Carboxymethyl)lysine Formation Research Articles

A DFT study of the carboxymethyl-phosphatidylethanolamine formation from glyoxal and phosphatidylethanolamine surface. Comparison with the formation of N(ε)-(carboxymethyl)lysine from glyoxal and L-lysine.

Mechanisms of the generation of carboxymethyl compounds Nε-(carboxymethyl)lysine (CML) and carboxymethyl-phosphatidylethanolamine (CM-PE) from the reactions between glyoxal and L-lysine, and glyoxal and phosphatidylethanolamine (PE) were studied using the DFT method at the PBE/DNP level of theory. In order to study the reaction with PE, a periodic model of the PE surface was built. The starting surface model includes two molecules of PE, a molecule of monohydrated form of glyoxal, and five water molecules as explicit solvent that form a hydrogen bond network, which are involved in the reactions by stabilizing reaction intermediates and transition states and as proton-transfer carriers, important in all steps of reactions. Both reactions take place in three steps, namely, (1) carbino-diol-amine formation; (2) dehydration; and (3) rearrangement into carboxymethyl final products. The rate-limiting step for the formation of CML/CM-PE was the dehydration stage. The comparison of both reactions in their equivalent stages showed a catalytic role of the PE surface; it is highlighted in the case of dehydration step where its relative free energy barrier had a value of 5.3 kcal mol(-1) lower than that obtained in the L-lysine-glyoxal system. This study gives insights into the active role of the phospholipid surface in some chemical reactions that occur above it. Our results also give support to consider the pathway of formation of CML and CM-PE from the reactions between glyoxal and L-lysine, and glyoxal and PE as an alternative pathway for generation of these advanced glycation end-products (AGEs).

Inhibitory effect of lotus seedpod oligomeric procyanidins on advanced glycation end product formation in a lactose–lysine model system

Industrial food processing induces protein glycation modifications and toxic advanced glycation end products (AGEs) which affect human health. Therefore, it is of interest to monitor AGEs in food processing. The present study was carried out to investigate the influence of lotus seedpod oligomeric procyanidin (LSOPC) concentrations, solution pH value and metal ions on AGE formation by heat treatment of lactose–lysine model solutions. Nε-(carboxymethyl) lysine (CML), as one of the common AGEs was also determined by HPLC–MS/MS in this experiment. The results showed that LSOPC can inhibit the formation of AGEs effectively at higher concentrations, lower temperature, and it can reverse the promotion function of metal ions because of its high inhibition activity. Also, LSOPC can inhibit CML formation in the Maillard reaction as well. These results indicated that LSOPC could be used as functional food ingredients to inhibit AGE formation.

Investigation of variations in the acrylamide and N(ε) -(carboxymethyl) lysine contents in cookies during baking.

Baking processing is indispensable to determine special sensory prosperities of cookies and induces the formation of some beneficial components such as antioxidants. However, the formation of some Maillard reaction-derived chemical hazards, such as acrylamide (AA) and N(ε) -(carboxymethyl) lysine (CML) in cookies is also a significant consequence of baking processing from a food safety standpoint. This study investigated the effects of baking conditions on the formation of AA and CML, as well as the antioxidant activity (AOA) of cookies. Cookies were baked at various baking temperatures (155 to 230 °C) and times (1.5 to 31 min). AA and CML contents were determined by ultra-performance liquid chromatography-tandem mass spectrometry, respectively. The highest level of AA was obtained in the cookies baked at 155 °C/21 min and 205 °C/11 min (328.93 ± 3.10 μg/kg and 329.29 ± 5.29 μg/kg), while the highest level of CML was obtained in the cookies baked at 230 °C/1.5 min (118.05 ± 0.21 mg/kg). AA was prone to form at relatively low temperature range (155 to 205 °C), however, CML at relatively high temperature range (205 to 230 °C). The CML content was much higher than the AA content in the same set of cookies, by about 2 to 3 orders of magnitude. The AOA of cookies increased at more severe baking conditions. According to the AA and CML content, AOA and sensory properties of cookies, the temperature-time regime of 180 °C/16 min might be a compromised selection. However, only optimizing the baking condition was not enough for manufacture of high-quality cookies. Cookies, a kind of widely consumed bakery products in the world, contain some potentially harmful compounds, like acrylamide (AA) and N(ε) -(carboxymethyl) lysine (CML). AA in cookies has led to public health concern and several research efforts. But CML, another Maillard reaction-derived chemical hazard, has been neglected so far, even though its content is much higher than that of AA in cookies. The results contribute to further insight into the Maillard reaction and are useful for the selection of baking conditions to produce high-quality cookies with lower AA and CML contents, higher AOA, and better sensory properties.

