Toxic End Products Research Articles

Urinalysis of 4-hydroxynonenal, a marker of oxidative stress, using stir bar sorptive extraction–thermal desorption–gas chromatography/mass spectrometry

A simple and fast method for the measurement of 4-hydroxynonenal (4HNE), a highly toxic end-product of lipid peroxidation, in urine samples is described. The method combines stir bar sorptive extraction (SBSE) with two derivatization steps, followed by thermal desorption and GC/MS. 4HNE is derivatized in situ with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine and the oxime is extracted from the aqueous phase with SBSE. The 4HNE-oxime is further acylated by headspace derivatization prior to thermal desorption. Derivatization reactions and extraction were optimized in terms of reagent quantities, temperature and time. The method is linear over a concentration range of 0.5-5 ng mL(-1) with a correlation coefficient of 0.997. The limit of detection and limit of quantitation are 22 and 75 pg mL(-1) urine, respectively. The high sensitivity of the method allows the measurement of physiological concentrations of 4HNE in urine samples.

Messenger Molecules and Cell Death

Programmed cell death, also called apoptosis, participates not only in normal physiologic processes such as development of the immune system, but also in many diseases. A loss of normal cell death may occur in cancer, and excessive cell death is found in a variety of neurodegenerative conditions. We describe 3 distinct pathways that regulate cell death. First, bilirubin, often thought to be a toxic end product of heme metabolism, serves as a physiologic cytoprotectant that may attenuate multiple forms of morbidity. In a second pathway, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mediates a novel cell death cascade. Cytotoxic stimuli, via nitric oxide generation, lead to the binding of GAPDH to the protein Siah1, translocation of GAPDH-Siah1 to the nucleus, and ultimately cell death. Third, cytochrome c, released from mitochondria early in apoptosis, synergizes with inositol-1,4,5-triphosphate (IP3) to elicit massive cellular calcium release, resulting in cell death. These pathways may regulate cell survival in a variety of pathologic states and represent fertile targets for novel therapies.

Oxalate induced expression of monocyte chemoattractant protein-1 (MCP-1) in HK-2 cells involves reactive oxygen species

Oxalate is a toxic end product of metabolism largely because of its propensity to crystallize and form calcium oxalate, which is insoluble at physiologic pH and often deposits at very unfortunate sites, notably the kidneys. In the current study, we investigated the oxalate-induced injury and up-regulation of monocyte-chemoattractant protein-1 (MCP-1) in HK-2 cells, a proximal tubular epithelial cell line derived from normal human kidney. The cells were exposed to oxalate ions for different lengths of time. The culture media was tested for LDH release, a cell injury marker. mRNA was isolated from the cells and subjected to reverse transcriptase-polymerase chain reaction. The data showed that oxalate exposure resulted in cell injury in a time and concentration dependent manner. The MCP-1 mRNA increased following exposure to oxalate and was reduced upon treatment with free radical scavengers, catalase and superoxide dismutase. These data support the importance of reactive oxygen species in the induction of expression of MCP-1 in renal epithelial cells. To our knowledge, this is the first report of MCP-1 expression and its upregulation by oxalate exposure in HK-2 cells.

