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The Maillard Reaction of Ascorbic Acid with Amino Acids and Proteins - Identification of Products
The Maillard Reaction of Ascorbic Acid with Amino Acids and Proteins Identification of Products
-
Monika Pischetsrieder, Bernd Larisch, and Theodor Severin INSTITUT FUR LEBENSMITTELCHEMIE, LUDWIG-MAXIMILIANS-UVERSITAT m C H E N , SOPHIENSTR 10,80333 MUNICH, GERMANY Summary
The malent binding of L-ascorbic acid (AA) or its degradation products to proteins (protein ascorbylation) is of importance for food chemistry and medical science. The main reaction produa of mixtures of AA and alkylamines, such as propylamine or W-acetyllysine, was 3deoxy-3-(alkylamino)ascorbic acid (3-DAA). When Ldehydroascorbic acid @HA), the primary oxidation product of AA, was reacted, several products could be detected using HPLC-DAD. The four major compounds were identified as Zdeoxy-2(alky1amino)ascorbic acid (ZDAA), 3-DAA. oxalic acid monoalkylamide (OMA)and o d i c acid dialkylamide (ODA). In the presence of arginine derivatives and oxygen, the main degradation product of AA was identified as $4441 ,Z~ydroxy-3-pro~liden)-3-imidazolin-5-on-2-yI)-~-o~~ne @PI). Since OMA was a major product when DHA was reacted with lysine derivatives, it was speculated that OMA may also be formed as an ascoxbylation product of proteins. Thus, OMA-modifiedprotein was
synthesized and a polyclonal antibody was produced that was highly specific for OMA. In a competitive ELISA, ascorbylated protein inhiited anhhdy binding, indicating that OMA is formed as an ascorbylation product of proteins. Oxygen is necessary for the generation of OMA. The antibody did not show crossreactivity with several proteins that had been glycosylated with other carbohydrates,including AGE-protein.
Introduction
During the Maillard reaction, Eree amino acids or side chains of proteins react with reducing sugars, resulting in the formation of a variety of products. These compounds are formed during processing and storage of foodstuffs and in vivo. They have a major influence on the quality of food and on many processes in vivo. Recently, it was discovered that in addition to sugars, L-ascorbic acid (AA) can undergo a Maillard-type reaction.’In particular, protein ascorbylation, the covalent binding of AA or its degradation products to proteins, deserves attention. This reaction leads to various changes of the physical and physiological properties of proteins, such as browning, formation of fluoresent compounds,2 protein cross linking’ and protein pre~ipitation.~ It has been suggested that the Maillard reaction of AA has antinutritional effects: and causes discolouration’ and off-flavour formation6 during food processing. Furthermore, there is strong evidence that under oxidative conditions, protein ascorbylation can also occur in vivo,’ resulting in undesirable physiological effects, such as cataract formation.’ However, little is known about reaction mechanisms that lead to protein ascorbylation and products have not been identified. Thus, the objective of this study was to elucidate the structure of ascorbylation products. First, alkylamines and amino acid derivatives were reacted with AA under various conditions and the major products were identified. In a second step, immunological methods were used to show that oxalic acid monoamide (OM),one of the main products of the reaction of AA with alkylamine, is also formed during protein ascorbylation.
I08
The Maillard Reaction in Foods and Medicine
Materials and Methods
3-Deoxy-3-(alkylamino)ascorbicacid (3-DAA) 3-DAA was isolated from a reaction mixture of AA and propylamine or N“-acetyllysine by preparative HPLC and the structure was determined by spectroscopic data.‘ 2- Deoxy-2-(alkylamino)ascorbic acid (2-DAA), Oxalic acid monoalkylamide ( O M ) , and Oxalic acid dialkylamide (ODA) The products were isolated as described before.’ L-Dehydroascorbic acid (DHA) was heated with propylamine or IT-acetyllysine and the compounds were isolated by preparative HPLC. Identification of the products was achieved by interpretation of spectroscopic data and comparison of chromatographic and spectroscopic properties with those of synthesized reference compounds. N”4ce~l-N‘-(4-(1,2-dihy&~~-3-propyliden)-3-imidazolin-5-on-2-y~-~-ornithine (DPI) DPI was prepared by the reaction of DHA with W-acetylarginine and purified using preparative HPLC. lo Identification was achieved with the help of spectroscopic data. Analysis of OM-protein by ELJSA Immunological assays were performed as described before. ” OM-protein was synthesized by the reaction of oxalic acid bis(N-hydroxysuccinimide) ester with proteins and polyclonal anti-OMA antibody was obtained by injecting OMA-protein into two rabbits. The antibody was characterized by noncompetitive ELISA, and cross-reactivity with various compounds was determined in a competitive assay.
