[New metod for quantification of UHMWPE wear particles around joint replacements]. / Nová metoda kvantifikace oterových cástic UHMWPE v okolí kloubních náhrad.

PURPOSE OF THE STUDY: The orthopaedic community has unanimously adopted the view that ultra high molecular weight polyethylene (UHMWPE) wear particles are a very frequent cause of aseptic implant loosening. Some studies have tried to provide objective evidence for this.We have found descriptions of particle distribution or morphology, but no report that would objectively cor- relate the number of particles in zones surrounding an implant with the extent of damage to these zones. The aim of this study was to develop a method allowing us to evaluate a number of samples with polyethylene abrasive wear large enough to find association between the extent of damage around a THA and the number of biologically active UHMWPE wear particles, 0.1 to 10 microm in size. MATERIAL AND METHODS: In 28 patients undergoing revision total hip arthroplasty (THA) at the 1st Orthopaedic Clinic, 1st Faculty of Medicine, Char- les University, we took samples of typical osteoaggressive granuloma from defined zones around the implant; the zones corresponded to those described by Gruen and DeLee. The extent of tissue damage in each zone was evaluated on the basis of pre-operative radiographs and by the extent of osteolysis and damage to soft tissues actually observed during revision THA. The volume of wear particles in each zone was assessed by the IRc method developed by us; this is based on a quantitative evaluation of infrared spectra. To verify the methodology, a comparison between tissue damage and the number of particles in each zone was made in three randomly selected patients. RESULTS: We introduced a method of detailed orthopaedic evaluation which enabled us to categorize zones around a revised THA according to the extent of damaged tissue. As a result, a series of zones ranked by the extent of damaged tissue, or an "orthopaedist's statement" (OS), was obtained. At the same time we adopted a method, based on infrared spectroscopy and termed IRc, by which the number of particles in the samples of damaged tissues and osteoaggressive granulomas collected from the area around a revised THA was determined.The results of evaluation were presented as numerical data that, in a defined way, were converted into a series of zones ranked according to the number of wear particles, i.e., the "result of measurement" (RM). In this study we verified the methods described above and made a comparison of OSs and RMs for three randomly selected patients. The very good agreement found confirmed the reliability of both methods which will soon be used to evaluate a group of patients large enough to provide statistically significant results. DISCUSSION: The IRc method determines a total volume of UHMWPE wear particles, 0.1 to 10 microm in size, which are generally considered to be most biologically active. This study suggests that the distribution of particles around a THA is uneven and that relation between tissue damage and the number of wear particles in individual zones surrounding a THA does exist. The major conclusion from the orthopaedic point of view is a confirmation of the assumption that UHMWPE wear particles are one of the chief causes of THA failure. Although this fact is generally accepted, studies correlating the number of particles with tissue damage and osteolysis in individual zones are very scarce. CONCLUSIONS: The quick and simple IRc method offers a possibility to quantify polyethylene wear particles in soft tissues. The number of 0.1 to 10 microm wear polyethylene particles correlated with pre-operative radiographic findings and orthopaedic evaluation of revision THAs in three randomly selected patients. The confirmed correlation between the extent of tissue damage in individual zones surrounding a THA and the volume of wear particles detected in these zones supports the view that UHMWPE wear particles are one of the main causes of THA failure.

[Method for assessment of distribution of UHMWPE wear particles in periprosthetic tissues in total hip arthroplasty]. / Metodika sledování distribuce oterových cástic UHMWPE v okolních tkáních u TEP kycelního kloubu.