Comparative Gut Microflora, Metabolic Challenges, and Potential Opportunities

Bacteria colonize practically every habitat in the nature. Bacterial community of the gastrointestinal tract (GIT) is one of the major metabolic tissues of animals. Still, its species composition is not known, because methods that have been previously used for bacterial analysis do not capture the species present. The bacterial community within the GIT not only has intracommunity interactions, but it also interacts with the host tissues. For the host, it is essential to tolerate commensal bacteria and to recognize and fight intestinal pathogens. In addition to recognized pathogens, there are many functional bacterial groups, which produce metabolites considered harmful for the host. Such functional bacterial groups include protein-fermenting bacteria that produce toxic end products in their amino acid metabolism, which is why a high-protein diet is often considered unhealthy. Carbohydrates are preferred substrates for gastrointestinal bacteria, but in the distal colon, they may become depleted, and putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of slowly fermenting dietary fibers.Microbial communities in the GIT of different animals have developed hand-in-hand with their digestion strategy. In monogastric animals, bacterial fermentation has concentrated in the lower GIT, whereas in ruminants bacteria are responsible for the initial digestion of the diet. Rumen-like sites with intense bacterial fermentation are found in most animals, but no other group of animals totally depends on bacteria. The response of rumen bacteria to feed components can be evaluated by an in vitro rumen simulation, which measures relevant fermentation parameters, such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). In monogastric animals, it is essential that the host has measures to prevent bacterial competition for substrates in regions in which absorption takes place; the use of prophylactic antibiotics for production animals has aided this.Availability of good methods for monitoring the total bacterial communities and their metabolism is a prerequisite for informative studies in the future.

Effects of flooding on downstream processes of glycolysis and fermentation in roots of Melaleuca cajuputi seedlings

We investigated the energy metabolism in roots of flooded Melaleuca cajuputi Powell, a tropical flood-tolerant tree species, by measuring adenylate concentrations and activities of glycolytic and fermentative enzymes under flooded conditions. Adenylate energy charge (AEC) decreased slightly to 0.72 on the second day of flooding and recovered to around 0.8 by the fourth day of flooding. Activities of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) increased initially and then decreased to the control level after 14 days of flooding. On the other hand, activities of pyruvate kinase (EC 2.7.1.40), phosphoenolpyruvate phosphatase (EC 3.1.3.2), and a series of phosphoenolpyruvate carboxylase (EC 4.1.1.31), malate dehydrogenase (EC 1.1.1.37), and NADP dependent malic enzyme (EC 1.1.1.40), which can convert PEP into pyruvate, were not induced in flooded roots throughout the experiment. These results suggest that neither the downstream reactions of glycolysis nor ATP production via glycolysis was enhanced by flooding, whereas alcohol fermentation was enhanced. With the low ATP yield of the glycolysis–alcohol fermentation pathway and no induction of glycolytic enzymes, the glycolysis–alcohol fermentation pathway itself contributes little to ATP production in flooded roots of M. cajuputi. These physiological responses of M. cajuputi to flooding may have the advantages of surviving flooded conditions because they can avoid exhaustion of sugar and accumulation of ethanol, a toxic end product of alcohol fermentation.

Expression of Four Glutamine Synthetase Genes in the Early Stages of Development of Rainbow Trout (Oncorhynchus mykiss) in Relationship to Nitrogen Excretion

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.

Feed-forward Regulation of Bile Acid Detoxification by CYP3A4: STUDIES IN HUMANIZED TRANSGENIC MICE

Bile acids are potentially toxic end products of cholesterol metabolism and their concentrations must be tightly regulated. Homeostasis is maintained by both feed-forward regulation and feedback regulation. We used humanized transgenic mice incorporating 13 kb of the 5' regulatory flanking sequence of CYP3A4 linked to a lacZ reporter gene to explore the in vivo relationship between bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis after bile duct ligation (BDL). Male transgenic mice were subjected to BDL or sham surgery prior to sacrifice on days 3, 6, and 10, and others were injected with intraperitoneal lithocholic acid (LCA) or vehicle alone. BDL resulted in marked hepatic activation of the CYP3A4/lacZ transgene in pericentral hepatocytes, with an 80-fold increase in transgene activation by day 10. Individual bile acids were quantified by liquid chromatography/mass spectrometry. Serum 6beta-hydroxylated bile acids were increased following BDL, confirming the physiological relevance of endogenous Cyp3a induction to bile acid detoxification. Although concentrations of conjugated primary bile acids increased after BDL, there was no increase in LCA, a putative PXR ligand, indicating that this cannot be the only endogenous bile acid mediating this protective response. Moreover, in LCA-treated animals, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining showed hepatic activation of the CYP3A4 transgene only on the liver capsular surface, and minimal parenchymal induction, despite significant liver injury. This study demonstrates that CYP3A up-regulation is a significant in vivo adaptive response to cholestasis. However, this up-regulation is not dependent on increases in circulating LCA and the role of other bile acids as regulatory molecules requires further exploration.

Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II cells.

4-Hydroxy-trans-2,3-nonenal (HNE) is a toxic end product of lipid peroxidation. This multifunctional aldehyde reacts with proteins, phospholipids, and nucleic acids, consequently activating/inactivating enzymes, affecting signal transduction and gene expression. HNE is mainly detoxified by glutathione (GSH) conjugation. In our previous report, we showed that GSH conjugates of 4-hydroxynonenal (HNE-SG) are substrates of multidrug resistance-associated protein 2 (MRP2). MRP2 has been shown to export HNE-SG conjugates into the extracellular space. In the present study, the role of MRP2 in the detoxification of HNE was studied using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2. MRP2 reduced the intracellular accumulation of HNE-SG conjugate but unexpectedly increased the susceptibility of cells to HNE. The viability of cells was reduced to approximately 70% in the presence of 62.5 microM HNE in MDCK II cells expressing MRP2, whereas MDCK II cells remained unaffected. MRP2 accelerated the elimination of intracellular GSH via a conjugation reaction with HNE (half-life of GSH was 30.1 and 12.2 min for MDCK II cells and MDCK II cells expressing MRP2, respectively). Moreover, the consumption of GSH was unlimited in MDCK II cells expressing MRP2, finally resulting in necrosis. These results indicate that MRP2 has an adverse effect during the detoxification of HNE in MDCK II cells and suggest that expression of MRP2 may enhance the damage caused by oxidative stress.

New horizons in AGE research

The formation, persistence, and tissue toxicity of advanced glycation end products (AGEs) has emerged to be one of the most coherent mechanisms to explain the development of many of the pathological complications of diabetes and normal aging. In vivo and recent human clinical studies have served to underscore the pathophysiological importance of AGEs. Current research into the AGE pathway is proceeding along several fronts.

Reducing Household Waste: Japan Learns from Germany

The Generation of solid waste is a serious proble, worldwide, particularly among industrialized nations where the amount of solid waste generated continues to increase in both absolute and per capita terms.1 For the most part, muncipal governments are the bodies that must grapple with the various aspects of solid waste, including waste collection, transfer, and disposal. The rising cost and decreasing availability of landfills, as well as citizen opposition to proposed landfill sites, are making disposal increasingly problematic. Waste incineration is often employed to reduce the volume of waste sent to landfills, but this practice is not without its environmental costs—including, increased air pollution and the creation of a potentially toxic end product (incinerator ash).

Growth of the bacteriocin-producing Lactobacillus sakei strain CTC 494 in MRS broth is strongly reduced due to nutrient exhaustion: a nutrient depletion model for the growth of lactic acid bacteria.

Although commercial MRS broth has been designed to allow excellent growth of lactobacilli, most of these bacteria are still subjected to a self-inhibiting process. The most likely explanation is the accumulation of lactic acid or other toxic end products and the depletion of nutrients. In this study, the self-inhibition of Lactobacillus sakei CTC 494 was analyzed in a kinetic way, and a nutrient depletion model was set up to describe the growth inhibition process. This simple model has considerable advantages compared to commonly used descriptive models such as the logistic growth equation. It offers a better fit and a more realistic description of the growth data by taking into account both growth inhibition due to lactic acid production and changes in growth rates due to nutrient depletion. Depending on the fermentation conditions, in MRS broth there appears to be a strong decrease of the specific growth rate over time. Some undefined compounds present in the complex nitrogen source of MRS broth appear to be of crucial importance because of their limited availability. Moreover, nutrient availability affects bacteriocin production through its effect on cell growth as well as on the bacteriocin production per cell. A plateau value for the bacteriocin production by L. sakei CTC 494 was observed.

