Inhibitory effects of Ge-132 (carboxyethyl germanium sesquioxide) derivatives on enkephalin-degrading enzymes

Twenty-eight species of carboxyethyl germanium sesquioxide (Ge-132) derivatives were examined for their inhibitory effects on enkephalin-degrading enzymes that were purified from monkey brain, the longitudinal muscle layer of bovine small intestine, and human cerebrospinal fluid (CSF). A series of the sulfurized Ge-132 derivatives showed strong inhibition of these purified enzymes. The most effective ones were Ge-014 and Ge-107, which showed IC50 values of 60 and 100 micrograms/ml, respectively, for dipeptidylcarboxypeptidase from the longitudinal muscle layer. They also exhibited inhibitory activity against aminopeptidase from human CSF, the IC50 values being 450 and 440 micrograms/ml, respectively. Furthermore, these compounds showed inhibition of dipeptidylaminopeptidase from monkey brain and the longitudinal muscle layer of bovine small intestine. These compounds are expected to have analgesic effects due to their inhibition of the degradation of endogenous opioid peptides.     

Effects of a lactobacilli, oligosaccharide and organic germanium intake on the immune responses of mice

The organic germanium compound, Ge-132, has immune-modulating effects. We evaluated the symbiotic effects of Ge-132 with lactobacilli and oligosaccharide (LB/OS) on the immune responses of mice. The highest fecal IgA levels were observed in the mice receiving a low concentration of Ge-132 with LB/OS for 8 weeks. Our data suggest that LB/OS with a low concentration of Ge-132 stimulated the intestinal immunity.     

Protective effect of carboxyethylgermanium sesquioxide (Ge-132) on superoxide generation by60Co-irradiated leukocytes

The carboxyethylgermanium sesquioxide, Ge-132, is an organogermanium compound which has been shown to modulate leukocyte functions. In this study, we examined the effect of Ge-132 on the generation of superoxide radicals (O₂ ^–) either from leukocytes or in cell-free system, employing the highly sensitive 2-methyl-6-[p-methoxy-phenyl]-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA) -dependent chemiluminescence method and the specific electron spin resonance/spin trapping method, respectively. In addition, thein vitro protective effect of Ge-132 on the leukocytes irradiated with⁶⁰Co was studied.The incubation with Ge-132 resulted in an increase in basal O₂ ^– release of intact leukocytes, but had no effect on OZ generation from leukocytes stimulated with phorbol myristate acetate (PMA). Irradiation with⁶⁰Co decreased the Oz generation from leukocytes in a dose-dependent manner. Ge-132 had no effect on basal O₂ ^– release from⁶⁰Co-irradiated leukocytes, but it prevented the decrease in PMA-stimulated O₂ ^– generation by irradiated leukocytes. Ge-132 itself had no superoxide scavenging activity in cell-free system. On the other hand, higher concentrations of Ge-132 had decreasing effects on both basal O₂ ^–1 release and PMA-stimulated O₂ ^– generation from leukocytes, but they did not affect leukocyte viability. Above results indicate that 1) Ge-132 can stimulate the basal O₂ ^– release from leukocytes, 2) Ge-132 can prevent the decrease of O₂ ^– generation by⁶⁰Co-irradiated leukocytes, 3) in higher concentrations, Ge-132 may have a membrane stabilizing effect.     

Nephrotoxicity and neurotoxicity in humans from organogermanium compounds and germanium dioxide