Results and Discussion Although the Maillard reaction of AA is of great importance in food chemistry and medicine, little is known about reaction mechanisms and products. Amino acid analyses of ascorbylated proteins have revealed that AA attacks mainly lysine chains and, to a minor extent, arginine and histidine.I2 For the present study, free amino acids were heated with AA under various conditions and the reactions were monitored by HPLC. The major Maillard products of AA were isolated and their structures identified. Reaction of AA with +sine derivatives under nonoxidative conditions #en AA was heated with N“-acetyllysine, a major product, which had a W maximum at 278 run, could be detected by HPLC. The compound was isolated and its structure determined by spectroscopic data and chemical methods. It was found that the hydroxyl group in position 3 of AA had been substituted by the e-aminogroup of lysine, resulting in the formation of 3-deoxy-3-(N”-(N”-acetyllysin)-yl)-ascorbic acid (3-DAA) (Figure 1). 3DAA is readily formed during heating of a mixture of AA and W-acetyllysine under reflux, but also during incubation for several days at 37 “C. Reaction of AA with +sine derivatives under oxidative conditions When a mixture of AA and W-acetyllysine was reacted under oxidative conditions or when L-dehydroascorbic acid (DHA), the primary oxidation product of AA was used as a reactant, several products were detected by HPLC. Identification of the five main products was achieved by the use of propylamine as a model compound for W-acetyllysine. Later it was shown that analogous products are also formed as derivatives of W-acetyllysine.
109
Reaction Mechanism
OH DHA 4-
0
\\
0\\
0
//
c-c RH" NHR
c-c
~.
\
HO'
ODA
W
0
// \
NHR
HO
OMA
OH 2-DAA
R = e.g. propylamine, N*-acetyllysine
Figure 1. Reactions of AA with alkylamines and N"-acetyllysine Heating of DHA with propylamine leads to the formation of two minor products which were identified as AA and 3-DAA. It can be assumed that DHA is first reduced by reactive intermediates to give AA which reacts hrther to produce 3-DAA as described above. The three main products were isolated and their structures elucidated: 2-deoxy-2-propylaminoascorbic acid (2-DAA), oxalic acid monopropylamide (OMA), and oxalic acid dipropylamide (ODA) (Figure 1). It is likely that 2-DAA and 3-DAA have strong anti-oxidative and - in the presence of metal ions pro-oxidative properties, which even exceed those of AA.I3 Thus, their formation may be of importance in food processing and in vivo. Since ODA has two molecules of amine incorporated, it can be assumed that the ODA-protein adduct contributes to AA-induced protein cross linking.
-
Reaction of AA with arginine derivatives Incubation of AA with various arginine derivatives, such as IT-acetylarginine leads to the formation of a major product with characteristic W maxima at 238 and 283 nm. The compound was isolated and identified as N6-(4-(1,2-dihydroxy-3-propyliden)-3-imidazolinS-on-2-yl)-~-omithine @PI, Figure 2).1° The presence of oxygen was essential for the formation of DPI. Since DPI can also be formed from DHA and L-xylosone, it was concluded that AA is first oxidized and decarboxylated to give L-xylosone. Subsequently, Lxylosone condenses with the guanidinium group of arginine, and DPI is formed following dehydration.
The Maillard Reaction in Foods and Medicine
110
HO
KN 1
OH
HCOH
/
N“-acetylarginine
AA
$COH
DPI
Figure 2. Reaction of AA with W-acetylarginine With the formation of 2 - D f i 3 - D A q OMA, ODA, and DPI,5 Maillard products of AA have been isolated, which can be formed as derivatives of free lysine and arginine, or of the side chains of proteins. Assuming their generation during food processing or in vivo, such products can contribute to various effects of protein ascorbylation.
Immunochemical detecfion of O M as an ascorbylafionproducf of profeins
Since most of the above mentioned Maillard products are not stable under the conditions of acidic or alkaline hydrolysis, it is difficult to prove that they are also formed during protein ascorbylation. Consequently, a polyclonal antibody was raised against OMA-protein, because OMA is one of the main products of the Maillard reaction of AA with low molecular weight amines. The antibody was used in competitive and noncompetitive ELISAs. In a competitive ELISA, total binding inhibition was achieved by the addition of purified oxalic acid mono(NE-(N”-acetyllysin)-yl)amide. This result indicated that the antibody was highly specific against O M . Furthermore, products similar to OM-protein, such as acetylated and CML-protein, did not show cross-reactivity, confirming the specificity of the antibody (Figure 3).