Advanced glycation end-products: a review.

Advanced glycation end-products are a complex and heterogeneous group of compounds that have been implicated in diabetes related complications. At present it is not known if they are the cause or the consequence of the complications observed. We discuss the chemistry of advanced glycated end-product formation and their patho-biochemistry particularly in relation to the diabetic microvascular complications of retinopathy, neuropathy and nephropathy as well as their role in the accelerated vasculopathy observed in diabetes. The concept of carbonyl stress as a cause for advanced glycated end-product toxicity is mentioned. We discuss alterations in the concentrations of advanced glycated end-products in the body, particularly in relation to changes occurring with age, diabetes and its complications such as nephropathy. Problems relating to current methods of advanced glycated end-product detection and measurement are highlighted including the lack of a universally established method of detection or unit of measurement. Agents used for the treatment of advanced glycated end-product accumulation are reviewed, with an emphasis on the results of the recent phase III trials using aminoguanidine and diabetes related complications.

Rapid-rate thermophilic, single-stage and two-phase methane fermentation of synfuel-industry wastewaters

Thermophilic (55 degrees C) anaerobic digestion of wastewaters from wet carbonization of Minnesota peat was conducted in a simple, upflow digester at an HRT of one day to provide a methane yield of 0.20 SCM/kg VS added and a BOD5 reduction of 85%. Carbonization of Maine peat conducted for a much longer duration of thermal treatment produced recalcitrant and potentially toxic end products that reduced the biodegradability of wastewaters. Methane fermentation of the Maine-peat wet-carbonization waste was inhibited at an HRT of one day in the single-stage digester. However, two-phase digestion with separate acid-phase fermentation to promote hydrolytic degradation of toxic end products followed by separate methane fermentation at an HRT of 2 days exhibited a methane yield of 0.21 SCM/kg VS added and a BOD5 reduction of 76%. Volatile fatty acids profiles along the depths digesting cultures indicated that single-stage and two-phase digestion could be accomplished at one-half the reactor residence times (one day for single-stage and three days for two-phase) used in this research.

Metabolic Activities of the Gut Microflora in Relation to Cancer

The human large intestine is host to a diverse range of bacteria with numbers reaching 10 11 /ml of faecal material. This population plays a significant role in colonic metabolism and health. Undigested dietary substrates and endogenous residues are metabolised by the gut flora. Some of the products of this metabolism have been associated with carcinogenic processes such as tumour promotion (ammonia, secondary bile acids), mutagenesis (fecapenaenes) and carcinogenesis (N-nitroso compounds). Bacterial enzymes involved with carcinogen formation include β -glucuronidase, β -glycosidase, azoreductase, nitroreductase, nitrate reductase, the conversion of pre-carcinogen 2 amino-3-methyl-7H-imidazo[4,5-f]quinoline(IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one(7OHIQ). Protective effects of gut bacterial metabolism include carcinogen binding, detoxification of methylmercury and formation of lignans and isoflavones. Diet is known to play a role in gut microflora metabolism and cancer development. Studies have shown that gut bacterial enzymes are affected by diet and bacterial metabolism of dietary protein releases toxic products including ammonia, phenols and cresols. The interrelationships between diet, gut microflora metabolism and effects on the host are complicated however they may be important in the understanding of epidemiological associations between exogenous factors and colorectal cancer risk. Keywords: microflora metabolism, colon cancer, toxic end-products, detoxification.

