Emma Guagnellini

Matrix Metalloproteinase-1 and Matrix Metalloproteinase-3 Gene Promoter Polymorphisms Are Associated With Carotid Artery Stenosis

Background and Purpose— The matrix metalloproteinases (MMPs) are a family of enzymes that are important in the resorption of extracellular matrix and are involved in atherogenesis. Recently, 2 common polymorphisms on MMP-1 (1G/2G) and MMP-3 (5A/6A) gene promoters have been described. The aim of this study was to investigate a possible association between MMP polymorphisms and increased risk of internal carotid artery (ICA) stenosis. Methods— We studied 91 patients consecutively recruited for ICA stenosis who had undergone carotid endarterectomy and 133 subjects without ICA stenosis (controls). Polymorphic genotypes were determined by polymerase chain reaction and sequencing analysis. Results— The frequency of the 6A allele was significantly different between cases and controls: 0.62 and 0.50, respectively (odds ratio [OR], 1.58; 95% CI, 1.08 to 2.33;P =0.017). The frequency of 6A/6A genotype was significantly higher in cases with involvement of both carotids (OR, 3.13; 95% CI, 1.14 to 8.5;P =0.026) and in patients with stenosis >70% (OR, 2.55; 95% CI, 1.07 to 6.07;P =0.033). No significant differences were observed in MMP-1 distribution. Patients who were homozygous for both the 6A and 2G alleles had an elevated relative risk of ICA stenosis (OR, 2.66; 95% CI, 1.23 to 5.72;P =0.016). Multiple logistic regression analysis using the common risk factors and the 6A and 2G allele variants revealed that the 6A allele was an independent risk factor for ICA stenosis (P =0.049). When 6A/6A and 2G/2G were combined, the risk factor for ICA stenosis was 3-fold higher (OR, 3.31; 95% CI, 1.48 to 7.42;P =0.004). Conclusions— Homozygosity for the 6A allele of the MMP-3 promoter is associated with carotid stenosis and, in association with MMP-1 2G homozygosity, predicts an increased risk of ICA stenosis. Even if obtained from a relatively limited patient series, these results might have relevant implications for treatment of ICA stenosis and possibly prevention of carotid-related stroke.

Internal carotid artery occlusive disease and polymorphisms of fractalkine receptor CX3CR1: A genetic risk factor

Background and Purpose— Fractalkine (FKN), a chemokine expressed by inflamed endothelium, induces leukocyte adhesion and migration via the receptor CX3CR1. The polymorphisms V249I and T280M affect receptor expression and function. The role of FKN in atherosclerosis has been recently demonstrated. The aim of this study was to investigate a possible association between CX3CR1 polymorphisms and increased risk of internal carotid artery (ICA) occlusive disease. Methods— We studied 108 patients consecutively recruited for ICA occlusive disease, 84 of whom underwent operation for carotid endarterectomy, and 204 subjects without ICA occlusive disease (controls). Polymorphic genotypes were determined by polymerase chain reaction and sequencing analysis. Results— The adjusted odds ratio (OR) associated with the presence of the M280 (TM+MM versus TT genotype) was 0.55 (95% CI: 0.29 to 0.99; P = 0.037). Therefore, this allele is associated with a reduced risk of ICA occlusive disease. No significant differences were observed in I249 distribution. The frequency of I249 allele was significantly higher in cases of hard plaques, which are considered more stable than soft ones (OR: 0.38; 95% CI: 0.13 to 1.05; P = 0.037). Multiple logistic regression analysis using the common risk factors and the I249 and M280 allele variants revealed that the M280 allele was an independent risk factor for ICA stenosis (P = 0.047). Conclusion— The results show that the CX3CR1 M280 is an independent genetic risk factor for ICA occlusive disease and that I249 is involved in the stability of carotid plaques. Even if obtained from a relatively limited patient series, these results might have relevant implications for treatment of ICA stenosis and possibly prevention of carotid related stroke. Further prospective cross-sectional studies are needed to confirm these results.

Preparation of a Stable Liquid Material for Calibration and Quality Control for Lysosomal Enzymes in Plasma. Assay of Enzymes of Lysosomal Origin in Plasma, I.

