Systematical approach in evaluation of LC method for determination of raloxifene hydrochloride and its impurities employing experimental design.

Method validation presents a detailed investigation of analytical method and provision of the evidence that the method, when correctly applied, produces results that fit to the purpose. In order to achieve the method validation scope efficiently, experimental design presents a very useful tool. The greatest benefits of such approach could be seen in robustness testing through the provision of very useful data about the control of the chromatographic system during the routine application. In this paper, robustness testing of the LC method proposed for the determination of raloxifene hydrochloride and its four impurities was done employing Plackett-Burman design. Applying this design, the effect of five real factors (acetonitrile content, sodium dodecyl sulfate content, column temperature, pH of the mobile phase and flow rate) on the corresponding resolution factors was investigated through twelve experiments. Furthermore, the insignificance intervals for significant factors were calculated and the parameters for system suitability tests were defined. Eventually, the other validation parameters were tested and the effectiveness of the proposed analytical method with a high degree of accuracy was confirmed.

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities.

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.

The thyroid C cells of ovariectomized rats treated with estradiol.

The structure and function of thyroid C cells were studied in ovariectomized (Ovx) adult female rats without and after chronic treatment with estradiol dipropionate (EDP). A peroxidase-antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume, nuclei, and relative volume density were evaluated in comparison with sham-operated control rats using a stereological method. The number of C cells was calculated. CT content in the sera was determined by radioimmunoassay. Ovariectomy (Ovx) led to a 21% increase in body weight ( P<0.005), while treatment of Ovx rats with EDP decreased body weight by 25% ( P<0.01). The immunoreactivity for CT in C cells of the Ovx rats was markedly increased. Significant decreases in the volume of C cells (by 13%; P<0.05) and serum CT (by 45%) were recorded, while the C cell number increased by 59% ( P<0.05) in relation to the corresponding controls. The treatment of Ovx rats with EDP caused conspicuous degranulation of the C cells. The cellular volume was increased by 11% and serum CT by 36% in comparison with Ovx animals. At the same time a decrease in C cell number by 29% ( P<0.05) was evident. It may be concluded that estradiol deficiency after Ovx reduced the synthesis and release of CT, while chronic treatment of these animals with EDP had a positive effect on the secretory activity of thyroid C cells.

Epidemiology of end-stage renal disease and current status of hemodialysis in Yugoslavia.

Maintenance hemodialysis (HD) in Yugoslavia started in the sixties and followed the dialysis trends in the Western Europe. However, in the last decade the development of renal replacement therapy (RRT) slowed down. In this report the epidemiology of ESRD from 1997-1999 and the survey of the status of HD treatment in Yugoslavia in 1999 are presented. Epidemiological data are obtained by the annual center questionnaires (response rate: 92.6 -94.2%). The survey of HD status is based on a specific questionnaire and covered 2108 patients (65%). At the end of 1999 there were 56 RRT centers in Yugoslavia treating 3939 patients: 3232 (82%) patients by HD, 248 (6.3%) by peritoneal dialysis, and 459 (11.7%) living with transplanted kidney. In a three year period, incidence of ESRD ranged from 108-128 pmp, point prevalence from 435-463 pmp and mortality rate from 20.7-17.9. Numerous refugee patients were treated over the last 10 years. Main causes of ESRD were glomerulonephritis (30%); Balkan nephropathy represented 11% and diabetic nephropathy 7% of all primary renal diseases. Cardiovascular and cerebrovascular diseases were the most common causes of death of RRT patients. Most centers are overcrowded and HD machines are worn out. Mean Kt/V was 1.19+/-0.08, mean URR% 58.8+/-7.4. The shortage of drugs prevented adequate management: 83% of HD patients had hemoglobin level less than 100 g/L but only 10.3 -17.8% were treated with rHuEpo; 64.5% of patients had phosphate levels higher than 1.7 mmol/L but only 33.5% used phosphate binders; 47% of patients had hypertension despite the antihypertensive therapy. The prevalence of hepatitis B remained unchanged (about 14%) in HD population during the last three years, but the prevalence of anti-HCV positive patients decreased (31-23%). In conclusion, there is a well developed dialysis service in Yugoslavia but insufficient conditions for adequate treatment.

[Surgical anatomy of the spleen with special emphasis on its segmental architecture]. / Hirurska anatomija slezine sa posebnim osvrtom na njenu segmentnu gradju.

The authors have analyzed several aspects of the surgical anatomy of spleen, commencing with historical data, topography, peritoneal ligaments, variations in shape, embryology and accessory spleens and venous system of the spleen. The mode of splenic artery branching, variations of polar arteries, and intra- and extraparenchymatous arterial anastomoses were thoroughly analyzed. It was shown that the spleen in most cases consists of five vascular territories (segments) clearly demarcated from each other, stressing the practical significance of splenic anatomy in segmental dearterialization of the spleen.

Effects of pH on protein association: modification of the proton-linkage model and experimental verification of the modified model in the case of cytochrome c and plastocyanin.