On the use of micro-electrodes in fish pond sediments

The surface layer of fish pond sediments is characterized by very high microbial activity. Oxygen uptake by the microbes at the sediment surface leaves little oxygen to penetrate the sediment. In marine and freshwater lake sediments, oxygen only penetrated a few millimeters. In this study, oxygen penetration into the sediment layer in fish ponds and simulated fish ponds was measured with an oxygen micro-electrode. Oxygen profiles determined with micro-electrodes showed that oxygen does not penetrate deeper than 1 mm under quiescent conditions in intensive and semi-intensive fish pond sediments. Sediment oxygen consumption (SOD) at approximately 25°C, calculated from measured oxygen profiles, ranged from 45 to 50 mg O 2/m 2 per h. Change in water temperature and the addition of formaldehyde showed that most of the oxygen consumption in the sediment surface layer is related to biological activity. Redox potential profiles of fish pond sediments measured with a micro-platinum electrode, showed reducing conditions conducive to sulfide formation at 2–4 mm below the sediment surface. Nitrate addition to the overlying water poised redox potential above 300 mV to a sediment depth of about 15 mm. Nitrate thus buffered the sediment–water interface against reducing conditions in the sediment and the possible diffusion of toxic reduced end products to the water.

(E)-4-Hydroxy-2-Nonenal May be Involved in the Pathogenesis of Parkinson’s Disease

( E)-4-Hydroxy-2-nonenal (HNE) is a toxic end-product of the free radical-stimulated peroxidation of phospholoipid-bound arachidonic acid in cell membranes. There is a growing body of evidence to suggest that free radicals may play an important role in the pathology of Parkinson’s disease. HNE is highly electrophilic and is conjugated to reduced glutathione (GSH) by glutathione S-transferase. The depletion of GSH in the substantia nigra of Parkinson’s patients and in the brainstem of mice treated with the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prompted this study on the concentrations of HNE in the cerebrospinal fluid (CSF) and plasma of Parkinson’s patients and the brainstem of mice treated with MPTP. HNE was identified and quantitated by a highly specific and sensitive method based on the gas chromatography-negative-ion chemical ionisation mass spectrometry of the O-pentafluorobenzyl oxime derivative using 9 D 3-4-hydroxy-2-nonenal as an internal standard. The mean concentration of HNE in the CSF of patients with Parkinson’s disease was 1.47 ± 0.76 μM (mean ± SD, n = 10), while the concentration in the CSF of a group of control patients was 0.38 ± 0.14 μM ( n = 10; p < .01). The mean concentration of HNE in the plasma of Parkinson’s patients was 0.68 ± 0.15 μM ( n = 20) and the concentration in the control group was 0.47 ± 0 12 μM ( n = 20; p < .05). The mean peak concentration of HNE in the brainstem of mice after a single s.c. dose of MPTP (40 mg/kg) was 3.62 ± 0.36 nM/g wet wt. ( n = 17) at 12 h while the control value was 0.45 ± 0.05 nM/g wet wt. ( n = 20; p < .05). The GSH concentration in the brainstem of MPTP-treated mice at 24 h. was 0.65 ± 0.03 μM/g wet wt. ( n = 14) and the control value was 1.25 ± 0.03 μM/g wet wt. ( n = 20; p < .01). The corresponding concentration of GSH-HNE-conjugate at 24 h was 0.32 ± 0.09 μM/g wet wt. ( n = 12) compared with a control value of 0.05 ± 0.02 ( n = 16; p < .01). After treatment with α-tocopherol (2.35 g/kg s.c. daily × 3) the mean concentration of HNE 12 hr. after MPTP injection was 0.89 ± 0.06 nM/g wet wt. ( n = 18). The HNE concentration in a group not treated with α-tocopherol prior to MPTP injection was 3.49 ± 0.09 nM/g wet wt. ( n = 14; p < .05). The concentration of GSH in the mice pretreated with α-tocopherol before MPTP injection was 1.14 ± 0.02 μM/g wet wt. ( n = 17) at 24 h compared to 0.61 ± 0.02 μM/g wet wt. ( n = 14) in the untreated mice ( p < .05). The direct injection of HNE (1, 10, 100, 1,000 μM) into the substantia nigra caused a dose dependent depletion of GSH in the brainstem of mice. The mean concentration of GSH 24 hr after the injection of 100 μM of HNE was 0.43 ± 0.22 μM/g wet wt. ( n = 4) compared with a control value of 1.48 ± 0.02 μM/g wet wt. ( n = 8; p < .05). The corresponding concentration of GSH-HNE-conjugate was 0.32 ± 0.12 μM/g wet wt. ( n = 4) while the control value was 0.04 ± 0.02 μM/g wet wt. ( n = 8). These data suggest that HNE may be a causeative neurotoxin in Parkinson’s disease and that HNE may also be involved in MPTP toxicity.