N ϵ-(carboxymethyl)lysine: A Review on Analytical Methods, Formation, and Occurrence in Processed Food, and Health Impact

Foods are often heat processed and may contain advanced glycation end products (AGE). One of the most widely studied AGE is N ϵ-(carboxymethyl)lysine (CML); nevertheless, knowledge on dietary CML is fragmentary. This study aimed to review current scientific knowledge on analytical methods to determine CML contents in food, chemical pathways of CML formation in food, occurrence of CML in food, and health implications of dietary exposure to CML. Chemical analyses of CML in food products are carried out by immunochemical assays and instrumental methods, but the former method may interfere with the food matrix. CML is formed in food through various chemical pathways, depending on food ingredients and processing conditions. The compound is present in many cooked foods, with relatively high concentrations in carbohydrate-rich foods and dairy products. Dietary CML is very likely to impair human health, but full cause-effect evidence is not available yet. More studies on metabolic effects and impact of food-derived CML on human health should be performed. Food production should be optimized to minimize CML concentrations, while maintaining acceptable microbiological safety and organoleptic properties of the final food product. To this end, more insights into effects of food composition and processing conditions on CML formation are necessary.

Hydroxyl radical induced by lipid in Maillard reaction model system promotes diet-derived Nε-carboxymethyllysine formation

N(ε)-carboxymethyllysine (CML) is commonly found in food, and is considered as a potential hazard to human health. However, the effect of lipids on CML formation in Maillard reaction is still not clarified. In this study, the content of diet-derived CML and its key intermediates, epsilon-fructoselysine (FL) and glyoxal (GO), is determined with high performance liquid chromatography mass spectrum (HPLC-MS) in model system containing lipid compounds. According to the results, hydroxyl radical (OH) induced by Fenton reagent can promote the three pathways of CML formation. Moreover, in the Maillard reaction system, linoleic acid (Lin), oleic acid (Ole) and glycerol trioleate (Tri) can induce more OH·, which promotes CML formation. Their level of promoting CML formation is in the order of Ole>Lin>Tri. On the contrary, glycerol (Gly) can scavenge OH·, which inhibit the CML formation. Finally, it is proved that FL content and GO content decreases with heating time in model system, while CML content increases with heating time. Thus, it is concluded that in the Maillard reaction system lipids can induce more OH·, which promotes the conversion from FL and GO to CML. Our research may contribute to the development of inhibitory methods for diet-derived CML by scavenging OH·.

Simultaneous analysis of Nε-(carboxymethyl)lysine, reducing sugars, and lysine during the dairy thermal process

A new analytical method allowing the simultaneous quantification of Nε-(carboxymethyl)lysine (CML), lysine, and reducing sugars (glucose, lactose, and galactose) is described. It is based on high performance anion-exchange chromatography with pulsed amperometric electrochemical detection. This method demonstrated a low limit of quantification (0.385 to 0.866mg/L), excellent linear correlation (R2>0.997), and desired calibration range (3.125 to 25mg/L). In addition, lactose-lysine solutions containing sulfite (4 to 400mmol/L) were heated at 110°C for 2h. The results showed that sulfite inhibited the formation of CML and promoted the consumption of reducing sugars and lysine in the Maillard reaction model. The method proved to be useful for simultaneous analysis of CML, lysine, and reducing sugars (glucose, galactose, and lactose) in the Maillard reaction system. Moreover, sulfite was an effective inhibitor of CML formation.

