Lipoproteins in the DCCT/EDIC cohort:associations with diabetic nephropathy

Lipoproteins may contribute to diabetic nephropathy. Nuclear magnetic resonance (NMR) can quantify subclasses and mean particle size of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), and LDL particle concentration. The relationship between detailed lipoprotein analyses and diabetic nephropathy is of interest.

Serum lipoproteins in the diabetes control and complications trial/epidemiology of diabetes intervention and complications cohort:associations with gender and glycemia

To relate the nuclear magnetic resonance (NMR)-determined lipoprotein profile, conventional lipid and apolipoprotein measures, and in vitro oxidizibility of LDL with gender and glycemia in type 1 diabetes.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL.

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P <0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P <0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P <0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells

We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes.

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine:two products of nonenzymatic modification of aminophospholipids formed in vivo

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

Carboxymethylethanolamine, a biomarker of phospholipid modification during the maillard reaction in vivo

Nepsilon-(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, approximately 0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n = 22, p > 0.5). Levels of CML in erythrocyte membrane proteins were approximately 0.2 mmol/mol of lysine for both control and diabetic subjects (p > 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p = 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.

Lipoprotein glycation and its metabolic consequences

Glycation of lipoproteins is implicated in the development of the macro- and microvascular complications of diabetes, atherosclerosis in general, and other disease processes including aging. Enhanced glycation may have direct effects, and may also amplify the effects of oxidative stress on lipoproteins. Most studies have examined the effects of glycation of LDL, particularly with respect to its atherogenicity. Other lipoproteins are more difficult to study because their several apolipoproteins, being of varying age, are not uniformly exposed to glucose. Inhibition of the combined stresses of glycation and oxidation towards lipoproteins may have beneficial effects on health.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia reperfusion injury and rheumatoid arthritis. Chemical modification of amino acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative stress in vivo. The focus of the studies described here was initially to identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (HNE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays for the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diimine cross-link and the Michael addition product of HNE to lysine. Using these assays, we showed that the concentrations of all three compounds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine (CML) also increased during LDL oxidation, while that of its putative carbohydrate precursor the Amadori compound N epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating that CML is a marker of both glycoxidation and lipoxidation reactions. These results suggest that MDA and HNE adducts to lysine residues should serve as biomarkers of lipid modification resulting from lipid peroxidation reactions, while CML may serve as a biomarker of general oxidative stress resulting from both carbohydrate and lipid oxidation reactions.

Increased serum pigment epithelium derived factor levels in Type 2 diabetes patients

Serum PEDF levels (mean (S.D.)) were increased in 96 Type 2 diabetic vs. 54 non-diabetic subjects; 5.3 (2.8) vs. 3.2 (2.0)mug/ml, p

Immune complexes containing modified lipoproteins are related to the progression of internal carotid intima-media thickness in patients with type 1 diabetes

Modified lipoproteins induce autoimmune responses including the synthesis of autoantibodies with pro-inflammatory characteristics. Circulating modified lipoprotein autoantibodies combine with circulating antigens and form immune complexes (IC). We now report the results of a study investigating the role of circulating IC containing modified lipoproteins in the progression of carotid intima-media thickness (IMT) in patients enrolled in the Epidemiology of Diabetes Interventions and Complications (EDIC) Trial, a follow-up study of the Diabetes Control and Complications Trial (DCCT). This cohort includes 1229 patients with type 1 diabetes in whom B-mode ultrasonography of internal and common carotid arteries was performed in 1994-1996 and in 1998-2000. Conventional CHD risk factors, antibodies against modified forms of LDL and modified lipoprotein IC were determined in 1050 of these patients from blood collected in 1996-1998. Cholesterol and apolipoprotein B content of IC (surrogate markers of modified ApoB-rich lipoproteins) were significantly higher in patients who showed progression of the internal carotid IMT than in those showing no progression, regression or mild progression. Multivariate linear and logistic regression modeling using conventional and non-conventional risk factors showed that the cholesterol content of IC was a significant positive predictor of internal carotid IMT progression. In conclusion these data demonstrate that increased levels of modified ApoB-rich IC are associated with increased progression of internal carotid IMT in the DCCT/EDIC cohort of type 1 diabetes.