Ser128Arg gene polymorphism for E-selectin and severity of atherosclerotic arterial disease.

AIM: Genetic factors appear to be important in the pathogenesis of cardiovascular and cerebrovascular disease. Adhesion molecules like the members of the selectin family participate in the interaction between leukocytes and the endothelium. They are also involved in the pathogenesis of the atherosclerotic process. In E-selectin, exchange from serine to arginine (position 128, S128R) is correlated with early atherosclerosis. The aim of this study was to assess E-selectin Ser128Arg polymorphism in subjects with clinical and instrumental evidence of atherosclerosis and to analyze the correlations with clinical severity. METHODS: A total of 144 subjects (100 men and 44 women, mean age 72 years, range 48-78) with atherosclerotic disease in different vascular sites documented by angiography were studied; 138 volunteers were recruited as a control group. Whole blood was collected; DNA was extracted with a commercial kit and amplified with 2 primers. The PCR was performed by standard procedure. To assess the disease severity all patients were classified by an arbitrary clinical and angiographic score scale. RESULTS: The genotype distribution between patients and controls was different, although statistical significance was not achieved (p=0.06). In patients a significant difference in Arg allele frequency was observed between mild and severe atherosclerotic disease (OR 2.28; 95% CI 1.15-4.52; p=0.017). Four ho-mozygous cases for S128R were found in patients, none in controls. All these 4 patients had the highest severity score, that means a more severe atherosclerotic disease. CONCLUSION: Our study suggests that the E-selectin polymorphism may be associated with severity of atherosclerotic disease, but does not allow us to conclude that it is actually a risk factor for atherosclerosis.

Matrix metalloproteinase-1 promoter polymorphism 1G/2G is correlated with colorectal cancer invasiveness.

PURPOSE: Matrix metalloproteinase-1 (MMP-1) is likely to be involved in invasion and metastasis of several tumors by degrading the extracellular matrix. A single guanine insertion polymorphism (2G) in the MMP-1 promoter region creates an Ets binding site causing the elevation of transcriptional level and local expression of MMP-1. The aim of this study was to evaluate the impact of this 2G insertion type polymorphism on invasion and metastasis of colorectal cancer (CRC). EXPERIMENTAL DESIGN: We genotyped for this 1G/2G polymorphism 60 patients, who were operated on for CRC and followed for 6-30 months (median: 21). A control population of 164 age- and sex-matched tumor-free subjects was also genotyped for the same polymorphism. RESULTS: The proportion of 2G homozygotes was higher in the CRC group than in the controls (P = 0.014; odds ratio, 2.21; 95% confidence interval, 1.17-4.16). The CRC group was divided in a group without metastasis (M-) and a group that had developed metastasis (M+). At the time of diagnosis, 2G homozygotes were more represented in the M+ group than in M- (P = 0.0082; odds ratio, 4.73; 95% confidence interval, 1.46-15.26). The difference between M- patients and controls did not achieve statistical significance (P = 0.52). CONCLUSIONS: Our results suggest that the presence of 2G polymorphism at the MMP-1 promoter region may favor the growth and the metastatic process in CRC patients and could be looked at as a risk factor for a worse prognosis.

Homocysteine concentration in primary and systemic sclerosis associated Raynaud's phenomenon.

OBJECTIVE: To investigate whether patients with systemic sclerosis (SSc) have raised homocysteine (Hcy) plasma levels, thought to be an independent risk factor for vascular disease, and to study the relationship between Hcy and endothelial damage, and between Hcy and methylene-tetrahydrofolate reductase (MTHFR) genotypes, and patients' vitamin nutritional status, which are among the more frequent causes of hyperhomocysteinemia. METHODS: We measured Hcy, von Willebrand factor (vWF), folic acid, and vitamin B12 plasma levels and analyzed the frequencies of MTHFR mutations in 30 patients with SSc and 12 patients with primary Raynaud's phenomenon (RP); 29 healthy subjects served as controls. RESULTS: Patients with SSc had higher Hcy and vWF concentrations than those with RP (p < 0.01 and p < 0.02, respectively) or controls (p < 0.02 and p < 0.0001, respectively). Folic acid and vitamin B12 were lower in SSc than in RP (p < 0.01 and p < 0.02, respectively) or controls (p < 0.05). MTHFR genotype did not influence Hcy, folate, or vitamin B12 concentrations, but patients homozygous for the mutant gene had higher vWF levels. CONCLUSION: Patients with SSc, but not those with RP, had significantly higher Hcy and vWF plasma levels. Nutritional rather than inherited factors seem to have a pathogenic role in SSc hyperhomocysteinemia.

