Metabolism of 3-deoxyglucosone,an intermediate compound in the Maillard reaction,administered orally or intravenously to rats

Tha Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [¹⁴C]3DG was synthesized from [¹⁴C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [¹⁴C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO₂ were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [¹⁴C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO₂ were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [¹⁴C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and ¹³C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.     

3-脱氧葡糖松代谢酶产生菌的筛选及产酶条件

从霉菌和酵母中筛选到一株酵母（Saccharomycescerevisiae231）,该菌株具有能够代谢3-脱氧葡糖松（3－deoxyglucosone）的酶,且活性较高。研究了该菌株的最适产酶条件：培养温度28℃,培养基起始pH7.0,培养时间12h,碳源、氮源分别为蔗糖、牛肉膏,添加KH₂PO₄、Ca（H₂PO₄）₂·H₂O能促进产酶。     

Immunochemical detection of 3-deoxyglucosone in serum

3-Deoxyglucosone (3-DG) is a metabolite of glucose that is thought to lead to the production of advanced glycation end products in diabetes. The previous assay for 3-DG in serum was based on a multi-step protocol, including derivatization, extraction, HPLC separation, and detection. In the current studies, we established a monoclonal antibody that recognizes the 3-DG-derivative, which is generated by the reaction of 3-DG and a 2,3-diamino-benzene derivative. Attachment of a biotin moiety to the 2,3-diamino-benzene ring via a linker allowed development of a highly sensitive chemiluminescent enzyme immunoassay for 3-DG equivalents. Unlike the previous assay, this method does not require extraction of 3-DG derivatives from serum. Treatment of 3-DG in serum with the DAB-link-biotin produced a quinoxaline derivative, which was specifically recognized by the monoclonal antibody. Using this assay, we found that serum 3-DG was higher in streptozotocin-induced diabetic rats than in normal control rats (25+/-5.6 vs. 9.8+/-1.1 microg/L). This simple assay may allow the monitoring of conditions leading to the accumulation of advanced glycation end products and evaluation of the risk of complications in diabetic patients.     

Enzymatic synthesis of 2-amino- 3-hydroxy-1,6-hexanedicar☐ylic acid using serinehydroxymethyltransferase

The scale up of earlier work from these laboratories using the enzyme serinehydroxymethyltransferase has resulted in the use of this enzyme for the synthesis of 2-amino-3-hydroxy-1,6-hexanedicar☐ylic acid (7) on a preparative scale. This compound, which has been barely described in the literature, is potentially useful for the synthesis of carbocyclic β-lactams and carbocyclic nucleosides.     

Enzymatic synthesis of an orlistat intermediate using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

Methyl (R)-3-hydroxytetradeconoate ((R)-MHOT) is a crucial chiral intermediate for the chemical synthesis of the anti-obesity drug, orlistat. Here, (R)-MHOT was prepared from methyl 3-oxotetradecanoate (MOT) using a mutant of the short-chain dehydrogenase/reductase (SDR) from Novosphingobium aromaticivorans (NaSDR). Mutant NaSDR-G145A/I199L had a 3.23 times greater k_cat value than that of wild type toward MOT. The conditions for the expression of recombinant NaSDR-G145A/I199L were further investigated and obtained cells were used for gram-scale preparation of (R)-MHOT with 50 g/L of MOT. The target product was extracted and confirmed by gas chromatography; the enantiomeric excess value of (R)-MHOT was 99.0 %. Molecular docking analysis was used to reveal the molecular basis of the enhanced catalytic activity of NaSDR-G145A/I199L; NaSDR-G145A/I199L presented a more effective docking posture than NaSDR. This is the first reported use of SDR for preparing (R)-MHOT via the reduction of MOT. Our study provides a foundation for greener preparation of (R)-MHOT.     

Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.     

l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria

Summary We have studied the 3-mercaptopyruvate pathway (transamination pathway) ofl-cysteine metabolism in rat liver mitochondria.l-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. Whenl-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withl-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation froml-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatl-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.     

Differential diagnosis of (inherited) amino acid metabolism or transport disorders

Summary Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.     