+OMA-Lp -fCML-BSA -X-Acetyl-BSA
10%
0.m1
0,001
0.01 Inhibitor [mg/mll
0,1
1
Figure 3. ELISA competition curves to characterize the specificity of the anti-OMA antibody
111
Reaction Mechanisms 100%
80%
B
p 60%
E
r3
z
40%
20%
0% o.Ooo1
1
Figure 4. ELISA competition curves with ascorbylated proteins as inhibitors
Subsequently, ascorbylated protein was subjected to competitive ELISA, and dosedependent binding inhibition of up to 100 % was observed (Figure 4). This result shows that OMA represents an ascorbylation product of proteins. Formation of OMA from AA and proteins was highly dependent on the presence of oxygen. Protein which was incubated in the presence of DHA showed higher reactivity than protein which was treated with AA in the presence of oxygen. In the absence of oxygen, AA did not generate OMA. Glycated or AGE-protein (advanced glycation end-product) did not show cross-reactivity. In addition, OMA could not be detected on proteins, which had been glycosylated by other carbohydrates, such as fructose or lactose. Only ribosylated protein displayed significant cross-reactivity. Thus, it can be concluded that the antibody can be used to distinguish ascorbylated from glycated protein. References 1. B. Ortwerth and P. Olesen, Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction, Biochim. Biophys. Acta, 1988, 956, 10-22. 2. K. Bensch, J. Fleming and W. Lohmann, The role of ascorbic acid in senile cataract, Proc. Natl. Acad Sci. USA, 1985,82, 7193 7196. 3. B. Ortwerth, M. Feather and P. Olesen, The precipitation and cross-linking of lens crystallins by ascorbic acid, Exp. Eye Rex, 1988, 47, 155 - 168. 4. I. Ziderman, K. Gregorski, S.Lopez and M. Friedman, Thermal interaction of ascorbic
-
acid and sodium ascorbate with proteins in relation to nonenzymatic browning and Maillard reactions in foods, J.Agric. Food Chem., 1989,37, 1480 1486. 5. S. Rogacheva, M. Kuntcheva, I. Panchev and T. Obretenov, L-Ascorbic acid in nonenzymatic reactions I. Reaction with glycine, 2. Lebensm. Unters. Forsch., 1995,
-
200,52 - 58. 6. H. Sakurai, K. Ishii, H. Nguyen, Z. Reblova, H.Valentova and J. Pokorny,
Condensation reactions of dehydroascorbic acid with aspartame, in ‘Chemical Reactions in Foods 111 Proceedings’, J. Velisek and J. Davidek (eds), Czech. Chemical Society, Prague, 1996, p. 112.
-
nte Maillard Reaction in Foods and Medicine
112
7. J. Hunt, Ascorbic acid in diabetes mellitus, in 'Subcellular Biochemistry, Vol. 25.
Ascorbic acid: Biochemistry and Biomedical Cell Biology', J.R. Harris (ed.), Plenum Press, New York, 1996, p. 369. 8. M. Pischetsrieder, B. Larisch and Th. Severin, Reaction of ascorbic acid with aliphatic amines, J. Agric. FoodChem., 1995,43, 3004 - 3006. 9. B. Larisch, M. Pischetsrieder and Th. Severin, Reactions of dehydrascorbic acid with primary aliphatic amines including N"-acetyllysine, J. Agric. Food Chem., 1996, 44,
-
1630 1634. 10. M. Pischetsrieder, Reaction of L-ascorbic acid with L-arginine derivatives, J. Agric. FoodChem., 1996,442081 - 2085.
1 1. M. Pischetsrieder, B. Larisch and W. Seidel, Immunochemical detection of oxalic acid
monoamides which are formed during the oxidative reaction of L-ascorbic acid and proteins, J. Agric. Food Chem., 1997, 45, 2070 - 2075. 12. S. Slight, M. Feather and B. Ortwerth, Glycation of lens proteins by the oxidation products of ascorbic acid, Biochim. Biophys. Acta, 1990, 1038, 367 - 374. 13. M. Pischetsrieder and Th. Severin, New aspects on the Maillard reaction - formation of aminoreductones from sugars and L-ascorbic acid, Recenl Res. Devel. in Agriculiural & Food Chem., 1997, 1,29 - 37.
