Products of advanced glycation in patients with type 2 diabetes and vascular disease.

BACKGROUND: Non-enzymatic glycation leading to advanced glycation endproduct (AGE) formation is thought to contribute to vascular pathology. In the present study, AGEs and anti-AGE antibodies in free and immune complex-bound form were assayed in the serum of diabetic (DMCAD) (n = 69) and nondiabetic (n = 78) patients with coronary artery disease (CAD) and in control subjects (n = 47) free from vascular disease. METHODS: A blocking enzyme-linked immunosorbent assay (ELISA) was used to test immunoreactivity against AGE epitope(s) and a competitive ELISA was used to measure total AGE content. RESULTS: Anti-AGE immunoreactivity was significantly higher in diabetic than in control subjects (P = 0.045). Although a wide range of anti-AGE antibody titres were observed in nondiabetic CAD patients, there was no significant difference from those of control subjects. Both diabetic and nondiabetic CAD patients had a higher concentration of circulating immune complexes containing the AGE moiety as antigen than did control subjects (DMCAD versus control, P = 0.041; CAD versus control, P = 0.047). Study patients showed a positive correlation between serum AGE and AGE-immune complexes (DM, r = 0.29, P = 0.014; CAD, r = 0.26, P = 0.019), whereas no such correlation was recorded in controls (r = 0.08, P = 0.89). CONCLUSION: To our knowledge, this is the first study demonstrating increased AGE-immune complexes in patients with CAD, either with or without diabetes, suggesting that AGE-immune complexes might be involved in the atherosclerotic process, either as the result of it or as part of the pathophysiologic process.

Soluble LDL-immune complexes in type 2 diabetes and vascular disease.

BACKGROUND AND AIMS: The oxidative modification of LDL has been shown to affect its clearance and to exert cytotoxic and immunogenic effects. The objective of our study was to analyse markers of LDL oxidation-soluble LDL containing immune complexes (LDL-ICs) in type 2 diabetes with micro- and macrovascular disease. PATIENTS AND METHODS: The study included 69 diabetic patients with coronary artery disease (DM + CAD), 78 non-diabetics with CAD, 47 controls, and 27 diabetics with nephropathy and 36 free from complications. OxLDL antibodies and advanced glycated end-products were measured by ELISA, and LDL-IC apo B content after PEG precipitation. RESULTS: Determination of a broad range of oxLDL antibody activity in all study groups showed no significant differences. In contrast, the content of apo B, a component of the antigen moiety of oxLDL-ICs, was higher in CAD and diabetes (+ CAD) than in LDL-ICs isolated from controls (p < 0.001). LDL-ICs did not differ between patients with CAD + DM and CAD patients free from diabetes. LDL-IC levels in diabetic patients with or without microangiopathy were significantly higher than in healthy volunteers (PEG-apo B 0.278 +/- 0.107 vs. 0.165 +/- 105 g/l, p < 0.002; PEG-IgG 151.7 +/- 76 vs. 115.4 +/- 62 g/l, p < 0.05). However, there was no significant difference in the level of circulating LDL-ICs between the subgroup of diabetic patients with nephropathy/retinopathy and patients free of microvascular disease (Ab-oxLDL 27.7 +/- 10.4 vs. 27.1 +/- 9.3 AU, NS; PEG-apo B 0.324 +/- 0.111 vs. 0.287 +/- 0.124 g/l, NS; PEG-IgG 1.68 +/- 0.68 vs. 1.42 +/- 0.80 g/l, NS). There was a statistically significant positive correlation between AGE content and LDL-ICs (r = 0.35, p < 0.009). A significant but inverse correlation was recorded between triglyceride concentration and level of LDL-ICs in DM + CAD (r = - 0.32, p < 0.016) and CAD patients (r = - 0.35, p < 0.002). A highly significant negative correlation between triglycerides and circulating LDL-ICs (r = - 0.54, p < 0.039) was observed in patients with early nephropathy, but not in those with physiological proteinuria. It is known that at a high triglyceride level in type 2 diabetes, the majority of LDL are small and dense, thus being more susceptible to oxidative modification. This could be a possible mechanism explaining why more LDL-ICs, with a level inversely correlating with triglyceride concentration, are generated in diabetes. CONCLUSION: The increased level of circulating LDL-ICs is a risk factor for the general population, including those with diabetes. Our results suggested the contribution of LDL-ICs to the development of atherosclerosis to probably be more significant than the direct contribution of oxLDLAb itself.