Several lysosomal enzymes present in human plasma (N-acetyl-beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-galactosidase, alpha-L-fucosidase, alpha-mannosidase, beta-glucosidase) were maintained in a fully active state for at least 8 months by the addition of ethylene glycol (300 milligrams final concentration) to freshly prepared plasma and storage at -20 degrees C. Pools of human plasma from healthy humans, stabilized and stored as above, and containing a low, medium or high content of the above enzymes, were used to establish the analytical imprecision (within-run, day-to-day and total imprecision) of the fluorimetric assay. Ten replicates in ten different analytical series, covering a period of two months, were performed. The total imprecision (expressed as coefficient of variation) was in general lower than 10%; in a few cases, particularly plasma samples with a low enzyme content, the total imprecision was 18%. The isozymes A, B, I1, and I2 of N-acetyl-beta-glucosaminidase displayed the same stability upon storage as the unfractionated enzyme. It is concluded that pools of human plasma containing known amounts of lysosomal enzymes, stabilized by the addition of 300 micrograms ethylene glycol and stored at -20 degrees C, are suitable liquid materials for calibration and quality control for the assay of the same enzymes.

Serum enzymes of lysosomal origin as indicators of the metabolic control in non-insulin-dependent diabetics

SummarySeveral lysosomal enzymes (β-N-acetyl-D-glucosaminidase, β-D-glucuronidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, α-L-fucosidase and α-D-mannosidase) were determined in the serum of 54 non-insulin-dependent diabetics with different degrees of metabolic control and without complications and in 18 non-insulin-dependent diabetics with complications. The serum levels of β-N-acetyl-D-glucosaminidase, β-D-glucuronidase, α-D-galactosidase, and α-D-mannosidase were significantly (p<0.01) higher in the diabetics without complications. The levels of β-N-acetyl-D-glucosaminidase and β-D-glucuronidase were inversely proportional to the degree of metabolic control, in a statistically significant manner. Moreover the levels of these enzymes decreased to normal values during a 2-month period of controlled oral hypoglycemic drug-diet therapy resulting in metabolic compensation. The presence of complications was indicated by a further increase of serum β-N-acetyl-D-glucosaminidase and β-D-glucuronidase; however the portion of lysosomal enzyme activities due to complications remained unchanged after controlled therapy aimed at compensating the metabolism. The conclusion is drawn that in non-insulin-dependent diabetics, as already shown for insulin dependent-diabetics, serum lysosomal enzymes, especially β-N-acetyl-D-glucosaminidase and β-D-glucuronidase, are good intraindividual indicators of the metabolic control of the disease.

Behaviour of several enzymes of lysosomal origin in human plasma during whole blood storage

Adriana Lombard0 ‘, Giancarlo Goi a, Emma Guagnellini b, Alessandra Fabi a, Gianalfredo Sciorelli ‘, Albert0 B. Burlina d and Guido Tettamanti a** U Department of Biological Chemisrty, The Medical School, University of Milan, Milan h Clinical Chemistry Laboraiory, Bassini Hospital, Cinisello Balsamo, Milan, ’ Immunohaematologv Division, National Institute o/Cancer, Milan and’ Departmenr of Paediatrics. University of Verona, Verona (Italy)

Automated Fluorimetric Assay Procedure for Glucohydrolases Using a Routine Centrifugal Analyser Assay of Enzymes of Lysosomal Origin in Plasma, II

The manual fluorimetric procedure, considered as a reference method for the determination of N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and beta-D-galactosidase in human plasma, was automated as a routine method, using the IL Monarch centrifugal analyser. Using a liquid standard with a known enzyme content, the automated assay correlated fairly well with the reference manual method (r values very close to 1). Its analytical imprecision was much lower than that of the manual method. The automated assay of N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and beta-D-galactosidase gave coefficients of variation of 5.7-6.9, 3.6-5.0 and 3.8-4.2%, respectively, detection limits of 4, 2 and 1 mU/l plasma respectively, and linear responses of up to 73, 8.4 and 0.9 U/l of plasma respectively. Furthermore, the method required only small volumes of undiluted plasma (4-10 microliters). This method appears to be reliable, sensitive, simple enough for routine analyses and as cost effective as the most common routine serum enzyme assays.