Ultraviolet B radiation enhances glycation of human serum albumin and the formation of CML in vitro

Non‐enzymatic glycation of human serum albumin (HSA) is a major contributor to the pathology of diabetes. Exposure to ultraviolet B (UVB) irradiation is known to perturb protein structure and generate reactive oxygen species (ROS) which might accelerate protein glycation. The objective of this study was to evaluate the effect of UVB irradiation on the glycation level of HSA by D‐glucose and the formation of carboxymethyl lysine (CML), an Advanced Glycation Endproducts (AGEs) used as a biomarker of oxidatively damaged tissues. Compared to the untreated HSA, UVB exposed HSA showed a more rapid increase in fluorescence intensity over time, as well as enhanced peak areas of AGEs in the HPLC elution profile. Circular dichroism spectrum revealed a more extended loss in protein secondary structure when glycation had taken place under exposure to UVB. More D‐glucose adducts were found on UVB treated HSA, generating a higher mass shift of protein in MALDI profile. Compared to samples in the dark, reactions exposure to UVB produced about double amount of CML within 60 hours of incubation. In conclusion, UVB radiation accelerates the glycation level of HSA and promotes the formation of AGEs, which may be stimulated by the generation of ROS by UV radiation. This study warrants further investigation as there have been few reports on the correlation between the UV radiation of proteins and their enhanced glycation by a reducing sugar.

Formation and Inhibition of Nε-(Carboxymethyl)lysine in Saccharide-Lysine Model Systems during Microwave Heating

Nε-(carboxymethyl) lysine (CML) is the most abundant advanced glycation end product (AGE), and frequently selected as an AGEs marker in laboratory studies. In this paper, the formation and inhibition of Nε-(carboxymethyl)lysine in saccharide-lysine model systems during microwave heating have been studied. The microwave heating treatment significantly promoted the formation of CML during Maillard reactions, which was related to the reaction temperature, time and type of saccharide. The order of CML formation for different saccharides was lactose > glucose > sucrose. Then, the inhibition effect on CML by five inhibitors was further examined. According to the results, ascorbic acid and tocopherol did not affect inhibition of CML, in contrast, thiamin, rutin and quercetin inhibited CML formation, and the inhibitory effects were concentration dependent.

Okara Promoted Acrylamide and Carboxymethyl-lysine Formation in Bakery Products

Soybeans are widely used in bakery products because of their technological advantages and, recently, soybean-containing products have been marketed as functional foods thanks to several health benefits. Okara is a soybean-based ingredient obtained after elimination of the water-soluble component from ground soybeans. In this paper the effect of okara addition to bakery products on the formation of some potentially harmful Maillard reaction products was evaluated. Cookies obtained by replacing 15% of wheat flour with okara showed a visible browning increase and a more intense Maillard reaction development as shown by higher concentrations of 5-hydroxymethyl-2-furaldehyde (HMF) (+100%), acrylamide (+60%), and carboxymethyl-lysine (CML) (+400%) with respect to the control. This phenomenon could be related to the presence in okara of about 50% of insoluble dietary fiber: the fiber reduces water activity during cooking, thus promoting Maillard reaction. To confirm this hypothesis, cookies obtained by replacing 7% of wheat flour with three different types of dietary fiber (cellulose, chitosan, and pea fiber) were prepared: these experimental cookies showed higher Maillard reaction product concentration with respect to the control and, in particular, HMF and CML values were directly related to the fiber water-holding capacity (WHC). To extend the observation to the food market, a sampling of soybean-containing commercial bakery products was analyzed by comparing the concentrations of Maillard reaction products with those of similar bakery products without soy. Soybean-containing samples showed higher concentrations of acrylamide and CML than corresponding controls.

Formation of Nε-(Carboxymethyl)lysine in Saccharide-Lysine Model Systems by Different Heat Treatments

Abstract Advanced glycation endproducts (AGEs) form when proteins are heated with reducing sugars. Nε-(Carboxymethyl) lysine (CML), as one of the common AGEs studied in consumed foods, was determined by HPLC-MS/MS in this experiment. The aim of this work was to evaluate the performance of forming CML incubated by different heat treatments in saccharide - lysine model systems. Different heating treatment such as water heating (W- heating), drying oven heating (D- heating) and microwave heating (M- heating) in saccharide-lysine model systems can affect the production of CML. M-heating method showed higher formation of CML capacity than W-heating method and D-heating method. The higher temperature and higher molar ratio of saccharide to lysine can increase the CML content. The order of reactivity for the formation of CML was lactose > glucose > sucrose.