果蔬制品中的美拉德反应及其控制方法研究进展

美拉德反应是一种普遍存在于果蔬制品加工和贮藏过程中的非酶促类褐变反应,其反应机制复杂,会引起褐变,破坏产品感官品质,降低食用价值,还会生成有毒有害物质,危害人体健康。本文对果蔬制品中美拉德反应的历程、影响因素和抑制手段进行归纳总结,对目前研究存在的问题进行展望,以期为果蔬制品中美拉德反应的控制提供参考。     

细菌3-脱氧葡糖醛酮代谢酶的纯化及性质研究

细菌Bacillus sp.2粗酶液通过(NH₄)₂SO₄分级分离、Q Sepharose FF、Sephadex G-100(Ⅰ)、Hydroxyapatite和Sephadex G-100(Ⅱ)柱层析分离,纯化了一种以NADPH为辅酶的3-脱氧葡糖醛酮(3-DG)代谢酶,定性为2-羰基醛还原酶.纯化酶的比活力为63.75 U/mg,在SDS-聚丙烯酰胺凝胶上显示一条蛋白质带.该酶分子质量约为32 ku,酶反应最适pH约为6.2, 在pH 5～8, 温度25～30℃之间酶保持稳定;该酶对3-DG的K_m为2.3 mmol/L.添加适量的EDTA、巯基乙醇或二硫苏糖醇能明显提高酶的活性;而碘乙酸、N-乙基顺丁烯二酰亚胺抑制酶的活性.     

Enzymatic resolution of 3-(3-indolyl)butyric acid: a key intermediate for indolmycin synthesis

Both enantiomers of 3-(3-indolyl)butyric acid, a key intermediate of indolmycin, were successfully prepared by lipase-catalysed enantioselective hydrolysis. Of the enzymes examined, Pseudomonas fluorescens lipase (lipase AK) showed the best enantioselectivity and highest reactivity for the hydrolysis of (±)-trifluoroethyl 3-(3-indolyl)butyrate. Under optimal conditions, optical resolution was completed in one enzyme-catalysed step, the S-acid and unreacted R-ester being obtained in high optical purity.     

In vivo monitoring of serum protein cross linking in patients with diabetes mellitus. Evidence for pharmacological modification of immunoglobulin G cross links

Summary It is well known that increased cross linking of proteins due to nonenzymatic glycosylation occurs in diabetic animals and humans leading to accumulation of proteins (e.g. collagen). This in turn is strongly associated with diabetic long term complications.We developed a noninvasive method for studying in vivo cross linking and its pharmacological inhibition by L-arginine in a blind placebo controlled study with crossing over of two treatment periods of three months each.Glycemic control was assessed by determining blood glucose, HbA1c, fructosamine, and total glycosylated hemoglobin. The patients were randomly assigned to two treatment groups A (n = 14) and B (n = 16). 20 healthy volunteers served as controls. Treatment consisted of two daily dosages of 1 g L-arginine free base. Cross linking of a human serum protein (IgG) was assessed by SDS polyacrylamide gel electrophoresis and subsequent Western blotting.Diabetic patients showed a statistically increased number of cross links compared to normal controls (Group A: 3.6 vs 2.0 bands, group B: 3.8 vs 2.0 bands). L-arginine led to a significant reduction of cross links in both treatment groups (Group A: 3.6 to 2.1 bands, group B: 3.8 to 2.5 bands).The described noninvasive method for assessing in vivo cross linking requires onlyµl amounts of serum and could serve to monitor protein cross linking in patients with diabetes mellitus.     