-
Monika Pischetsrieder, Bernd Larisch, and Theodor Severin INSTITUT FUR LEBENSMITTELCHEMIE, LUDWIG-MAXIMILIANS-UVERSITAT m C H E N , SOPHIENSTR 10,80333 MUNICH, GERMANY Summary
The malent binding of L-ascorbic acid (AA) or its degradation products to proteins (protein ascorbylation) is of importance for food chemistry and medical science. The main reaction produa of mixtures of AA and alkylamines, such as propylamine or W-acetyllysine, was 3deoxy-3-(alkylamino)ascorbic acid (3-DAA). When Ldehydroascorbic acid @HA), the primary oxidation product of AA, was reacted, several products could be detected using HPLC-DAD. The four major compounds were identified as Zdeoxy-2(alky1amino)ascorbic acid (ZDAA), 3-DAA. oxalic acid monoalkylamide (OMA)and o d i c acid dialkylamide (ODA). In the presence of arginine derivatives and oxygen, the main degradation product of AA was identified as $4441 ,Z~ydroxy-3-pro~liden)-3-imidazolin-5-on-2-yI)-~-o~~ne @PI). Since OMA was a major product when DHA was reacted with lysine derivatives, it was speculated that OMA may also be formed as an ascoxbylation product of proteins. Thus, OMA-modifiedprotein was
synthesized and a polyclonal antibody was produced that was highly specific for OMA. In a competitive ELISA, ascorbylated protein inhiited anhhdy binding, indicating that OMA is formed as an ascorbylation product of proteins. Oxygen is necessary for the generation of OMA. The antibody did not show crossreactivity with several proteins that had been glycosylated with other carbohydrates,including AGE-protein.
Introduction
During the Maillard reaction, Eree amino acids or side chains of proteins react with reducing sugars, resulting in the formation of a variety of products. These compounds are formed during processing and storage of foodstuffs and in vivo. They have a major influence on the quality of food and on many processes in vivo. Recently, it was discovered that in addition to sugars, L-ascorbic acid (AA) can undergo a Maillard-type reaction.’In particular, protein ascorbylation, the covalent binding of AA or its degradation products to proteins, deserves attention. This reaction leads to various changes of the physical and physiological properties of proteins, such as browning, formation of fluoresent compounds,2 protein cross linking’ and protein pre~ipitation.~ It has been suggested that the Maillard reaction of AA has antinutritional effects: and causes discolouration’ and off-flavour formation6 during food processing. Furthermore, there is strong evidence that under oxidative conditions, protein ascorbylation can also occur in vivo,’ resulting in undesirable physiological effects, such as cataract formation.’ However, little is known about reaction mechanisms that lead to protein ascorbylation and products have not been identified. Thus, the objective of this study was to elucidate the structure of ascorbylation products. First, alkylamines and amino acid derivatives were reacted with AA under various conditions and the major products were identified. In a second step, immunological methods were used to show that oxalic acid monoamide (OM),one of the main products of the reaction of AA with alkylamine, is also formed during protein ascorbylation.
I08
The Maillard Reaction in Foods and Medicine
Materials and Methods
3-Deoxy-3-(alkylamino)ascorbicacid (3-DAA) 3-DAA was isolated from a reaction mixture of AA and propylamine or N“-acetyllysine by preparative HPLC and the structure was determined by spectroscopic data.‘ 2- Deoxy-2-(alkylamino)ascorbic acid (2-DAA), Oxalic acid monoalkylamide ( O M ) , and Oxalic acid dialkylamide (ODA) The products were isolated as described before.’ L-Dehydroascorbic acid (DHA) was heated with propylamine or IT-acetyllysine and the compounds were isolated by preparative HPLC. Identification of the products was achieved by interpretation of spectroscopic data and comparison of chromatographic and spectroscopic properties with those of synthesized reference compounds. N”4ce~l-N‘-(4-(1,2-dihy&~~-3-propyliden)-3-imidazolin-5-on-2-y~-~-ornithine (DPI) DPI was prepared by the reaction of DHA with W-acetylarginine and purified using preparative HPLC. lo Identification was achieved with the help of spectroscopic data. Analysis of OM-protein by ELJSA Immunological assays were performed as described before. ” OM-protein was synthesized by the reaction of oxalic acid bis(N-hydroxysuccinimide) ester with proteins and polyclonal anti-OMA antibody was obtained by injecting OMA-protein into two rabbits. The antibody was characterized by noncompetitive ELISA, and cross-reactivity with various compounds was determined in a competitive assay.