Immunological studies on glycated human IgG

AimsTo study the immunogenicity of advanced glycation end product (AGE) modified IgG (AGE-IgG) in experimental animals. Main methodsHuman IgG was subjected to in vitro glycation with glucose and the formation of Nε-(carboxymethyl)lysine (CML) was evaluated by high performance liquid chromatography (HPLC). The immunogenicity of native and AGE-IgG was investigated by raising polyclonal antibodies against them in rabbits. The induced antibodies were purified on a Protein-A agarose affinity column. Specific binding of antibodies was screened by competitive inhibition assay and band shift assay. Cross reactions of induced antibodies with various proteins or amino acids and their glycated conformers were ascertained by competitive inhibition ELISA. Key findingsWe detected the CML formation in AGE-IgG. The AGE-IgG was found to be highly immunogenic due to the generation of neo-epitopes on it. Affinity purified antibodies exhibited high degree of specific binding with AGE-IgG in comparison to the native IgG. Antibodies against AGE-IgG exhibited diverse antigen binding characteristics and the glycated conformers of various proteins and amino acids were found to be effective inhibitors of antibody-immunogen interaction in cross reaction studies. Band shift assay reiterated the results obtained by direct binding and competitive inhibition assay. SignificanceThe induced antibodies against AGE-IgG resembled the diverse antigen binding characteristics of autoantibodies found in rheumatoid arthritis (RA). IgG modified by AGEs under oxidative stress presents unique neo-epitopes which may be one of the factors for the induction of autoantibodies in RA patients.

Determination of Advanced Glycation Endproducts by LC-MS/MS in Raw and Roasted Almonds (Prunus dulcis)

A sensitive and reliable LC-(ESI)MS/MS method was developed and validated for the simultaneous analysis of five common advanced glycation endproducts (AGEs) after enzymatic digestion in raw and roasted almonds. AGEs included carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), pyralline (Pyr), argpyrimidine (Arg-p), and pentosidine (Pento-s). This method allows accurate quantitation of free and AGE-protein adducts of target AGEs. Results indicate that CML and CEL are found in both raw and roasted almonds. Pyr was identified for the first time in roasted almonds and accounted for 64.4% of free plus bound measured AGEs. Arg-p and Pento-s were below the limit of detection in all almond samples tested. Free AGEs accounted for 1.3-26.8% of free plus bound measured AGEs, indicating that protein-bound forms predominate. The roasting process significantly increased CML, CEL, and Pyr formation, but no significant correlation was observed between these AGEs and roasting temperature.

Endogenous formation of Nε-(carboxymethyl)lysine is increased in fatty livers and induces inflammatory markers in an in vitro model of hepatic steatosis

Increased lipid peroxidation and inflammation are major factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A lipoxidation product that could play a role in the induction of hepatic inflammation is N(ε)-(carboxymethyl)lysine (CML). The aim of the present study was to investigate the relationship between steatosis and CML and to study the role of CML in hepatic inflammation. We included 74 obese individuals, which were categorized into 3 groups according to the grade of hepatic steatosis. CML accumulation in liver biopsies was assessed by immunohistochemistry and plasma CML levels were measured by mass spectrometry. Plasma CML levels were also determined in the hepatic artery, portal, and hepatic vein of 22 individuals, and CML fluxes across the liver were calculated. Hepatocyte cell lines were used to study CML formation during intracellular lipid accumulation and the effect of CML on pro-inflammatory cytokine expression. Gene expression levels of the inflammatory markers were determined in liver biopsies of the obese individuals. CML accumulation was significantly associated with the grade of hepatic steatosis, the grade of hepatic inflammation, and gene expression levels of inflammatory markers PAI-1, IL-8, and CRP. Analysis of CML fluxes showed no release/uptake of CML by the liver. Lipid accumulation in hepatocytes, induced by incubation with fatty acids, was associated with increased CML formation and expression of the receptor for advanced glycation endproducts (RAGE), PAI-1, IL-8, IL-6, and CRP. Pyridoxamine and aminoguanidine inhibited the endogenous CML formation and the increased RAGE, PAI-1, IL-8, IL-6, and CRP expression. Incubation of hepatocytes with CML-albumin increased the expression of RAGE, PAI-1, and IL-6, which was inhibited by an antibody against RAGE. Accumulation of CML and a CML-upregulated RAGE-dependent inflammatory response in steatotic livers may play an important role in hepatic steatosis and in the pathogenesis of NAFLD.