PURPOSE OF THE STUDY: Aseptic loosening of implants is the main complication affecting the longevity of joint prostheses. The highest proportion of loosening occurs due to osteolysis produced by the presence of ultra-high molecular weight polyethylene (UHMWPE) wear particles smaller than 1 microm. These can be identified by microscopic, spectroscopic or light-scattering methods. Here we describe our method for counting wear particles, based on the principle of light scattering. MATERIAL AND METHODS: Between 2002 and 2004, we collected samples of polyethylene granuloma in 19 patients who underwent revision total hip arthroplasty (THA) for aseptic loosening. The samples were obtained from strictly defined areas corresponding to the radiographic presentation of periprosthetic zones describes by Gruen and DeLee in THA. The frozen samples were lyophilized and subjected to delipidation and hydrolyzation procedures in 65 % HNO3. The top part of solution containing wear particles was blended with isopropanol, and the mixture was filtered through a 10-microm polycarbon membrane. Subsequently, the filtrate was filtered through a 0.1-microm membrane. Membranes with trapped particles, 0.1 to 1.0 microm in size, were sent for particle characterization and quantification. The number of wear particles was measured by the method based on light scattering with calibration (LSC), using a Beckman Coulter LS230 analyzer that can express particle size distribution in a given volume in percent. The method was based on the fact that each particle reflects rays that can be measured. The medium measured contained an unknown number of UHMWPE particles and a known number of calibration glass beads varying in size. The number of UHMWPE particles was calculated from the known number of calibration beads. RESULTS: Because the collected samples were also used to develop the method, comprehensive data was obtained in six patients only. Particle distribution recorded in the periprosthetic zones in THA varied greatly; up to a five-fold difference in particle concentration was observed between the zones. In five of the six patients, the highest particle concentration was found in zone III. DISCUSSION: Seeking a method that would be exact, quick and cheap and would eliminate particle aggregation remains the subject of study for researchers cooperating with clinical practice. At present methods based on weighing isolated particles are used most frequently. We developed the LSC method that, for quantification, utilizes the ability of particles to disperse light, and allows us to calculate the real numbers of UHMWPE wear particles in a medium containing a known number of calibration particles. Although this is an indirect method, it gives more accurate results than the direct weighing of particles. The advantages of the LSC method involve less demand on sample purity, greater speed and low limits of detection. The method is useful for statistical evaluation of a larger number of samples. The variation in particle distribution in THA found in this study is in agreement with the relevant literature data; it is also in agreement with our assumptions and clinical findings. CONCLUSIONS: The authors developed an original method for assessment of UHMWPE wear particles in tissue samples, which is quicker than the methods so far used. In the periprosthetic tissues studied, particles about 1 microm in size were detected; their numbers (about 1010 particles per gram dry tissue) are in agreement with the literature data. The distribution of particles in periprosthetic zones in THA was uneven. The highest number of particles was found in the neighboring zone III and zone 7, as described by Gruen and DeLee. Key words: wear, polyethylene, total hip arthroplasty, light scattering, aseptic loosening, wear particles.

Studies on phytohemagglutinins. XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins.

The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.

Studies on phytohemagglutinins. XXVII. A study of the pea lectin binding site.

Under defined mild conditions the reaction of the pea lectin with 2-nitrophenylsulfenyl chloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis and separation of the tryptic peptides yields only one homogeneous yellow peptide containing the modified tryptophan residue. The isolated peptide has the following sequence (NPS, nitrophenylsulfenyl): HAsp-Val-Val-Pro-Glu-(2-NPS-Trp)-Val-ArgOH. The octapeptide is either directly a part of the pea lectin binding site or it plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the octapeptide isolated from the pea lectin is almost identical with that part of the peptide chain of concanavalin A near to which the location of the sugar binding site is supposed to be.

Nitric oxide generation from extracellularly applied NG-hydroxy-L-arginine in LPS-activated RAW 264 macrophages.

Lipopolysaccharide (LPS)-activated but not control RAW 264 macrophages produced nitric oxide (NO) from extracellularly-applied NG-hydroxy-L-arginine (L-NOHA) in a concentration-dependent manner, as measured by EPR spin trapping and assays for NO2- and NO3-. This production was inhibited by NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, NO-synthase inhibitors, as well as by L-lysine, a competitor for the y+ amino acid carrier system. No significant differences were found between L-NOHA and L-arginine with respect to the rate of NO production and the effects of inhibitors. These results provide evidence that extracellular L-NOHA can enter LPS-activated RAW 264 macrophages via a cationic amino acid carrier system and be metabolized to NO by NO-synthase. The data also suggest that no alternative pathway exists for NO production from L-NOHA in non-activated RAW 264 macrophages.

Possible mechanism of nitric oxide production from N(G)-hydroxy-L-arginine or hydroxylamine by superoxide ion.

It has been speculated the N(G)-hydroxy-L-arginine (OH-L-Arg), which is an intermediate in NO production from L-arginine, may be converted to NO by superoxide ion. However, there is still no direct evidence for this conversion. In the present study this was investigated using superoxide ion generated either in acellular or cellular systems. It was found that OH-L-Arg and hydroxylamine were converted to nitrite and nitrate apparently via NO by superoxide ion in aqueous solution. Arginine remained unaffected. These changes were observed during reaction of chemical substances as well as in a biological system (zymosan-activated macrophages in culture). Superoxide dismutase prevented this transformation. OH-L-Arg was also spontaneously hydrolysed to hydroxylamine and L-citrulline, however this occurred at pH > 9 only. Activated microsomes (containing different isoforms of cytochrome P450) were unable to replace NO-synthase in its ability to produce OH-L-Arg from L-arginine. These data support the hypothesis that a pathway alterative to the well-known synthesis of NO by NO-synthase via OH-L-Arg exists. This pathway may involve the production of OH-L-Arg by NO-synthase and decomposition of OH-L-Arg to NO by the action of superoxide ion. Alternatively, hydrolysis of OH-L-Arg to hydroxylamine may occur followed by its oxidation to NO, again by superoxide ion.

