Simple colorimetric methods for determination of sub-milligram amounts of ultra-high molecular weight polyethylene wear particles.

New colorimetric methods are described for determination of sub-milligram amounts of ultra-high molecular weight polyethylene (UHMWPE) wear particles. These methods are based on the irreversible binding of the fluorescein-conjugated bovine serum albumin or the hydrophobic dye Oil Red O to wear particles. UHMWPE particles bind both substances from their solutions and thus decrease the absorbance of these solutions. The decrease is linearly dependent on the amount of added wear particles in the sub-milligram range suitable for practical use. The newly developed method offers improved accuracy and precision compared to Fourier transformed infrared spectroscopy (Slouf M, et al. Quantification of UHMWPE wear in periprosthetic tissues of hip arthoplasty: description of a new method based on IR and comparison with radiographic appearance. Wear 2008;265:674-684.).

Binding of proteins to ultra high molecular weight polyethylene wear particles as a possible mechanism of macrophage and lymphocyte activation.

Binding of five human plasma proteins (IgG, serum albumin, α(1)-acid glycoprotein, holo-transferrin, α(1)-antitrypsin) to ultra high molecular weight polyethylene wear particles (0.1-10 µm) isolated from hip periprosthetic tissues was studied in vitro. All tested plasma proteins were bound to wear particles in a similar way indicating irreversible binding. Analogous interaction was found also between GUR 4120 particles (diameter ∼250 µm) and two tested plasma proteins (human serum albumin and α(1)-acid glycoprotein). The binding was not affected by pH of a buffer or the isoelectric point of bound proteins; thus it was apparently of clearly hydrophobic nature. We hypothesize that the binding causes some unfolding of the bound proteins, thus exposing new determinants with which sensitive cells may react. This could be a mechanism by which polyethylene wear particles trigger, for example, macrophages activity and thence initiate aseptic inflammation and cause the failure of total joint replacements. Results can contribute to the choice of a convenient construction type of prostheses.

Artificial lipid-protein complexes accelerate cholesterol crystallisation in model bile.

Cholesterol gallstone disease is one of the major health problems in the world. Substances which can affect the crystallisation of cholesterol from human bile have been given considerable attention. Various substances (among them natural lipid-protein complexes) have been tested for cholesterol crystallisation-promoting activity. Various artificial lipid-albumin complexes have been prepared of which taurodeoxycholate-human serum albumin-calcium ions (TDC-HSA-Ca(2+)) had the highest cholesterol crystallisation-promoting activity. This cholesterol crystallisation-promoting activity is similar to that for the lipid-protein complex isolated from native human bile [concanavalin A nonbinding fraction (con A(-) fraction)]. Addition of cholesterol to the TDC-HSA-Ca(2+) complex further increased the cholesterol crystallisation-promoting activity whereas the addition of lecithin had an opposite effect. The interaction of individual components of the TDC-HSA-Ca(2+) complex was followed using several methods. A new effect of Ca(2+) ions (increase in the number of binding sites for bile salts) on the interaction of TDC with HSA was found by equilibrium dialysis. Interaction of TDC with albumin and Ca(2+) did not induce any modification of the secondary structure of albumin. The results of fluorescence spectroscopy may indicate that TDC is at least partially bound to not essentially fatty acid free HSA somehow via admixtures, probably fatty acids. Difference absorption spectrum of the TDC-HSA-Ca(2+)-cholesterol complex was very similar to that of the "natural" lipid-protein complex (con A(-) fraction). From the three drugs with different albumin binding characteristics, only sulphadimethoxin had an observable effect on the cholesterol crystallisation-promoting activity. The action of the TDC-HSA-Ca(2+) complex decreased significantly after the addition of sulphadimethoxin. The addition of TDC modified the absorption spectrum of the sulphadimethoxin-HSA-Ca(2+) complex. It can be suggested that the complex of HSA with bile salts (TDC mainly) and Ca(2+) forms a nucleation centre for cholesterol crystallisation in bile.

[Pronuclear and antinuclear factors in the pathogenesis of cholesterol cholelithiasis]. / Pronukleacní a antinukleacní faktory v patogenezi cholesterolové cholelitiázy.

Contrary to hitherto published results, the authors provided evidence of significant pronucleation activity in the protein fraction which is not linked to concanavaline A. Delipidation or proteolysis markedly reduce the pronucleation activity of this fraction. Albumin was identified as the main protein in this fraction. The lipid-protein complex formed by albumin and lipids had a high pronucleation and crystallization activity in relation to cholesterol. Calcium ions increased the crystalization activity. Complexes formed by proteins and lipids can be vectors of the main pronucleation activity in bile. In investigations of the main cholesterol fraction the authors provided evidence that only part of so-called pronucleation proteins is linked to vesicles--i.e. IgM, IgA and biliary glycoprotein BGP I and II. The authors assume that only proteins firmly linked to vesicles can participate in the process of cholesterol crystallization. Biliary glycoprotein BGP I and II was present in vesicles and when added into a model bile it presented a high pronucleation activity. Biliary glycoprotein is a new hitherto not identified pronucleation protein in bile.

Lipid-protein complexes as cholesterol pronucleating agents in human bile.

