Simultaneous detection of N-acetyl-L-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis

N-acetyl-l-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.     

Studies on human serum high density lipoproteins. Self-association of apolipoprotein A-I in aqueous solutions.

Human serum apolipoprotein A-I (apo-A-I), the major protein component of the human serum high density lipoproteins, was studied in aqueous solutions of differing ionic strength and pH by the techniques of sedimentation equilibrium ultracentrifugation and frontal analysis gel chromatography. The ultracentrifugal studies indicate the apo-A-I is a self-associating system that is dependent upon protein concentration, but relatively independent of the nature of the medium. The apparent weight average molecular weights obtained from solutions of initial apo-A-I concentration between 0.2 and 0.9 mg/ml were in the range of 3.0 to 16.7 x 10(4) (monomer molecular weight = 28,014). Of the several models of self-associated examined, that which gave the best theoretical fit was for the monomer-dimertetramer-octamer model. The self-association of apo-A-I in aqueous solutions was further documented by frontal analysis gel chromatography, which not only corroborated the ultracentrifugal results, but also indicated that the multiple species of apo-A-I in solution attain equilibrium rather rapidly. Besides having intrinsic importance, these results indicate that the solution properties of apo-A-I must be established before ligand binding studies are conducted and interpreted.     

Lipoprotein(a): its inheritance and molecular basis of its atherothrombotic role

Lipoprotein(a) or Lp(a), is a member of the plasma lipoproteins with general properties of LDL but with a protein moiety represented by apoB100 disulfide linked to apolipoprotein(a) or apo(a). Apo(a) is polymorphic in size; at present a total of 11 isoforms have been reported, but more are likely to be identified in view of the fact that at least 19 alleles of the apo(a) gene have recently been reported. There are remarkable variations in the plasma Lp(a) levels; but uncertainties still exist about the factors responsible for this variability. High plasma Lp(a) levels have been associated with an increased incidence of cardiovascular disease, mainly based on epidemiological evidence. Both atherogenic and thrombogenic potentials have been suggested; the first attributable to the LDL-like properties of Lp(a) and the other to the plasminogen-like characteristics of apo(a). From the mechanistic viewpoint in vitro studies suggest that the thrombogenic action may occur at the level of the endothelium whereas Lp(a) that localizes in the sub-endothelial intima is expected to undergo complexation with matrix components and favor the formation of the atherosclerotique plaque. How Lp(a) polymorphism relates to the postulated cardiovascular pathogenicity of this lipoprotein remains to be established.     

Properties of human free apolipoprotein(a) and lipoprotein(a) after either freezing or lyophilization in the presence and absence of cryopreservatives

Apolipoprotein(a), apo(a), the specific multikringle glycoprotein constituent of lipoprotein(a), Lp(a), occurs in the plasma mostly bound to apoB100-containing lipoproteins but also in a free form. Often the properties of these products are determined after storage in the cold; yet limited information is available on their stability at low temperatures. To shed light on this subject, we examined the effect of two parameters, freezing and lyophilization, in either the absence or the presence of cryopreservatives. Lp(a)s each having a single apo(a) size isoform containing either 14 or 17 kringle (K) IVs were isolated from the plasma of healthy donors by combining density gradient ultracentrifugation and lysine-Sepharose column chromatography using solutions containing both antioxidants and proteolytic inhibitors. Apo(a) was obtained from parent Lp(a) by a mild limited reductive procedure. Either freezing at -20 degrees C or lyophilization in the presence of 5% sucrose did not change the electrophoretic, immunochemical, and lysine-binding properties of Lp(a) including its ability to generate free apo(a). Irrespective of source, apo(a) remained stable when either frozen at -20 and -80 degrees C or lyophilized in the presence of 125 mM trehalose. In all cases, the absence of cryopreservatives caused the samples to aggregate irreversibly. Thawed or reconstituted samples of both free and bound apo(a) kept at 4 degrees C under sterile conditions in the presence of antioxidants, proteolytic inhibitors, and cryopreservative exhibited no significant changes in properties within the time of observation. Both apo(a) isoforms gave comparable results. We conclude that apo(a), either free or bound, can be kept stable at low temperatures in the presence of appropriate cryopreservatives.     

Isolation and characterization of a dog serum lipoprotein having apolipoprotein A-I as its predominant protein constituent.

The serum high density lipoproteins (HDL) of normolipemic dogs (beagles) were isolated in the density range of p 1.063 to 1.21 g/ml, and characterized in terms of composition and physical properties (flotation and diffusion coefficients, partial specific volume, molecular weight, electrophoretic mobility, ultraviolet absorption, and circular dichroism). The results indicated that canine HDL is a relatively homogeneous class with a molecular weight of about 230 000 and general properties similar to those reported for human HDL. After delipidation, the resulting apolipoprotein, apo-HDL, was fractionated by Sephadex G-200 column chromatography in urea or guanidine hydrochloride solutions. About 90% of the apo-HDL consisted of a protein with a molecular weight of about 28 000, similar in amino acid composition to human apolipoprotein A-I and having the same NH2 terminus (aspartic acid) and COOH terminus (glutamine) and no carbohydrates. Two other proteins were isolated, one having an apparent mol wt of 55 000 and representing, at least in part, an aggregate of apolipoprotein A-I and the other component with a mol wt of 8000, not yet characterized. The results indicate that canine HDL, as an intact complex, has general physical properties that lie between those reported for human HDL2 and HDL3, and that it differs compositionally from the human products mainly in its predominant content of apo-A-I. These findings together with evidence for the relatively homogeneous nature of the canine HDL provide new prospects for unraveling the relationship between polypeptide composition and HDL structure.     

Physicochemical characterization of Rhesus low density lipoproteins.

The serum low density lipoprotein (LDL; p 1.019-1.050 g/ml) of the normal Macaca mulatta monkey (rhesus), kept on a low-fat Purina primate chow diet, was isolated by ultracentrifugal flotation, and its physicochemical properties were compared with those previously reported for human LDL. Rhesus LDL was found to be chemically similar to human LDL. The values for the sedimentation (S25, w-O) and diffusion (D25,w-O) coefficients were 7.09 S and 2.50 times 10- minus-7 cm-2 sec- minus-1, respectively. The intrinsic viscosity was 3.40 ml g- minus-1. The partial specific volume of rhesus LDL, determined in an Anton Paar precision density meter, was 0.960 ml g- minus-1. Molecular weights, calculated from a combination of S-O and D-O and of S-O and [n], were in agreement with the weight-average molecular weight, Mw, of 2.29 times 10-6 obtained by high-speed sedimentation equilibrium. In addition, a Z-average molecular weight, Mz, of 2.73 times 10-6 was calculated because curvature in the graphs of log c vs. r-2 indicated that rhesus LDL was heterogeneous. From the frictional ratio of 1.02, a maximum hydration of 0.1 g of H2O/g of lipoprotein was obtained. On electron micrographs, rhesus LDL appeared spherical with a mean diameter of 196 A, which was substantiated by hydrodynamic analysis.