Results and Discussion Although the Maillard reaction of AA is of great importance in food chemistry and medicine, little is known about reaction mechanisms and products. Amino acid analyses of ascorbylated proteins have revealed that AA attacks mainly lysine chains and, to a minor extent, arginine and histidine.I2 For the present study, free amino acids were heated with AA under various conditions and the reactions were monitored by HPLC. The major Maillard products of AA were isolated and their structures identified. Reaction of AA with +sine derivatives under nonoxidative conditions #en AA was heated with N“-acetyllysine, a major product, which had a W maximum at 278 run, could be detected by HPLC. The compound was isolated and its structure determined by spectroscopic data and chemical methods. It was found that the hydroxyl group in position 3 of AA had been substituted by the e-aminogroup of lysine, resulting in the formation of 3-deoxy-3-(N”-(N”-acetyllysin)-yl)-ascorbic acid (3-DAA) (Figure 1). 3DAA is readily formed during heating of a mixture of AA and W-acetyllysine under reflux, but also during incubation for several days at 37 “C. Reaction of AA with +sine derivatives under oxidative conditions When a mixture of AA and W-acetyllysine was reacted under oxidative conditions or when L-dehydroascorbic acid (DHA), the primary oxidation product of AA was used as a reactant, several products were detected by HPLC. Identification of the five main products was achieved by the use of propylamine as a model compound for W-acetyllysine. Later it was shown that analogous products are also formed as derivatives of W-acetyllysine.
109
Reaction Mechanism
OH DHA 4-
0
\\
0\\
0
//
c-c RH" NHR
c-c
~.
\
HO'
ODA
W
0
// \
NHR
HO
OMA
OH 2-DAA
R = e.g. propylamine, N*-acetyllysine
Figure 1. Reactions of AA with alkylamines and N"-acetyllysine Heating of DHA with propylamine leads to the formation of two minor products which were identified as AA and 3-DAA. It can be assumed that DHA is first reduced by reactive intermediates to give AA which reacts hrther to produce 3-DAA as described above. The three main products were isolated and their structures elucidated: 2-deoxy-2-propylaminoascorbic acid (2-DAA), oxalic acid monopropylamide (OMA), and oxalic acid dipropylamide (ODA) (Figure 1). It is likely that 2-DAA and 3-DAA have strong anti-oxidative and - in the presence of metal ions pro-oxidative properties, which even exceed those of AA.I3 Thus, their formation may be of importance in food processing and in vivo. Since ODA has two molecules of amine incorporated, it can be assumed that the ODA-protein adduct contributes to AA-induced protein cross linking.
-
Reaction of AA with arginine derivatives Incubation of AA with various arginine derivatives, such as IT-acetylarginine leads to the formation of a major product with characteristic W maxima at 238 and 283 nm. The compound was isolated and identified as N6-(4-(1,2-dihydroxy-3-propyliden)-3-imidazolinS-on-2-yl)-~-omithine @PI, Figure 2).1° The presence of oxygen was essential for the formation of DPI. Since DPI can also be formed from DHA and L-xylosone, it was concluded that AA is first oxidized and decarboxylated to give L-xylosone. Subsequently, Lxylosone condenses with the guanidinium group of arginine, and DPI is formed following dehydration.
The Maillard Reaction in Foods and Medicine
110
HO
KN 1
OH
HCOH
/
N“-acetylarginine
AA
$COH
DPI
Figure 2. Reaction of AA with W-acetylarginine With the formation of 2 - D f i 3 - D A q OMA, ODA, and DPI,5 Maillard products of AA have been isolated, which can be formed as derivatives of free lysine and arginine, or of the side chains of proteins. Assuming their generation during food processing or in vivo, such products can contribute to various effects of protein ascorbylation.
Immunochemical detecfion of O M as an ascorbylafionproducf of profeins
Since most of the above mentioned Maillard products are not stable under the conditions of acidic or alkaline hydrolysis, it is difficult to prove that they are also formed during protein ascorbylation. Consequently, a polyclonal antibody was raised against OMA-protein, because OMA is one of the main products of the Maillard reaction of AA with low molecular weight amines. The antibody was used in competitive and noncompetitive ELISAs. In a competitive ELISA, total binding inhibition was achieved by the addition of purified oxalic acid mono(NE-(N”-acetyllysin)-yl)amide. This result indicated that the antibody was highly specific against O M . Furthermore, products similar to OM-protein, such as acetylated and CML-protein, did not show cross-reactivity, confirming the specificity of the antibody (Figure 3).