Lipoprotein lipase gene polymorphism and lipid profile in patients with hypertriglyceridemia.

AIM: To assess lipid profile and the genotype distribution of lipoprotein lipase gene polymorphism at Pvu II polymorphic site within the intron between exons 6 and 7 in patients with hypertriglyceridemia. METHODS: Pvu II polymorphism was determined in 116 hypertriglyceridemic patients and 50 normolipidemic controls from Zagreb, Croatia. DNA was extracted from peripheral blood mononuclear cells. Polymerase chain reaction was used for amplification of 6th intron, which was then restricted with Pvu II-restriction endonuclease. Serum lipid and lipoprotein fractions were determined by standard enzymatic methods. Cholesterol concentrations in HDL subfractions, HDL2 and HDL3, were determined after precipitation with polyethyleneglycol. Apolipoproteins (apo) A-I and B were determined by immunonephelometry. RESULTS: Triglycerides showed a positive correlation with total cholesterol (r=0.222, 95% CI=0.041-0.389, p=0.017) and inverse correlation with HDL-cholesterol (r= -0.278, 95% CI= -0.449 to -0.088, p=0.005), especially with HDL3-cholesterol (r= -0.333, 95% CI= -0.497 to -0.147, p=0.001). The respective frequencies for genotypes /, +/, and +/+ were 22, 58, and 36 in the patient group, and 17, 17, and 16 in the control group. Serum triglycerides in the patient group, expressed as median in mmol/L, were 3.30 (range, 2.60-10.90), 3.60 (range, 2.50-21.50), and 3.99 (range, 2.50-15.56), respectively. Serum concentration of triglycerides differed significantly between the +/+ and / genotype (p=0.043). CONCLUSION: There is an association between genetic variation at the locus for lipoprotein lipase and high serum triglyceride levels. This might prove useful in the detection of individuals susceptible to the development of hypertriglyceridemia, as well as a marker in the analysis of this genetic defect in patient families.

A novel missense mutation C127R (FH Zagreb) in the LDL-receptor gene.

We employed the analysis of single-strand conformation polymorphisms to identify mutations in exon 4 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Three familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns. DNA sequencing revealed that the abnormal pattern of exon 4A was due to heterozygosity (T/C) at nucleotide 442. Nucleotide 442 is the first base of codon 127, and the T-->C mutation (C127R) changes this codon from CysTGT to ArgCGT. Abnormal patterns of exon 4B were due to heterozygosity (A/G) at nucleotide 662: nucleotide 662 is the second base of codon 200, and the A-->G mutation (D200G) changes this codon from AspGAC to GlyGGC. Mutation D200G was previously described as FH Padova, but mutation C127R (FH Zagreb) has not been reported previously. This novel mutation was confirmed by restriction endonuclease analysis with Dsa I. The screening of 420 familial hypercholesterolemia heterozygotes suggests that C127R and D200G account for about 0.7% of mutations causing familial hypercholesterolemia in Croatia.

Deletions in the SMN and NAIP genes in patients with spinal muscular atrophy in Croatia.

Two genes, i.e. survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) have been mapped to the SMA region of chromosome 5q13. Both genes are frequently deleted or truncated in SMA patients. We have studied 26 patients with SMA types I-III, 29 first relatives, and 14 subjects with mild adult-onset type IV. DNA deletion genotypes were determined by PCR techniques amplifying exons 7 and 8 of SMN, and exon 5 of NAIP gene which distinguish SMN and NAIP telomeric copy from a non-pathogenic gene homologue as a centromeric copy. Results revealed the homozygous deletions of exon 7 and 8 of the SMN gene and exon 5 of the NAIP gene in 3/3 infants with SMA I and in 1/20 with SMA type II. Exons 7 and 8 of the SMN gene were homozygously deleted in 10/20 and only exon 7 in 6/20 children with SMA type II. The overall percentage of deletion cases observed was 77% in children with SMA types I-III. Adult patients with type IV SMA showed no homozygous deletion of exons 7, 8 and 5 of the SMN and NAIP genes. Also, all relatives had both a telomeric and centromeric SMN and NAIP copy. Deletion analysis of SMN and NAIP genes are a significant diagnostic tool, because there are clinical entities resembling SMA which most likely have another pathogenetic background.