There is no known biological requirement for germanium (Ge), germanates, or any organogermanium compound. Ge deficiency has not been demonstrated in any animal. The estimated average dietary intake of Ge in humans is 1.5 mg/d. Ge is widely distributed in edible foods, all of which, with few exceptions, contain less than 5 ppm Ge, since higher levels are toxic to most plants. Ingestion of Ge compounds has been shown to produce toxic effects in experimental animals. In recent years inorganic germanium salts and novel organogermanium compounds, such as carboxyethyl germanium sesquioxide (Ge-132) and lactate-citrate-germanate (Ge lactate citrate) have been sold as "nutritional supplements" in some countries for their purported immunomodulatory effects or as health-producing elixirs, resulting in intakes of Ge significantly exceeding the estimated average dietary intake. Since 1982, there have been 18 reported cases of acute renal dysfunction or failure, including two deaths, linked to oral intake of Ge elixirs containing germanium dioxide (GeO2) or Ge-132. In these cases, biopsies show vacuolar degeneration in renal tubular epithelial cells, without proteinuria or hematuria, in the absence of glomerular changes. Serum creatinine levels have been well above 400 mumol/L in such patients. In 17 of 18 cases, accumulated elemental Ge intakes reportedly ranged between 16 to 328 g over a 4-36 mo period, or between 100 to 2000 times the average estimated dietary intake for human. In surviving patients, renal function improved after discontinuation of Ge supplementation. However, in no case was recovery complete. One organogermanium compound, an azaspiran organogermanium compound, 2-aza-8-germanspiro[4,5] decane-2-propamine-8,8-diethyl-N,N-dimethyl dichloride (spirogermanium), has been found to cause both neurotoxicity and pulmonary toxicity in phase I and II studies examining its chemotherapeutic potential as an antitumor drug in the treatment of various malignancies. In cancer patients given the drug spirogermanium, 40% experienced marked, yet transient neurotoxicity. Two patients suffered from pulmonary toxicity. Results of phases I and II human cancer trials for spirogermanium have not been favorable, with the exception of moderate benefits for three types of malignancies. It is recommended that patients exposed to long-term (greater than 3 mo) Ge supplementation at levels well above the estimated daily intake be medically supervised and monitored for potential renal-, pulmonary- or neurotoxicity. Further study regarding the mechanism of Ge-induced nephrotoxicity in human is warranted.     

Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins

The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of dimers and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, dimers and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions atpH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazoloneN -(5-methyl-4-imidazolon-2-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.Abbreviations AGE advanced glycation endproduct - BSA bovine serum albumin - HSA human serum albumin - MG-SA methylglyoxal-modified serum albumin - MG-BSA methylglyoxal-modified bovine serum albumin - MG-HSA methylglyoxal-modified human serum albumin - AGE-SA AGE-modified serum albumin - AGE-BSA AGE-modified bovine serum albumin - AGE-HSA AGE-modified human serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - HPLC high-performance liquid chromatography - FFI 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole     

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge-132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat- or acid-treatment, suggesting that the induced IFN is of gamma-nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge-132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge-132, neither induction of IFN nor augmentation of NK activity occurred, and X-ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge-132 are mediated by the induced IFN.     

Decreased plasma superoxide scavenging activity in immunological disorders — carboxyethylgermanium sesquioxide (Ge-132) as a promoter of prednisolone

Summary We investigated so-called superoxide scavenging activity (SSA) of plasma in patients with several immunological disorders, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyo-dermatomyositis (PM), progressive systemic sclerosis (PSS), myasthenia gravis (MG) and autoimmune thyroid disease (AT), using the electron paramagnetic resonance/spin trapping technique. Since carboxyethylgermanium sesquioxide, Ge-132, has been reported to modulate both the immune response and leukocyte functions, we have studiedin vivo effect of Ge-132 on plasma SSA and other laboratory parameters in these disorders. The plasma SSA was significantly lower in RA, SLE, PM and PSS, but not in MG and AT, as compared with that in healthy controls. An inverse correlation was observed between plasma SSA and parameters such as erythrocytes sedimentation rates, absolute number of leukocytes, C-reactive protein and serum globulin levels. Furthermore, plasma SSA was significantly decreased in rheumatoid factor-positive patients as compared to negative patients. No correlation was observed between plasma SSA and factors such as ages, sex of patients or the other laboratory parameters, such as serum albumin, triglyceride, cholesterol, hemoglobin and serum iron levels. Patients treated with prednisolone, especially ones with RA, showed an increase of plasma SSA. It appears that Ge-132 promotes prednisolone effects. Our results indicate that a decrease in plasma SSA is not disease specific, but inversely correlates with the severity and activity of inflammation. The methodology to measure plasma SSA presented in this work provides a helpful tool for determining the actual activity of the diseases as well asin vivo studies of antiinflammatory agents.     