+OMA-Lp -fCML-BSA -X-Acetyl-BSA
10%
0.m1
0,001
0.01 Inhibitor [mg/mll
0,1
1
Figure 3. ELISA competition curves to characterize the specificity of the anti-OMA antibody
111
Reaction Mechanisms 100%
80%
B
p 60%
E
r3
z
40%
20%
0% o.Ooo1
1
Figure 4. ELISA competition curves with ascorbylated proteins as inhibitors
Subsequently, ascorbylated protein was subjected to competitive ELISA, and dosedependent binding inhibition of up to 100 % was observed (Figure 4). This result shows that OMA represents an ascorbylation product of proteins. Formation of OMA from AA and proteins was highly dependent on the presence of oxygen. Protein which was incubated in the presence of DHA showed higher reactivity than protein which was treated with AA in the presence of oxygen. In the absence of oxygen, AA did not generate OMA. Glycated or AGE-protein (advanced glycation end-product) did not show cross-reactivity. In addition, OMA could not be detected on proteins, which had been glycosylated by other carbohydrates, such as fructose or lactose. Only ribosylated protein displayed significant cross-reactivity. Thus, it can be concluded that the antibody can be used to distinguish ascorbylated from glycated protein. References 1. B. Ortwerth and P. Olesen, Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction, Biochim. Biophys. Acta, 1988, 956, 10-22. 2. K. Bensch, J. Fleming and W. Lohmann, The role of ascorbic acid in senile cataract, Proc. Natl. Acad Sci. USA, 1985,82, 7193 7196. 3. B. Ortwerth, M. Feather and P. Olesen, The precipitation and cross-linking of lens crystallins by ascorbic acid, Exp. Eye Rex, 1988, 47, 155 - 168. 4. I. Ziderman, K. Gregorski, S.Lopez and M. Friedman, Thermal interaction of ascorbic
-
acid and sodium ascorbate with proteins in relation to nonenzymatic browning and Maillard reactions in foods, J.Agric. Food Chem., 1989,37, 1480 1486. 5. S. Rogacheva, M. Kuntcheva, I. Panchev and T. Obretenov, L-Ascorbic acid in nonenzymatic reactions I. Reaction with glycine, 2. Lebensm. Unters. Forsch., 1995,
-
200,52 - 58. 6. H. Sakurai, K. Ishii, H. Nguyen, Z. Reblova, H.Valentova and J. Pokorny,
Condensation reactions of dehydroascorbic acid with aspartame, in ‘Chemical Reactions in Foods 111 Proceedings’, J. Velisek and J. Davidek (eds), Czech. Chemical Society, Prague, 1996, p. 112.
-
nte Maillard Reaction in Foods and Medicine
112
7. J. Hunt, Ascorbic acid in diabetes mellitus, in 'Subcellular Biochemistry, Vol. 25.
Ascorbic acid: Biochemistry and Biomedical Cell Biology', J.R. Harris (ed.), Plenum Press, New York, 1996, p. 369. 8. M. Pischetsrieder, B. Larisch and Th. Severin, Reaction of ascorbic acid with aliphatic amines, J. Agric. FoodChem., 1995,43, 3004 - 3006. 9. B. Larisch, M. Pischetsrieder and Th. Severin, Reactions of dehydrascorbic acid with primary aliphatic amines including N"-acetyllysine, J. Agric. Food Chem., 1996, 44,
-
1630 1634. 10. M. Pischetsrieder, Reaction of L-ascorbic acid with L-arginine derivatives, J. Agric. FoodChem., 1996,442081 - 2085.
1 1. M. Pischetsrieder, B. Larisch and W. Seidel, Immunochemical detection of oxalic acid
monoamides which are formed during the oxidative reaction of L-ascorbic acid and proteins, J. Agric. Food Chem., 1997, 45, 2070 - 2075. 12. S. Slight, M. Feather and B. Ortwerth, Glycation of lens proteins by the oxidation products of ascorbic acid, Biochim. Biophys. Acta, 1990, 1038, 367 - 374. 13. M. Pischetsrieder and Th. Severin, New aspects on the Maillard reaction - formation of aminoreductones from sugars and L-ascorbic acid, Recenl Res. Devel. in Agriculiural & Food Chem., 1997, 1,29 - 37.