Probing the metabolism of genetically-engineered mammalian cells by heat flux

With the onset of the commercial production of target proteins by hybridoma and genetically engineered cells, there is a pressing requirement for biosensors to monitor on-line and in real-time their growth in culture. In terms of irreversible thermodynamics, most of the Gibbs energy provided in substrates for this process is dissipated as heat, with only a relatively small quantity being conserved in biomass and the target proteins. Calorimetry would appear, therefore, to be a strong contender for a metabolic probe. A flow microcalorimeter was modified for use with animal cells and tested using CHO 320 cells which produce the heterologous glycoprotein dimer, Interferon-γ. Preliminary results showed that heat flow did not follow the increase in cell-number concentration and it was realised that this technique must be combined with one to assess biomass on-line accurately to reflect metabolic activity. Dielectric spectroscopy was chosen because capacitance measurements of the culture measures the volume fraction of viable cells and not the dead ones. A commercial version, the viable cell monitor, was validated by parallel measurements of the volume of viable cells by flow cytometry. The combined probe showed that heat flux was a function of the specific growth rate which, in turn, was related to the fluxes for glucose and glutamine utilisation and possibly to the accumulation of toxic end products, lactate and ammonia. The calorimetric-respirometric (CR) ratio was highly negative during growth in the fully aerobic conditions, indicating simultaneous anaerobic metabolism during growth. The CR ratio gave a value consistent with solely oxidative processes once there was no growth. This might indicate that ATP demand was greater than could be satisfied by oxidative processes, but this was not the case since the cells were respiring at only ca. 60% of capacity, as judged by uncoupling with FCCP. It appears likely that lactate production was due to the need to produce biosynthetic precursors which were not supplied in the medium. A better medium design would probably decrease this requirement and thereby reduce the accumulation of toxic end products. The heat flux probe was shown to be invaluable in exploring the metabolism of CHO 320 cells grown in batch culture.

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached sta- tionary phase, and had a most probable number of viable cells that was greater than 10 6 /ml. When the cel- lobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cello- biose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 · 10 3 cells/ml. The stationary- phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Met- hylglyoxal, a potentially toxic end-product of carbohy- drate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supple- mented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent Km of cellotriose for- mation was 15-fold lower than the Km of glucose pro- duction (0.7 mM compared to 10 mM).

In Vitro Kinetic Studies of Formation of Antigenic Advanced Glycation End Products (AGEs): NOVEL INHIBITION OF POST-AMADORI GLYCATION PATHWAYS

Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products ("AGEs") and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.

Thiamine Pyrophosphate and Pyridoxamine Inhibit the Formation of Antigenic Advanced Glycation End-Products: Comparison with Aminoguanidine

Nonenzymatic glycation of proteins by glucose leading to the formation of toxic and immunogenic advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus, aging, and, possibly, neurodegenerative diseases such as Alzheimer's. We tested thein vitroinhibition of antigenic AGE formation on bovine serum albumin, ribonuclease A, and human hemoglobin by various vitamin B1and B6derivatives. Among the inhibitors,pyridoxamine and thiamine pyrophosphate potently inhibited AGE formation and were more effective than aminoguanidine,suggesting that these two compounds may have novel therapeutic potential in preventing vascular complications of diabetes. An unexpected finding was that aminoguanidine inhibited the late kinetic stages of glycation much more weakly than the early phase.