Among the various substances which accelerate the formation of cholesterol crystals in cholesterol supersaturated bile are proteins obtained from the bile by affinity chromatography on con A-Sepharose. Several such con A binding proteins have been identified and shown to mediate acceleration of cholesterol crystal formation in vitro. However, the major protein fraction, which does not bind con A, has been studied rarely. Investigation of the effect of this latter bile protein fraction on cholesterol crystallization is the aim of this study. Contrary to results published to date, the con A nonbinding protein fraction exerted a higher cholesterol crystallization promoting activity than the con A binding fraction. Delipidation as well as proteolytic degradation sharply decreased the activity of both fractions. Albumin was identified as the main component of the con A nonbinding fraction. A lipid-protein complex formed from the lipid and albumin possessed a very high cholesterol crystallization promoting activity whereas albumin or the lipid alone showed much lower activity. Bivalent ions, especially Mn2+ and Ca2+, increased the promoting activity of the lipid-protein complex. Thus, albumin and other bile protein can bind noncovalently biliary lipid material and such lipid-protein complexes may act as the main cholesterol crystallization promoter in the human bile.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. V. Alternative method for preparation of serum albumin-methotrexate derivative.

An alternative method for the preparation of human serum albumin-methotrexate derivative (HSA-MTX) using N-hydroxysuccinimide ester of methotrexate (MTX) was compared with that using the water-soluble carbodiimide (WSC) assistance. The prepared derivative was tested to find the advantages and drawbacks of this method. The N-hydroxysuccinimide ester method is more laborious than that using WSC but some reasons speak in favor of this method. The formation of protein-protein conjugates during the reaction was negligible and under specific conditions more MTX molecules could be bound to protein than by the WSC method. However, some disadvantages of this method were found: A laborious preparation, a small degree of denaturation of protein during the synthesis and, moreover, the binding of MTX to protein did not always take place through NH2-groups of protein but through some other groups (--OH, --SH) as well. These couplings were not so stable as an amidic (or peptidic) bond. In water environment they were hydrolyzed and MTX was slowly released. If there is no difference in a small fraction of protein-protein conjugates, the WSC-assisted method possesses evident advantages over the active MTX ester one, mainly because of the simple preparation of the stable derivative.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. III. Human serum albumin-methotrexate derivative, its preparation and basic testing.

The preparation and a more detailed characterization of human serum albumin-methotrexate derivative (HSA-MTX) is described. The synthesis of the derivative was performed by means of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (in methoiodide form) (WSC). Results of many experiments showed that, on the average, about 26 molecules of methotrexate (MTX) were coupled to one molecule of human serum albumin (HSA). The relative molecular weight of the formed derivative was estimated by gel chromatography on Sepharose 6B or on high-pressure liquid chromatography (HPLC) on SWK column, respectively. From the obtained data it follows that a considerable part of the HSA-MTX derivative formed protein-protein conjugate (up to about 4 X Mr HSA), nevertheless the derivative retains its good solubility as a native albumin. In order to eliminate the possibility of influencing the cytostatic activity of the derivative with byproducts of its synthesis, human serum albumin-folic acid derivative (HSA-FA) was prepared and tested by the same method. All demonstrated experiments proved that MTX was the only compound possessing the cytostatic activity. During the experimental therapy of Gardner lymphosarcoma (LSG) the following was found: (1) The intratumorous application of the drug was the most effective way of administration. (2) Any type of administration of the HSA-MTX derivative exerted a better effect than the same way of administration of free MTX. (3) The comparison of two (repeated) administrations of both drugs showed clearly that the HSA-MTX derivative was more efficient than free MTX. After HSA-MTX derivative treatment all animals survived without tumor. (4) For the estimation of the toxicity of the HSA-MTX derivative, three times and five times repeated intraperitoneal administration was performed. It was concluded that although the derivative was more toxic than free MTX, its therapeutic activity was better. After the elimination of the toxic manifestation of the HSA-MTX derivative by a suitable arrangement of drug doses, five times higher efficacy of the derivative was reached, as compared with free MTX. (5) The therapy by the HSA-FA derivative did not exhibit any therapeutic effect. The reason why HSA was used as a macromolecular carrier for cytostatics is discussed.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. IV. Therapy of murine melanoma B16 by human serum albumin-methotrexate derivative.

The influence of methotrexate bound to human serum albumin (HSA-MTX) and of free methotrexate (MTX) on B16 melanoma growth, dissemination and survival time of tumor-bearing animals was investigated. It was found that the growth of tumor was slower after therapy with the HSA-MTX derivative than after free MTX treatment. The reduction in tumor size recorded on day 21 after tumor transplantation was more significantly pronounced after HSA-MTX derivative therapy than in case of free MTX treatment. Contrary to our expectation there was no proportional difference in life span prolongation after therapy with these drugs. Comparing metastatic dissemination, the number and size of pulmonary metastatic colonies after HSA-MTX administration was more significantly reduced than after free MTX therapy.

Changes in metabolism, food and water intake of C3H mice following transplantation of Gardner lymphosarcoma (LSG).

Mice bearing progressing Gardner lymphosarcoma (LSG) increasingly gained in weight in the course of LSG development but their food and water consumption was reduced in comparison with control animals. During the neoplastic growth, oxygen consumption was decreased and the proportion of the metabolically active part (i.e. dried matter) declined. The body weight of tumour-bearing mice successfully cured with methotrexate (MTX) (30 mg/kg body weight, administered on days 1 and 3) was substantially lower than those of non-treated tumourous animals and was similar to that of intact controls. The food and water consumption of mice treated with MTX and that of control animals was similar. Values of the metabolic rate observed in animals treated with MTX were comparable to those recorded in the control group.