JMARS News Letter Vol. 1, April 22, 2006 Maillard Reaction Society (IMARS) Japan Maillard Reaction Society) The Maillard Reaction and Free Radicals:
Discovery of the Namiki Pathway
Nagoya University I graduated from university (in Tokyo, 1945) in the great confusion just after the Second World War and fortunately found work at the Institute of Physical and Chemical Research. The Institute was established in 1917 as the first general research institute in Japan and achieved much remarkable success for work on, e. g., vitamins and nuclear physics. The institute is now called RIKEN and is one of the world's leading research institutes. However, in those days it had greatly suffered from war damage and most laboratories had been burnt and left without research facilities and budget. At first, I studied on development of new antibiotics, and then started a laboratory working on radiation biology in 1956 and performed studies on protective substances in food and biological materials for radiation lethal effect. This work aimed to protect biologically important substances such as DNA in the living body from damage caused by active oxygen radicals formed by irradiation using radical scavengers and antioxidants. Later, I transferred to a laboratory of food chemistry at Nagoya University (1966) and continued my studies on antioxidants in various health foods such as sesame and tea which offer protection from active oxygen hazards in living systems including aging, carcinogenesis and others. These studies have now developed into a world-wide trend of the studies on functional foods. Through these studies, I became familiar with the chemistry and biology of free radicals (hereafter referred to as radicals). On the other hand, studies on the Maillard reaction were performed as an important task of food chemistry, particularly on elucidation of its reaction mechanism at JMARS News Letter Vol. 1, April 22, 2006 the initial stage. The mechanism of the Maillard structures of sugars used for the reaction except for the reaction had been proposed by J.E.Hodge (1953 and C-3 compounds and varied with the structures of the 1967) and had been introduced in all textbooks and amino group in the amino compounds. In addition, assumed to be the only theory (Fig. 1). However, I analysis of the hyperfine structure revealed that the believed that there must be some other mechanisms at radical might be derived from a pyrazine compound. work in the Maillard reaction other than that of Hodge Synthesis of the presumed pyrazine compound and its and challenged to elucidate the mechanism. Another determination showed complete consistency in ESR subject that remained to be elucidated was the spectra. Thus, it was demonstrated that a pyrazinium differences in the Maillard reaction between processing cation radical compound formed generally in the initial food and biological systems. Generally, foods show an stage of the Maillard reaction. Determination of the acidic pH range and have a slightly higher pH than chemical structure of an unknown intermediate radical neutral in living matter and this difference greatly in the reaction mixture by analysis of ESR spectrum is influences the reactions. The major reaction in the very rare, and the reason why these radical compounds living body involves a side chain amino group in are present so stably in the reaction mixture is not yet protein. This reaction called glycation and it is somewhat different from the reactions of sugars with Spectral analysis of the pyrazine skeleton suggested amino acids in the food system. that two nitrogen atoms in the pyrazine skeleton may be The key to the elucidation of the mechanism of the derived from amino acid, and two carbon atoms (C-2) Maillard reaction is a radical reaction. ESR (Electron are presumed to be derived from the sugar. This Spin Resonance) became popular in 1960s and the compound is hardly formed according to the presence of stable radicals in carbonated organic mechanism of Hodge, so we tried to identify a substances and roasted coffee beans became known. A precursor compound of the radical product in the long-lived radical was also observed in the melanoidin reaction mixture. After comprehensive analysis of the of the sugar and amino acid reaction. I hypothesized the isolated products formed at an early stage of the formation of a radical product different from the stable reaction, we confirmed a production of the precursor radicals in these polymers in the initial stage of the compound having a nitrogen atom in a C-2 compound Maillard reaction and tried to verify this in ESR prior to the formation of the Amadori compound and proved the formation process of the pyrazinium radical However, a trace amount of radicals that developed (Fig. 1). This is quite different from the mechanism of during the reaction has generally very unstable and Hodge and a new reaction mechanism that does not hardly detectable. Therefore, a radical trapping method, involve the Amadori rearrangement in the Maillard a rapid flow method and a very low temperature reaction was clearly found (1975). determination method was generally used, but these The characteristic feature of the new found reaction methods encountered many difficulties in practice. mechanism was the identification of an unstable The observed signal merely showed the presence of a intermediate at the center of the ongoing reaction with radical if succeeded and no structural information on the ESR spectrum elucidating the actual condition in radical product could be obtained given the level of the new flow of reaction. This procedure was different radical chemistry in those days. from the conventional procedures of the time. I was at a loss as to how to elucidate this difficult In 1982, I presented the results of the new reaction problem. One day, I proposed to Dr. Tateki Hayashi, a mechanism at a symposium of the American Chemical colleague and an expert in ESR, the measurement of an Society, 3) and received many responses and questions ESR spectrum of a warmed reaction mixture of a sugar from the audience. One member of the audience and an amino acid as is. After a while, Dr. Hayashi asked whether the mechanism would also occur in vivo, returned from the ESR laboratory saying that the ESR and I replied that it was possible. As described above, imaging had succeeded. Surprisingly, the chart revealed the mechanism of Hodge proceeds in a lower pH a spectrum with a fine structure beyond imagination environment and may face some problems at higher pH. because the measurement was carried out in an open However, the new mechanism was advantageous in a test tube containing the sugar-amino acid mixture higher pH range. I learned later that the man who asked heated in a water bath without removal of oxygen or the question was Dr. V.M.Monnier who later studied the freezing. I wondered whether the spectrum really two mechanisms and found that both proceeded equally showed the radical formed by an amino-carbonyl at about pH 7.2 and named the new mechanism the reaction of a sugar-amino acid and tried the reaction "Namiki pathway." 4) B.Crammerer et al. chemically with another sugar-amino acid system, obtaining a studied the mechanism and reported that the Namiki similarly fine spectrum for the mixture after only about pathway is predominant at pH 7.0 or over and that 10 minutes of heating with no browning. Thus we Hodge's mechanism proceeds at about 40% or less.5) demonstrated the deceptively difficult general The Namiki pathway is now recognized throughout the formation of a considerably stable radical compound world and is often cited in the scientific literature.2) different from the radical of melanoidins.1,3) One major difference between the two mechanisms Another surprising feature was that the determination is the influence of pH. Glucose is liable to take a ring of the spectrum of the reaction mixture at ordinary structure with less of a carbonyl structure at neutral or temperature showed a hyperfine structure. Radicals in lower pH and it is difficult to react with amino group. solid or crystal state sometimes show hyperfine However, the probability of an open ring form increases structures, but the observation of the spectrum of the at pH 7 or over and tends to form a Schiff's base reaction solution as is was very rare and their hyperfine together with the formation of a highly reactive C-2 structural patterns were not so different from the carbonyl-nitrogen compound by the cleavage of the