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2′-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.     

Analytical product study of germanium-containing medicine by different ICP-MS applications.

For several years organo-germanium containing medicine has been used for special treatments of e.g. cancer and AIDS. The active substances contain germanium as beta-carboxyethylgermanium sesquioxide ((GeCH2CH2COOH)203/"Ge-132"), spirogermanium, germanium-lactate-citrate or unspecified forms. For humans, germanium is not essential and in general the toxicity of the mentioned organo-germanium compounds is low. Acute and chronic toxic effects of inorganic germanium dioxide have been demonstrated. It is obvious that especially inorganic germanium has a higher potential of negative effects. Therefore, a widespread analytical product control is indispensable. Inductively coupled plasma mass spectrometry (ICP-MS) is the preferred technique and different applications were developed for controlling various parameters: (i) A speciation method using high performance liquid chromatography (HPLC) coupled with quadrupole (Q-) ICP-MS was developed for the identification of organo-germanium species in medicine. (ii) The nuclear magnetic resonance (NMR) technique was applied to confirm the molecular structure and to determine the molecule concentration. (iii) The total concentration of germanium in the medicine was determined in the diluted sample by high resolution (HR-) ICP-MS. (iv) For a general overview, a multi-element screening method of 56 elements with HR-ICP-MS was developed. The semi-quantitative mode was used for quantification and elements of higher abundance are reported. (v) Investigations about matrix-based interferences on masses of isotopes, which are generally determinable without remarkable problems. Isotopes like e.g. 85Rb, 88Sr, 89y, 90Zr, 93Nb and the isotopes of Ba are strongly interfered by different Ge-based molecules and need to be analysed in a higher resolution mode than used for other common matrices.     

Mutagen sensitivity and mutability in lentil

Summary Seeds of two cultivars, each of macrosperma and microsperma varietal groups of lentil were mutagenised with gamma-rays and NMU to determine their mutagen sensitivity and mutability. The increasing doeses of gamma-rays and NMU decreased germination, root and shoot length, pollen fertility and plant survival, but increased the occurrence of leaf spots. The root system was found to be more sensitive to both mutagens than the shoot. The macrosperma varietal group was more sensitive to both the mutagens than microsperma group. In microsperma group, variety Pusa-1 was more sensitive to both the mutagens than L-259, while in the macrosperma group L-1491 showed more sensitivity to the mutagens than L-1492. Radio-sensitivity corresponded positively with chemosensitivity in both varietal groups. There was a positive relationship between radio- and chemo-sensitivity of the genotypes and their mutability. The results also revealed the existence of a parallelism between radiomutability and chemo-mutability. Due to saturation in the mutational events and vigour of both diplontic and haplontic selection in the biological material, the mutation frequency either decreased or remained constant at higher doses of the mutagens.     

Ultrastructural localization of gonadotrophic hormones in the porcine pituitary using the immunoperoxidase technique

Summary Using specific antibodies against subunits of porcine LH and FSH, the pituitary cells which produce these two gonadotrophins were identified in the porcine pituitary at the ultrastructural level using the immunoperoxidase technique. It was clearly shown that most of the gonadotrophic cells are responsible for the production of both FSH and LH. However, some cells, only positive for FSH or LH, indicate that the concentrations of FSH and LH vary from cell to cell. At the ultrastructural level, the FSH/LH cells contain one class of secretory granules strongly positive for both FSH and LH as well as large negatively stained dense bodies. These findings indicate that the one cell-one hormone concept may not apply to gonadotrophic hormones; a FSH/LH cell cannot be distinguished from a LH or a FSH cell without immunocytochemical identification of its hormonal content.Abbreviations p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - p-FSH porcine follicule stimulating hormone - p-FSH porcine FSH subunit - b-TSH bovine thyrotropic hormone     