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.

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.

Diaphorase can metabolize some vasorelaxants to NO and eliminate NO scavenging effect of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO).

Diaphorase was studied as a possible oxidoreductase participating in NO production from some vasorelaxants. In the presence of NADH or NADPH, diaphorase can convert selected NO donors, glycerol trinitrate (GTN) and formaldoxime (FAL) to nitrites and nitrates with NO as an intermediate. This activity of diaphorase was inhibited by diphenyleneiodonium (DPI) (inhibitor of some NADPH-dependent flavoprotein oxidoreductases), while it remained uninhibited by NG-nitro-L-arginine methyl ester (inhibitor of NO synthase) 7-Ethoxyresorufin (inhibitor of cytochrome P-450 1A1 and cytochrome P-450 NADPH-dependent reductase) inhibited the conversion of GTN only. Existence of NO as an intermediate of the reaction was supported by results of electron paramagnetic resonance spectroscopy. In addition to its ability to affect the above mentioned NO donors, diaphorase was able to reduce 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and thus to eliminate its NO scavenging effect. This activity of diaphorase could also be inhibited by DPI. The reaction of diaphorase with GTN and PTIO was not affected by superoxide dismutase (SOD) or catalase. Reaction of FAL with diaphorase was lowered with SOD by 38 % indicating the partial participation of superoxide anion probably generated by the reaction of diaphorase with NADH or NADPH. Catalase had no effect. Diaphorase could apparently be one of the enzymes participating in the metabolism of studied NO donors to NO. The easy reduction and consequent elimination of PTIO by diaphorase could affect its use as an NO scavenger in biological tissues.

Phosphorylation of 68 kDa neurofilament proteins has no significant effect on their assembly.

Native low molecular weight neurofilaments (NF-L) from bovine spinal cord with original phosphate content of 0.4 moles of phosphate per 1 mol of protein were phosphorylated with cyclic AMP dependent protein kinase and protein kinase C. In a similar way recombinant mouse NF-L proteins which did not contain any phosphate were phosphorylated with the same enzymes in both, the assembled and disassembled forms. The final phosphate content in both types of NF-L proteins reached about 4 moles of phosphate per 1 mol of protein. This phosphorylation had no effect on the assembly of NF-L into filaments as observed by electron microscopy.

The effect of partial deglycosylation on the structure of alpha1-acid glycoprotein.

Changes in structure of alpha1-acid glycoprotein were followed after deglycosylation with neuraminidase, peptide N-glycohydrolase F or with a mixture of exoglycosidases. Partially deglycosylated preparations of alpha1-acid glycoprotein free of sialic acids, one complete saccharide component, sialic acids and one saccharide component and sialic acids and some of the external saccharides were obtained. The effect of these changes in saccharide components on the glycoprotein structure was studied by temperature perturbation difference spectroscopy, fluorescence spectroscopy, fourth-derivative of absorption spectra and spectra of CD. Partial deglycosylation resulted in transformation of the molecule to a more compact state in which phenylalanyl residues were even more buried, tyrosyl residues became more uniform and tryptophyl residues were less exposed. The content of ordered secondary structures decreased. The thermal stability of the molecule was not significantly affected. Removal of one of the five saccharide components from the native molecule had apparently deeper effect than total desialyzation of the glycoprotein.

Possible metabolic pathways of conversion of formaldoxime and glyceryl trinitrate to NO.

The oxidation of N-hydroxylated compounds may result in production of nitrogen oxides, including nitric oxide (NO). Oxidation may be independent on NO-synthase. Production of nitrites and nitrates via NO from formaldoxime and glyceryl trinitrate was studied and compared. Superoxide ion, ions Fe2+ and Fe3+, methemoglobin and methemoglobin + NADPH + methylene blue, oxyhemoglobin and oxyhemoglobin + NADPH + methylene blue in the presence of atmospheric oxygen were used as oxidoreductive agents. Formaldoxime (triformaxime) was chosen as a newly recognized atypical cyclic oxime which can be converted to NO and glyceryl trinitrate as a well-known NO donor of quite different structure. From the oxidoreductive agents used, glyceryl trinitrate was not converted to nitrites or nitrates by Fe2+ or Fe3+ and by methemoglobin alone. Formaldoxime was resistant to the action of superoxide ion and methemoglobin alone. Importance of these possible metabolic pathways for production of NO from examined vasodilators is discussed.

[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.

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.).