JMARS News Letter Vol. 1, April 22, 2006 Schiff's base by a reverse aldol reaction caused by an formed biochemically without the Maillard reaction and anion and leads to browning via the Namiki pathway. its influence in the living body is the focus of much In a lower pH range, the Amadori rearrangement occurs attention. The influence of the Maillard reaction of such by H+ as in the reaction with amino acid to form low molecular weight carbonyls in the living body will 3-deoxyglucosone (3DG) as stated by Hodge. This become more important. product with an active dicarbonyl compound also takes In any case, the effects of the Maillard reaction vary on a cyclic structure and exists with comparative depending on the conditions. In foods the reaction stability in foods. proceeds usually under serious conditions such as One more important feature of the Namiki pathway heating or long-term storage at lower pH causing is the presence of a radical in the reaction product discoloration, development of roast flavors or burned which reacts with oxygen and probably gives an active smells and the formation of pyrolyzate carcinogens, oxygen species. I have observed a very weak while the reaction under mild conditions in living body chemiluminescence in the early stage of the Maillard induces the accumulation of slight changes in living reaction and assumed it to be caused by the reaction of material causing serious damage in physiological the radical product and oxygen. G.T.Wondrak also functions related to aging and carcinogenesis. The proved the generation of chemiluminescence based on reaction mechanism of Hodge may be dominant in the the Namiki pathway and further proved by ESR the former system and that of the Namiki pathway in living formation of a pyrazinium radical bridge between -amino groups of lysine in a protein followed by the Extensive investigations over a long period of time formation of active oxygens by the reaction of the on the radical reactions and a will to meet scientific pyrazinium radical with oxygen.6) It is possible that the challenges led to the discovery of a previously opposite reaction of the pyrazinium radical with the unknown mechanism other than the conventional active oxygens may occur, so further studies on the mechanism of the Maillard reaction, and the influence of oxygen in the Maillard reaction should be development of a new scientific field. Studies in science are often like climbing a mountain for the first T. Hofmann studied the Maillard reaction in detail time, and no breakthrough can be accomplished without at pH 7.0 and proved once again our mechanism careful preparation and a spirit of challenge. Successful involving the formation of a pyrazinium radical studies involving close cooperation in chemistry, intermediate from a sugar through glyoxal. Furthermore, biology and medicine are eagerly awaited. he named the intermolecular pyrazinium radical bridge of lysine molecules the "CROSSPY radical" and References
elucidated its high reactivity with SH and OH Namiki, M, A New Development of the Maillard compound in food and living matter.7) These facts show Reaction and Glycation. SEIKAGAKU (The various possible influences of the Maillard reaction Journal of Biochemistry), 75 (1) 37-42, (2003)
product and functional substances in the living body Rizzi, G.P., Free Radicals in the Maillard Reaction. and future studies are awaited. Food Reviews Intern., 19, 375-395 (2003)
One more influence of the Namiki pathway is the Namiki, M., Hayashi, T., The Maillard Reaction in re-presentation of the importance of a low molecular Food and Nutrition, ACS Symp. Ser., 215, 21-46
weight carbonyl compound. As described above, glucose takes on a ring structure with hardly any Glomb, M.A. and Monnier, V. M., J. Biol. Chem. reactivity of a carbonyl group. It is thus the most stable 270, 10017-10026 (1995)
sugar in the Maillard reaction. In contrast, low Crammerer, B., and Kroch, L.W., Food Chem., 57,
molecular weight dicarbonyl compounds such as glycol aldehyde, glyceraldehyde, glyoxal and methyl glyoxal Wondrak, G.T., Pier, T. and Tressel, R., J. Biolumi. have experimentally been proved to have several Chemilumi. 10, 277-284 (1995)
hundred or thousand times reactivity compared with Hofmann, T., Bore, W., and Stettmaier, K., J. glucose. Compounds such as methyl glyoxal may be Agric. Food Chem. 47, 391-396 (1999)

Fig. 1: Main pathway of the
Maillard reaction.

JMARS News Letter Vol. 1, April 22, 2006 COST-IMARS Joint Workshop Napoli, May 24-27, 2006 II. 9th International Maillard Symposium Munich, September 1-5, 2007 JMARS News Letter Vol. 1, April 22, 2006


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