The conformation of bovine serum albumin adsorbed to the surface of single all‐dielectric nanoparticles following light‐induced heating

Interaction between nanoparticles and biomolecules leads to the formation of biocompatible or bioadverse complexes. Despite the rapid development of nanotechnologies for biology and medicine, relatively little is known about the structure of such complexes. Here, we report on the changes in conformation of a blood protein (bovine serum albumin) adsorbed on the surface of single all‐dielectric nanoparticles (silicon and germanium) following light‐induced heating to 640 K. This protein is considerably more resistant to heat when adsorbed on the nanoparticle than when in solution or in the solid state. Intriguingly, with germanium nanoparticles this heat resistance is more pronounced than with silicon. These observations will facilitate biocompatible usage of all‐dielectric nanoparticles.      

Aminoguanidine and metformin prevent the reduced rate of HDL-mediated cell cholesterol efflux induced by formation of advanced glycation end products

OBJECTIVE: The mechanisms whereby advanced glycation end products (AGE) contribute to atherogenesis in diabetes mellitus are not fully understood. In this study we analyzed in vitro the influence of advanced glycated albumin (AGE-albumin) as well as the role of the AGE inhibitors--aminoguanidine (AMG) and metformin (MF)--on the cell cholesterol efflux. METHODS: HDL3 and albumin-mediated cholesterol efflux was measured in mouse peritoneal macrophages and in SR-BI transfected cells that had been treated along time with dicarbonyl sugars or AGE-albumin, both in the presence or in the absence of AMG and MF. 125I-HDL3 cell binding and 125I-AGE-albumin cell degradation were measured. Carboxymethyllysine (CML) formation and SR-BI expressions were determined by immunoblot. RESULTS: AGE-albumin efficiently trapped cell cholesterol but impaired the HDL-mediated cell cholesterol efflux by decreasing HDL binding to the cell surface and inducing intracellular glycoxidation, without interfering with the SR-BI expression. Cell treatment with dicarbonyl sugars also disrupted the HDL-mediated cell cholesterol efflux, but this was prevented by AMG and MF that reduced CML formation. CONCLUSIONS: By adversely impairing the HDL-mediated cell cholesterol removal rate, AGE-albumin and cell glycoxidation could facilitate the development of premature atherosclerosis in diabetes mellitus (DM) and in other diseases associated with carbonyl and oxidative stress like in chronic uremia. Thus, drugs that prevent AGE formation may be useful to correct disturbances in cell cholesterol transport.     

Alpha-tocopherol in the retinal outer segment of bovine eyes

Summary -Tocopherol was identified in lipid extracts of bovine retinal outer segment (ROS) preparations. Positive identification was obtained by the thin layer chromatographic characteristics of the tocopherol form and its oxidation product -tocopherylquinone, and by the ultraviolet spectrum of the oxidized and KBH₄-reduced form of the tocopherylquinone. In the ROS preparations used, -tocopherol chromanol was the predominant species, the quinone form accounting for 25% or less of the total. The concentration of -tocopherol in the ROS preparations was about 0.1 mole -tocopherol per mole rhodopsin, or about 1 nmole/mg, protein. Mitochondria from bovine retina contained about 0.4 nmole -tocopherol per mg protein.     

The ratio of AGE to sRAGE independently associated with albuminuria in hypertensive patients

Background

Soluble receptor for advanced glycation end-products (sRAGE) and advanced glycation end-products (AGE) have been associated with risks of cardiovascular disease. Because sRAGE is regarded as a scavenger to AGE, we hypothesized that the ratio of AGE to sRAGE (AGE/sRAGE) is associated with albuminuria in hypertensive patients.