Control of the Maillard reaction by ferulic acid

This study investigated how ferulic acid (FA) affects the formation of certain Maillard reaction products (MRPs), i.e., early MRPs, fluorescent and non-fluorescent advanced glycation end products (AGEs), and melanoidins in model systems. Glycation mixtures were prepared containing soy glycinin or bovine serum albumin (final concentration 10mg/ml) and fructose (222mM) in 0.2% KOH in the presence or absence of FA (12.95mM) and incubated at 60°C for 60min. The extent of the MR was estimated by analysis of free amino groups, the incorporation of sugar into the protein backbone as well as the formation of Nϵ-(carboxymethyl)lysine (CML), fluorescent AGEs (λexc=337nm, λem=350–550nm) and melanoidins (absorbance at 420nm). Formation of CML and fluorescent AGEs was reduced by nearly 90% by the addition of FA while early MRPs and melanoidins were inhibited to a lesser extent (∼10% and 28%, respectively) compared to AGE formation. A controlled formation of early MRPs was achieved by use of FA, and it is a new finding. To the best of our knowledge, for the first time the use of FA as a reliable means of obtaining novel glycoprotein preparations containing low amounts of AGEs, with the potential to be used as functional food ingredients, is proposed.

Ex vivo detection of histone H1 modified with advanced glycation end products

A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes.

Natural compounds containing a catechol group enhance the formation of Nε-(carboxymethyl)lysine of the Maillard reaction

Inhibition of advanced glycation end-product (AGE) formation is a potential strategy for the prevention of clinical diabetes complications. Screening for new AGE inhibitors revealed several natural compounds that inhibited the formation of Nε-(carboxymethyl)lysine (CML), a major antigenic AGE structure, whereas natural compounds containing a catechol group, such as gallic acid and epicatechin, significantly enhanced CML formation. A similar enhancing effect was also observed by culturing THP-1 macrophages in the presence of catechol compounds. Although 4-methylcatechol significantly enhanced CML formation from glycated HSA (gHSA), a model for Amadori proteins, analogues of catechol such as 5-methylresorcinol and methylhydroquinone showed no enhancing effect. Even though 1mM 4-methylcatechol, epicatechin, and gallic acid significantly enhanced CML formation from gHSA, it was significantly inhibited by decreasing their concentration. The enhancing effect of 1mM catechol compounds was inhibited in the presence of the glutathione peroxidase system, thus demonstrating that hydrogen peroxide generated from catechol compounds plays an important role in the enhancement of CML formation. Furthermore, administration of 500mg/kg/day epicatechin to STZ-induced diabetic mice for 45days enhanced CML accumulation at the surface area of gastric epithelial cells in the stomach. This study provides the first evidence that high amounts of catechol-containing structures enhance oxidative stress, thus leading to enhanced CML formation, and this phenomenon may explain the paradoxical effect that some flavonoids have on redox status.

Effect of Inhibitor Compounds on Nε-(Carboxymethyl)lysine (CML) and Nε-(Carboxyethyl)lysine (CEL) Formation in Model Foods

The possible adverse effects on health of diet-derived advanced glycation endproducts (AGEs) and advanced lipoxidation endproducts (ALEs) is of current interest. This study had the objective of determining the effects of the addition of AGE/ALE inhibitors and different types of sugar and cooking oil on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods (sponge cakes). The cake baked using glucose produced the highest level of CML (2.07±0.24 mmol/mol lysine), whereas the cake baked using fructose produced the highest concentration of CEL (25.1±0.15 mmol/mol lysine). There were no significant differences between CML concentrations formed in the cakes prepared using different types of cooking oil, but significant differences (P<0.001) were observed between the cakes prepared using different proportions of cooking oil. The cakes containing oil generated greater concentrations of CML than sucrose. α-Tocopherol and rutin did not inhibit CML and CEL formation. In contrast, ferulic acid and thiamin, thiamin monophosphate, and thiamin pyrophosphate reduced CML and CEL formation.

Immunochemical and Mass Spectrometric Analysis of Nε-(Carboxymethyl)lysine Content of AGE−BSA Systems Prepared with and without Selected Antiglycation Agents

The present study was designed to compare surface plasmon resonance (SPR) biosensor, enzyme-linked immunosorbent assay (ELISA), and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for the analysis of Nε-(carboxymethyl)lysine (CML) in glucose-bovine serum albumin (BSA) model systems and to investigate the possible inhibitory effect of selected compounds (α-tocopherol, ferulic acid, rutin, thiamin, thiamin monophosphate, and thiamin pyrophosphate) on CML formation. The reported levels of CML detected were dependent upon the method of analysis employed. The highest reported concentrations were obtained with the SPR biosensor, whereas the lowest were found by ELISA. However, a high correlation was observed between these two immunochemical procedures. CML concentrations were dependent upon the type and concentration of the candidate CML inhibitor. All inhibitory compounds investigated, with the exception of α-tocopherol, decreased the level of CML formation in the glucose-BSA system.