Measurement of ionized magnesium with AVL 988/4 electrolyte analyzer: preliminary analytical and clinical results.

Only free magnesium has biological activity: technology for measuring the ionized fraction of magnesium is now available via ion-selective electrodes. We have evaluated an instrument (AVL 988/4) which determines ionized magnesium (cMg2+) with an ion-selective electrode based on the ionophore ETH 7025. The selectivity of the electrode is adequate for the ions normally present in plasma, except for calcium: the interference is automatically corrected by simultaneous measurements of calcium with compensation for the calcium interference to the magnesium signal. We have first verified possible interference caused by sampling procedures: known silicon interference has been avoided by use of glass tubes (BD Vacutainer with no additive, code 7626); heparin interference has been measured and found significant above 20 UI.mL-1 of plasma. Instrument evaluation according to NCCLS protocol gives the following imprecision results on 20 replicated analyses: cMg2+ (mmol.L-1) 1.29, 0.76, 0.23, CVs% (within-run) 0.67, 0.67, 3.00 and CVs% (between-run) 4.06, 3.91, 5.89 respectively. Linearity (in the range 0.23-1.60 mmol.L-1) was: measured cMg2+ = 0.981.(calculated cMg2+) + 0.009 mmol/L; r = 0.999. In healthy adults (n = 103) cMg2+ was in the range 0.46-0.74 mmol.L-1 (with a mean of 0.60 mmol/L and normal distribution). These values represent 57% to 84% of serum total magnesium concentration (TMg) (mean 71%). pH dependence of cMg2+ is present, usually to a lower extent with respect to cCa2+, but it seems different in patients with real or in vitro provoked acidosis and in hemodialyzed patients. Citrate interference on ionized magnesium measurements was found both in vitro and in vivo, whilst that due to lactate was demonstrated only in vitro. On a wide range of cMg2+ (n = 100), a good correlation is obtained both with TMg and ultrafiltrable Mg (UFMg): cMg2+ = 0.723.TMg + 0.008 mmol.L-1, r = 0.978; cMg2+ = 0.912.UFMg + 0.10 mmol.L-1, r = 0.968, respectively. The ionized magnesium in ultrafiltrate was found 25% lower than that in serum. The lifespan of the electrode, evaluated on the basis of both time from installation and on number of measured samples, was estimated longer than 4 months and able to analyze more than 1500 samples, whichever comes first. The four electrodes we used during 18 months behaved all the same way. The correlation between measurements performed in whole blood (WB-cMg2+) and in the corresponding serum (S-cMg2+) was excellent: WB-cMg2+ = 0.954.S-cMg2+ +0.02 mmol.L-1; r = 0.998; n = 60.

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.

Ion effects in measurement of sodium and ionized calcium in direct potentiometry.

In some instruments that measure sodium directly in whole blood, plasma, or serum using ion selective electrodes (direct potentiometry), the higher the ionic strength of the solution, the lower is the sodium recovery in serum, as predicted by theory. The same could be expected for ionized calcium. When measuring the recovery of serum sodium on indirect potentiometric instrument and by flame photometry, which determine concentration in prediluted samples, and on direct potentiometric instruments, we observed that two out of the three direct potentiometry instruments showed a decreased recovery of sodium, as the ionic strength was increased, while on all the other instruments the recovery was complete. No effect of increased ionic strength was noted on the ionized calcium measurements in serum on all the instruments tested. Analysing pure aqueous solutions of sodium and calcium chloride with increased ionic strength on the same instruments, the sodium recovery was always complete or positive, and the same was true for ionized calcium. We postulate some effect of ionic strength on the salt bridge of the measuring systems, which is different when analysing serum or pure aqueous solutions.

Incomplete recovery of sodium determined in direct potentiometry in blood samples added with heparin salts.