Methods

In this cross-sectional study, a total of 104 patients with essential hypertension were recruited. Hypertension was defined as a systolic blood pressure?≥?140 mmHg, a diastolic blood pressure?≥?90 mmHg, or use of antihypertensive treatment. Albuminuria was defined as albumin excretion rate ≧ 20 μg/min. Multivariate logistic regression analyses were performed to evaluate the association between AGE/sRAGE and albuminuria.

Results

Among the 104 patients, 30 (28.8%) patients had albuminuria and 74 (71.2%) patients did not. Patients with albuminuria had higher AGE (2.15 vs. 1.71 μg/mL), lower sRAGE (424.5 vs. 492.5 pg/ml) and higher AGE/sRAGE (3.79 vs. 3.29 μg/pg) than those without albuminuria. Multivariate logistic regression model revealed that AGE/sRAGE (OR?=?1.131, 95% CI?=?1.001–1.278, P?=?0.048) was independently associated with albuminuria. There was no significant relationship between AGE and sRAGE alone with albuminuria.

Conclusion

This study suggests that the ratio of AGE to sRAGE may be a surrogate biomarker for microvascular injury. Further prospective studies of the prognostic value of the ratio in relation to microvasular injury are needed.

     

Inhibition of creatine kinase activity in vitro by ethylmalonic acid in cerebral cortex of young rats

Short-chain acyl-CoA dehydrogenase deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of ethylmalonic (EMA) and methylsuccinic (MSA) acids and clinically by severe neurological symptoms. In the present study we investigated the in vitro effects of EMA and MSA on the activity of creatine kinase (CK) in homogenates from cerebral cortex, skeletal and cardiac muscle of rats. EMA significantly inhibited CK activity from cerebral cortex, but did not affect this activity in skeletal and cardiac muscle. Furthermore, MSA had no effect on this enzyme in all tested tissues. Glutathione (GSH), ascorbic acid and -tocopherol, and the nitric oxide synthase inhibitor L-NAME, did not affect the enzyme activity per se, but GSH fully prevented the inhibitory effect of EMA when co-incubated with EMA. In contrast, -tocopherol, ascorbic acid and L-NAME did not influence the inhibitory effect of the acid. The data suggest that inhibition of brain CK activity by EMA is possibly mediated by oxidation of essential groups of the enzyme, which are protected by the potent intracellular, endogenous, naturally occurring antioxidant GSH.     

Effect of germanium-132 on low-density lipoprotein oxidation and atherosclerosis in Kurosawa and Kusanagi hypercholesterolemic rabbits

Germanium-132 (Ge-132) was given at 200 mg/kg of body weight to 8-week-old Kurosawa and Kusanagi hypercholesterolemic (KHC) rabbits. Thirty-six weeks later, the susceptibility of plasma low-density lipoprotein to oxidation and the morphology of atherosclerosis in the aorta and coronary artery were investigated. Treatment with Ge-132 resulted in decreases in the oxidation rate and in the formation rate of thiobarbituric acid-reactive substances following copper-induced oxidation of LDL. Ge-132 is suggested to possess antioxidative properties, but this did not lead to any attenuation of atherosclerotic progression in the KHC rabbits.     

Enhancement effects of BSA and linoleic acid on hybridoma cell growth and antibody production

The effects of linoleic acid and bovine serum albumin on hybridoma cell growth and antibody production were investigated. In dish cultivation, linoleic acid on its own promoted cell growth when used at concentrations below 50 mg L^–1, but strongly inhibited growth at a concentration of 100 mg L^–1 on more. However, linoleic acid bound to bovine serum albumin did not inhibit cell growth, even at a concentration as high as 100 mg L^–1. Also, linoleic acid did not affect the specific antibody production rate, with or without bovine serum albumin. In order to elucidate the enhancement of antibody production by bovine serum albumin, fractions were prepared by ultrafiltration (98% molecular weight cut-offs, 50,000 and 17,000) and the effects of the fractionation on antibody production were studied in batch cultivation. The high-molecular-weight fraction (50,000) promoted antibody production whereas the low-molecular-weight fraction (17,000) inhibited it. In continuous cultivation, the high-molecular-weight fraction was also found to enhance antibody production.     