Inhibitory effect of fermentation byproducts on formation of advanced glycation end-products

Distilled residues (DR) of rice spirit and its derived vinegar produced a negligible inhibitory effect on advanced glycation end-products (AGE) formation. However, recycled DRs of rice spirit and barley spirit and their derived vinegars inhibited formation of N ε(carboxymethyl)lysine (CML), a major AGE. Unlike the aforementioned DR and derived vinegars, the DR of sweet potato spirit and vinegar, contained very little protein, lysine and arginine, but they most potently inhibited CML formation. The DRs of various spirits and vinegars showed similar free radical-scavenging activities.

Heat Treatment Increases the Level of AGEs in Human Blood

92 Levels of plasma pentosidine, which is one of the advanced glycation end-products (AGEs) of the Maillard reaction, are elevated by the pathogenesis of diabetes 1) and end-stage renal failure 2). A recent study suggested that because plasma pentosidine levels in patients with mild renal dysfunction increase before plasma creatinine levels increase, the measurement of plasma pentosidine level can be a useful clinical marker for the early diagnosis of beginning renal failure 3). Several medical laboratories in Japan have measured the levels of blood AGEs, such as Ne-(carboxymethyl)lysine (CML) and pentosidine, by a competitive enzyme-linked immunosorbent assay (ELISA). According to the authors’ protocol, plasma samples are incubated with proteases for anti-AGE antibodies to assess the intramolecular AGE structures, and are incubated again at 100°C for 15 min 3) to inactivate the protease. However, our previous study demonstrated that CML, a major antigenic AGE structure, is generated from the Amadori products by short-term heating in vitro, demonstrating that CML generated from Amadori products are an artifact of immunochemical detection by heating process 4). Furthermore, Miyata et al. 5) reported that substance(s) with a molecular weight of less than 5,000 Da that are abundantly present in uremic plasma exhibit enhanced pentosidine formation during in vitro incubation of uremic plasma at 37°C, thus strongly demonstrating that pentosidine is also generated by incubation in vitro. To confirm this notion, we conducted additional experiments to clarify whether the pentosidine content measured by high-performance liquid chromatography (HPLC) 6) in blood increases using the same procedure as that used by medical laboratories. As a result, the levels of pentosidine in patients with nondiabetic hemodialysis increased 1.2to 3-fold by heating at 100°C for 15 min compared to unheated samples. Moreover, the increased rate of pentosidine levels generated by the heating process was found to differ between patients. Therefore, the heating process as a pretreatment for pentosidine measurement by competitive ELISA appears to compromise the accuracy of the pentosidine concentration present in plasma. Taken together, previous reports 4,5) and the present study indicate that the level of pentosidine in clinical samples is overestimated by using a heating process. Heating enhances both the oxidative cleavage of Amadori products and the production of α-oxoaldehydes such as glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, which were indicated to be important precursors for AGE formation 4). We next measured the inhibitory effects of reduction of Amadori products on CML formation. Because CML is generated from the Amadori compounds by oxidative cleavage, whereas the Amadori compounds reduced into hexitol lysine and glucitol lysine are stable and do not easily generate CML, sodium cyanoborohydride (NaBH3CN) is used for the blood CML analysis using a competitive ELISA 7) to prevent CML formation during the heating process. However, the present study demonstrated that preincubation of glycated bovine serum albumin (glycated BSA), a model Amadori proteins, in the presence of 100 mM sodium borohydride (NaBH4), a stronger reducing agent than NaBH3CN, for 1 h did not inhibit CML formation induced by heating at 100°C for 15 min. From these data, it is likely that the conventional protocol used by medical laboratories which measures plasma pentosidine and CML after pretreatment may be in fact quantifying artifacts of heat treatment. Therefore, the establishment of a more reliable method for the quantification of AGEs in patient blood samples will clarify the physiological significance as well as the clinical usefulness of AGEs. Masako Nakano, Ryoji Nagai