The recent introduction on the market of apparatus for combined blood gas-electrolyte and ionized calcium analyses has raised the problem of whether the same sample can be submitted to all the analyses without generating problems for some analytes. The problem is basically connected to the common use of heparin as an anti-coagulant. To elucidate the possible effect of heparin on measurements we added pooled sera to tubes containing dried heparin/sodium chloride in increasing quantity. Our data demonstrate that, for one direct potentiometric instrument, the sodium recovery is reduced by a moderately high (70-130 IU ml-1) concentration of heparin or by the concomitant addition of the sodium ion which increases the ionic strength of the sample. The effect of the increased sodium chloride was studied separately, adding pooled sera to tubes containing dried sodium chloride in increasing quantity. The sodium recovery was reduced when the ionic strength of the sample was increased. We were unable to separate the negative effects on the sodium recovery due to heparin and due to the increased ionic strength in the sample.

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.

Multicentre evaluation of the Monarch (IL) clinical chemistry analyser.

A multicentre evaluation of the Monarch centrifugal analyser is reported. Precision, linearity and accuracy were assessed by comparison with routine methods. Calibration stability, photometric and dispensing accuracy, and carry-over related to samples and reagents were also evaluated. The overall performance of the instrument was good, showing an excellent photometric and dispensing accuracy, absence of sample-dependent carry-over, and almost negligible reagent carry-over. Good precision, linearity and correlation with routine methods were found for the parameters tested. The instrument is reliable and is now used as the routine clinical chemistry analyser in two of the three laboratories taking part in the evaluation.

Reliability of IL Monarch ion-selective electrode module for sodium, potassium, and chloride measurements.

We evaluated the IL Monarch random-access centrifugal analyzer for measurement of Na+, K+, and Cl- by an indirect potentiometric method. For different concentrations of control material, the total precision (CV) ranged between 0.82% and 1.14% for the three electrolytes; linearity was acceptable within a range of 103 to 215 mmol/L for Na+, 1.6-15.25 mmol/L for K+, and 80-173 mmol/L for Cl-. Data correlated well with those by flame photometry for Na+ and K+ and with those by coulometry for Cl-, both for various biological materials--sera, urines, dialysis fluids--and commercial control materials from various producers. Stability of the potentiometric signal was acceptable: daily variations were 0.2 mV for Na+, 0.05 mV for K+, and 0.03 mV for Cl-. Accordingly, we conclude that the system supplies reproducible and accurate results while being easy to use and requiring little maintenance. The use of indirect potentiometry offers results consistent with those obtained with traditional methods, and easily interpretable by clinical staff. However, better information about the actual ion activity in the tested sample for certain pathologies such as hyperlipemia and dysproteinemia could be obtained by methods involving direct potentiometry.

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

Several lysosomal enzymes (beta-N-acetyl-D-glucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase, alpha-L-fucosidase and alpha-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 beta-N-acetyl-D-glucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, and alpha-D-mannosidase were significantly (p less than 0.01) higher in the diabetics without complications. The levels of beta-N-acetyl-D-glucosaminidase and beta-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 beta-N-acetyl-D-glucosaminidase and beta-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 beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase, are good intraindividual indicators of the metabolic control of the disease.

Thrombin inhibitors in women on oral contraceptives.

Progressive antithrombin activity and the immunological levels of antithrombin III, alpha 2-macroglobulin, and alpha 1-antitrypsin were measured in women at various intervals during treatment with oral contraceptives and in a group of untreated women. No significant changes were observed in any of the above parameters. In both control and treated women, there was a positive statistical correlation between progressive antithrombin activity and antithrombin III immunological levels, whereas no correlation was found between the former and alpha 1-antitrypsin or alpha 2-macroglobulin immunological levels. This study questions the possibility of thrombin inhibitors being selective signs of hypercoagulability during treatment with oral contraceptives.

PIP: Clinical research was conducted into the progressive antithrombin activity of plasma and the concentrations of AT-3(antithrombin-3), alpha2-M(alpha2-macroglobulin), and alphal-AT(alphal-antitrypsin) of women taking OCs (oral contraceptives). Women at various intervals during low-dose OC therapy were tested and compared with women not taking OCs. No significant changes were observed in any of these 3 parameters. In both groups of women the progressive antithrombin activity of plasma decreased gradually during the menstrual cycle, with the low at day 21. In both groups, there was a positive correlation between progressive antithrombin activity and antithrombin-3 immunological levels, but no correlation was found between the former and either of the other 2 factors. Findings from this study do not support previous reports that OC treatment lowers AT-3 levels. AT-3 cannot be considered a selective sign of hyper-coagulability during OC treatment.