GM_3对小鼠腹腔巨噬细胞磷脂代谢转换率的影响

神经节苷脂ＧＭ_３对小鼠腹腔常驻巨噬细胞（Ｒ－Ｍ）和Ｇｅ－１３２体内激活的巨噬细胞（Ｇｅ－１３２－Ｍ）的磷脂代谢转换有显著的影响,当这两种Ｍ在体外用ＧＭ_３处理时,表现出［￣（３２）Ｐ］Ｐｉ和［￣３Ｈ］肌醇参入ＰＩ降低,参入ＰＩＰ、ＰＩＰ_２增加;但在［￣（３２）Ｐ］Ｐｉ和［￣３Ｈ］胆碱参入ＰＣ上,Ｒ－Ｍ与Ｇｅ－１３２－Ｍ不同,即ＧＭ_３促进同位素前体参入Ｒ－Ｍ的ＰＣ,抑制它们参入Ｇｅ－１３２－Ｍ的ＰＣ．以上结果表明ＧＭ３可能提高了ＰＩ或ＰＩＰ的磷酸激酶的活性,致使［￣（３２）Ｐ］ＰＩＰ和［￣（３２）Ｐ］ＰＩＰ_２增多,［￣（３２）Ｐ］ＰＩ减少．激活的Ｍ（Ｇｅ－１３２－Ｍ）本身ＰＣ代谢转换率较Ｒ－Ｍ高,当Ｇｅ－１３２－Ｍ再受ＧＭ_３刺激,ＰＣ代谢转换率降低,这提示ＧＭ_３对激活的Ｍ的ＰＣ代谢转换有调节作用．     

Identification of a novel advanced glycation end product derived from lactaldehyde

Advanced glycation end products (AGEs) are implicated in the development of diabetic complications via the receptor for AGEs (RAGE). We have reported that the 3-hydroxypyridinium (3HP)-containing AGEs derived from α-hydroxyaldehydes physically interact with RAGE and show cytotoxicity. Lactaldehyde (LA) is formed from a reaction between threonine and myeloperoxidase, but no LA-derived AGEs have been characterized. Here, we identify the structure and physiological effects of an AGE derived from LA. We isolated a novel 3HP derivative, 2-acetamido-6-(3-hydroxy-5-methyl-pyridin-1-ium-1-yl)hexanoate, named as N-acetyl-LAPL (lactaldehyde-derived pyridinium-type lysine adduct), from a mixture of LA with N^α-acetyl-L-lysine. LAPL was also detected in the LA-modified protein. LAPL elicited toxicity in PC12 neuronal cells, but the effect was suppressed by the soluble form of RAGE as a decoy receptor. Moreover, surface plasmon resonance-based analysis revealed that LAPL specifically binds to recombinant RAGE. These results indicate that LA generates an AGE containing the 3HP moiety and contributes to RAGE-dependent cytotoxicity.

Abbreviations: AGEs: advanced glycation end products; RAGE: receptor for advanced glycation end products; 3HP: 3-hydroxypyridinium; LA: lactaldehyde; LAPL: lactaldehyde-derived pyridinium-type lysine adduct; BSA: bovine serum albumin; GLAP: glyceraldehyde-derived pyridinium; MPO: myeloperoxidase; HFBA: heptafluorobutyric acid; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; LC-ESI-QTOF-MS: liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; NMR: nuclear magnetic resonance; LA-BSA: lactaldehyde-modified bovine serum albumin; PBS: phosphate buffered saline, GST, glutathione S-transferase; SPR: surface plasmon resonance; OP-lysine: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; GLO1: glyoxalase 1; MG, methylglyoxal