A frameshift mutation in the human fibrinogen Aalpha-chain gene (Aalpha(499)Ala frameshift stop) leading to dysfibrinogen San Giovanni Rotondo.

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala499 within the Aalpha-chain gene was identified, which predicted changes of the corresponding amino acids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (Aalpha[499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aalpha-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aalpha-chain was shown only in the propositus, who also carried normal Aalpha-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen. contained substantial amounts of albumin. Present findings confirm that truncated Aalpha-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

Determining sulfur-containing amino acids by capillary electrophoresis: a fast novel method for total homocyst(e)ine human plasma.

A high-performance capillary electrophoresis (HPCE) method based on laser-induced fluorescence detection is presented here. It enables the determination of sulfur-containing amino acids within 15 min. Fluorescence of sulfur-containing amino acids in plasma is linear over a range of 50-150 micromol/L for L-methionine, 5-100 micromol/L for L-homocysteine, and 50-200 micromol/L for L-cysteine. For homocysteine, we were able to detect 1 fmol injected, equivalent to a plasma concentration of 10 nmol/L. A similar sensitivity is present for cysteine, an even lower one being found for methionine. The intra- and interassay relative standard deviations are < 1%. High-performance liquid chromatography (HPLC) methods are commonly employed for quantifying blood concentrations of sulfur-containing amino acids. A comparative analysis of HPCE and HPLC quantitation of homocysteine has been carried out in 61 blood samples. Plasma concentrations measured by HPCE were in good agreement with those obtained employing an HPLC-based method, a satisfactory correlation being observed between the concentrations obtained by the two methods (r= 0.9972). Thus, the HPCE-based procedure presented here for the measurement of sulfur-containing amino acids in plasma is a simple, fast, accurate, and very sensitive method, suitable for routine determinations in clinical studies.

Fibrinogen plasma levels in an apparently healthy general population--relation to environmental and genetic determinants.

Elevated fibrinogen levels are an independent risk factor for cardiovascular ischemic disease. We investigated the relationship between cardiovascular ischemic risk factors, the fibrinogen Bbeta-chain G/A(-455) polymorphism and plasma fibrinogen levels in 989 apparently healthy subjects. Fibrinogen values were higher in subjects with C reactive protein (C-RP) >0.33 mg/dl, BMI >23.9 kg/m2, total cholesterol >4.84 mmol/l, triglycerides > 1.02 mmol/l, PAI-1 antigen >12.2 ng/ml, carriers of the A allele, first-degree relative history of coronary artery disease, or consuming >10 cigarettes per day (p<0.01). Men and ethanol drinkers showed lower plasma fibrinogen levels (p<0.01). The multivariate analysis confirmed the independent effect of C-RP, age, BMI, total cholesterol, gender, PAI-1, -455 G/A polymorphism, (p<0.05). BMI, total cholesterol, PAI-1, alcohol and smoking habit raised with the increase of age and differed between sexes. The A(-455) allele increasing effect was significant in women, especially in subjects aged <30 years, and in men aged <43 years. These results indicate that environmental factors contributed to a larger extent to fibrinogen variability, whereas the A(-455) allele was associated with a steeper increase in younger age quartiles.

PAI-1 plasma levels in a general population without clinical evidence of atherosclerosis: relation to environmental and genetic determinants.

Plasminogen activator inhibitor-1 (PAI-1) plasma levels have been consistently related to a polymorphism (4G/5G) of the PAI-1 gene. The renin-angiotensin pathway plays a role in the regulation of PAI-1 plasma levels. An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been related to plasma and cellular ACE levels. In 1032 employees (446 men and 586 women; 22 to 66 years old) of a hospital in southern Italy, we investigated the association between PAI-1 4G/5G and the ACE I/D gene variants and plasma PAI-1 antigen levels. None of the individuals enrolled had clinical evidence of atherosclerosis. In univariate analysis, PAI-1 levels were significantly higher in men (P<.001), alcohol drinkers (P<.001), smokers (P=.009), and homozygotes for the PAI-1 gene deletion allele (4G/4G) (P=.012). Multivariate analysis documented the independent effect on PAI-1 plasma levels of body mass index (P<.001), triglycerides (P<.001), sex (P<.001), PAI-1 4G/5G polymorphism (P=.019), smoking habit (P=.041), and ACE I/D genotype (P=.042). Thus, in addition to the markers of insulin resistance and smoking habit, gene variants of PAI-1 and ACE account for a significant portion of the between-individual variability of circulating PAI-1 antigen concentrations in a general population without clinical evidence of atherosclerosis.

[Impairment of diuresis in surgical intervention. Role of urinary prostaglandins]. / Contrazione della diuresi in corso di intervento chirurgico. Ruolo delle prostaglandine urinarie.

In 8 patients who underwent abdominal surgery for non-neoplastic reasons, we have evaluated some parameters of renal function (PRP, NaU, GFR and diuresis) plasma levels of PRA and ADH and urinary prostaglandins PGE2 and 6-keto-PGF1 alpha. In 4 patients we found that surgery per se was associated with enhancements of PRA, ADH and 6-keto-PGF1 alpha. In other 4 patients, Indomethacin, a specific inhibitor of prostaglandin synthesis was given and this was followed by impairment of natriuresis and RPF. These data confirm the central role of prostaglandins in the control of diuresis and natriuresis and suggest that use of drugs affecting prostaglandin synthesis should be avoided in patients who are undergoing surgery.