, Bains K, Moughan PJ (2012) Available lysine and digestible amino acid contents of proteinaceous foods of India. Br J Nutr 108:S59–S68CrossRefPubMedGoogle Scholar
  60. Rutherfurd SM, Fanning AC, Miller BJ, Moughan PJ (2015) Protein digestibility-corrected amino acid scores and digestible indispensable amino acid scores differentially describe protein quality in growing male rats. J Nutr (In Press)Google Scholar
  61. Schmitz M, Hagemeister H, Erbersdobler H (1991) Homoarginine labelling I suitable for determination of protein absorption in miniature pigs. J Nutr 121:1575–1580PubMedGoogle Scholar
  62. Stein HH, Sève B, Fuller MF, Moughan PJ, de Lange CFM (2007) Invited review: amino acid bioavailability and digestibility in pig feed ingredients. J Anim Sci 85:172–180CrossRefPubMedGoogle Scholar
  63. Torbatinejad NM, Rutherfurd SM, Moughan PJ (2005) Total and reactive lysine contents in selected cereal-based food products. J Agric Food Chem 53:4454–4458CrossRefPubMedGoogle Scholar
  64. Tran QD, van Lin CGJM, Hendriks WH, van der Poel AFB (2007) Lysine reactivity and starch gelatinization in extruded and pelleted canine diets. Anim Feed Sci Technol 138:162–168CrossRefGoogle Scholar
  65. Undi M, Moshtaghi SS, Wittenberg KM, Ingalis JR (1996) A comparative study on amino acid availability of moist heated canola meal for poultry vs. ruminants. Anim Feed Sci Technol 63:179–186CrossRefGoogle Scholar
  66. Yamaguchi M, Nakazawa T, Kuyama H et al (2005) High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry. Anal Chem 77:645–651CrossRefPubMedGoogle Scholar
  67. Zhang HL, Li DF, Qiao SY, Wang FL, Chen XJ, Thacker PA (2006) The effect of dietary homoarginine derived from guanidination of synthetic lysine on endogenous amino acid loss and apparent and true ileal amino acid digestibility in the pig. Anim Sci 82:23–30CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  1. 1.Riddet InstituteMassey UniversityPalmerston NorthNew Zealand

Personalised recommendations