The effect of ovariectomy on phenylalanine and tyrosine metabolism

Summary Although the regulatory activity of steroid hormones on amino acid metabolism has been described, no information is published on the effect of ovariectomy. We studied the influence of ovariectomy in Wistar rats determining the amino acids phenylalanine and tyrosine in liver, kidney, plasma and urine. 32 animals were used in the study, 12 animals were sham operated, 9 animals were ovariectomized and 11 rats were ovariectomized and supplemented with estradiol. No quantitative changes were detected comparing liver and kidney phenylalanine and tyrosine between the groups (sham operated rats liver phenylalanine 2,53nM/mg ± 1,07; liver tyrosine 1.95nM/mg ± 0.92; kidney phenylalanine 2.16nM/mg ± 0.53; kidney tyrosine 1.80nM/mg ± 0.39. Ovariectomized rats showed liver phenylalanine 3.07nM/mg ± 1.14; liver tyrosine 2.63nM/mg ± 1.01; kidney phenylalanine 2.30 nM/mg ± 0.74; kidney tyrosine 1.93nM/mg ± 0.63. Ovariectomized and estradiol supplemented rats presented with liver phenylalanine 2.84nM/mg ± 1.40; liver tyrosine 2.35nM/mg ± 1.28; kidney phenylalanine 1.91nM/mg ± 0.26, kidney tyrosine 1.67nM/mg ± 0.23.). When, however, the phenylalanine/tyrosine ratio in the liver was evaluated, ovariectomized rats showed a significant decrease of the quotient (p = 0.001). The phenylalanine/tyrosine ratio was restored by estradiol replacement. Our findings show that phenylalanine and tyrosine metabolism is under estradiol control. The effect on the metabolic changes could be mediated by enzyme systems as phenylalanine hydroxylase, tyrosine hydroxylase and tyrosine aminotransferase. Our results would be compatible with previous reports on the stimulatory effect of estradiol on these enzymes. The kidney phenylalanine/tyrosine ratio was unaffected by ovariectomy and/or estradiol replacement which can be easily explained by different pools, enzyme activities, filtration/reabsorption effects, etc.The urinary P/T ratio was decreased by ovariectomy and restored by estradiol replacement indicating endocrine control of renal reabsorption and secretion mechanisms.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

2,3-Dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) generated by the Maillard reaction in foods affect autonomic nervous activity and central nervous activity in human

ABSTRACT

This study investigated the effect of the odors generated by the glycine/glucose Maillard reaction and the potent odorants 2,3-dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) on the human mood and integrative physiological activity. The score of certain subjective moods, especially anger-hostility, and tension-anxiety were decreased significantly after inhalation of the Maillard reaction sample and DMHF, and fatigue-inertia mood was also significantly decreased by DMHF, suggesting a sedative effect of these odors on mood, while 3DP had no effect. Miosis rate and fingertip temperature increased significantly following inhalation of the odor from the Maillard reaction sample and both potent odorants, suggesting that the parasympathetic nervous system dominates through suppression of the sympathetic activity. The physiological relaxing effect of these odors was also confirmed by decreased flicker frequency value and decreased oxyhemoglobin in the prefrontal cortex.     

Carbohydrate polymers in food preservation: an integrated view of the Maillard reaction with special reference to discoveries of preserved foods in Sphagnum-dominated peat bogs

Well-preserved human bodies more than 2000 years old have been found in peat bogs derived mainly from sphagnum mosses. Preservation is correlated with the occurrence of -keto-carboxylate groups in a glycuronoglycan (‘sphagnan') that comprises 60% of the holocellulose in the hyaline cell walls of the mosses [Painter (1991b). Carbohydr. Polym., 15, 123–142]. There is now renewed interest in other biodegradable materials that have been found preserved in peat, including carcasses of domestic animals, loaves of bread, dried fruits, berries, and kegs of butter or cheese up to 1800 years old. This review attempts to correlate these examples of fortuitous preservation in peat with other, more familiar methods of food preservation that depend in the first instance upon the condensation of highly reactive carbonyl compounds with primary amino-groups or ammonia. The Maillard reaction inhibits microbial growth by sequestering ammonia, aminoacids and peptides, while the brown, polymeric end-products (‘melanoidins') inhibit by cross-linking polypeptide chains and sequestering essential, multivalent metal cations. These reactions could find broader or entirely new applications in food preservation.     

Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)

A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K_m values of 0.8-6.1 μM and 3.7-67 μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K_m = 96-390 μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.     

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Saccharide induced racemization of amino acids in the course of the Maillard reaction

Summary. The formation of D-amino acids on heating aqueous solutions of protein L-amino acids at pH 2.5 and pH 7.0 together with glucose, fructose or saccharose was investigated by enantioselective gas chromatography. The saccharide induced partial racemization (epimerisation) of L-amino acids is attributed to the Maillard reaction. Received October 1, 2001 Accepted October 2, 2001