Effects of pH on protein association are not well understood. To understand them better, we combine kinetic experiments, calculations of electrostatic properties, and a new theoretical treatment of pH effects. The familiar proton-linkage model, when used to analyze the dependence of the association constant K on pH, reveals little about the individual proteins. We modified this model to allow determination not only of the numbers of the H+ ions involved in the association but also of the pK(a) values, in both the separate and the associated proteins, of the side chains that are responsible for the dependence of K on pH. Some of these side chains have very similar pK(a) values, and we treat them as a group having a composite pK(a) value. Use of these composite pK(a) values greatly reduces the number of parameters and allows meaningful interpretation of the experimental results. We experimentally determined the variation of K in the interval 5.4 < or = pH < or = 9.0 for four diprotein complexes, those that the wild-type cytochrome c forms with the wild-type plastocyanin and its mutants Asp42Asn, Glu59Gln, and Glu60Gln. The excellent fittings of the experimental results to the modified model verified this model and revealed some unexpected and important properties of these prototypical redox metalloproteins. Protein association causes a decrease in the pK(a) values of the acidic side chains and an increase in the pK(a) values of the basic side chains. Upon association, three carboxylic side chains in wild-type plastocyanin each release a H+ ion. These side chains in free plastocyanin have an anomalously high composite pK(a) value, approximately 6.3. Upon association, five or six side chains in cytochrome c, likely those of lysine, each take up a H+ ion. Some of these side chains have anomalously low pK(a) values, less than 7.0. The unusual pK(a) values of the residues in the recognition patches of plastocyanin and cytochrome c may be significant for the biological functions of these proteins. Although each mutation in plastocyanin markedly, and differently, changed the dependence of K on pH, the model consistently gave excellent fittings. They showed decreased numbers of H+ ions released or taken up upon protein association and altered composite pK(a) values of the relevant side chains. Comparisons of the fitted composite pK(a) values with the theoretically calculated pK(a) values for plastocyanin indicated that Glu59 and Asp61 in the wild-type plastocyanin each release a H+ ion upon association with cytochrome c. Information of this kind cannot readily be obtained by spectroscopic methods. Our modification of the proton-linkage model is a general one, applicable also to ligands other than H+ ion and to processes other than association.

The role of cation-pi interactions in biomolecular association. Design of peptides favoring interactions between cationic and aromatic amino acid side chains.

Cation-pi interactions between amino acid side chains are increasingly being recognized as important structural and functional features of proteins and other biomolecules. Although these interactions have been found in static protein structures, they have not yet been detected in dynamic biomolecular systems. We determined, by (1)H NMR spectroscopic titrations, the energies of cation-pi interactions of the amino acid derivative AcLysOMe (1) with AcPheOEt (2) and with AcTyrOEt (3) in aqueous and three organic solvents. The interaction energy is substantial; it ranges from -2.1 to -3.4 kcal/mol and depends only slightly on the dielectric constant of the solvent. To assess the effects of auxiliary interactions and structural preorganization on formation of cation-pi interactions, we studied these interactions in the association of pentapeptides. Upon binding of the positively-charged peptide AcLysLysLysLysLysNH(2) (5) to the negatively-charged partner AcAspAspXAspAspNH(2) (6), in which X is Leu (6a), Tyr (6b), and Phe (6c), multiple interactions occur. Association of the two pentapeptides is dynamic. Free peptides and their complex are in fast exchange on the NMR time-scale, and 2D (1)H ROESY spectra of the complex of the two pentapeptides do not show intermolecular ROESY peaks. Perturbations of the chemical shifts indicated that the aromatic groups in peptides 6b and 6c were affected by the association with 5. The association constants K(A) for 5 with 6a and with 6b are nearly equal, (4.0 +/- 0.7) x 10(3) and (5.0 +/- 1.0) x 10(3) M(-)(1), respectively, while K(A) for 5 with 6c is larger, (8.3 +/- 1.3) x 10(3) M(-)(1). Molecular-dynamics (MD) simulations of the pentapeptide pairs confirmed that their association is dynamic and showed that cation-pi contacts between the two peptides are stereochemically possible. A transient complex between 5 and 6 with a prominent cation-pi interaction, obtained from MD simulations, was used as a template to design cyclic peptides C(X) featuring persistent cation-pi interactions. The cyclic peptide C(X) had a sequence in which X is Tyr, Phe, and Leu. The first two peptides do, but the third does not, contain the aromatic residue capable of interacting with a cationic Lys residue. This covalent construct offered conformational stability over the noncovalent complexes and allowed thorough studies by 2D NMR spectroscopy. Multiple conformations of the cyclic peptides C(Tyr) and C(Phe) are in slow exchange on the NMR time-scale. In one of these conformations, cation-pi interaction between Lys3 and Tyr9/Phe9 is clearly evident. Multiple NOEs between the side chains of residues 3 and 9 are observed; chemical-shift changes are consistent with the placement of the side chain of Lys3 over the aromatic ring. In contrast, the cyclic peptide C(Leu) showed no evidence for close approach of the side chains of Lys3 and Leu9. The cation-pi interaction persists in both DMSO and aqueous solvents. When the disulfide bond in the cyclic peptide C(Phe) was removed, the cation-pi interaction in the acyclic peptide AC(Phe) remained. To test the reliability of the pK(a) criterion for the existence of cation-pi interactions, we determined residue-specific pK(a) values of all four Lys side chains in all three cyclic peptides C(X). While NOE cross-peaks and perturbations of the chemical shifts clearly show the existence of the cation-pi interaction, pK(a) values of Lys3 in C(Tyr) and in C(Phe) differ only marginally from those values of other lysines in these dynamic peptides. Our experimental results with dynamic peptide systems highlight the role of cation-pi interactions in both intermolecular recognition at the protein-protein interface and intramolecular processes such as protein folding.

Enantioselective aminolysis of an alpha-chloroester catalyzed by Candida cylindracea lipase encapsulated in sol-gel silica glass.

[reaction: see text] Lipase from Candida cylindracea (CcL) encapsulated in porous silica glass by a sol-gel method catalyzes enantioselective aminolysis of ethyl 2-chloropropionate. A silica matrix enhances the enzyme activity, i.e., improves the yield. The scope and limitations of the aminolysis reaction were investigated, and dynamic kinetic resolution of the ester was achieved. Encapsulated lipase remains active when used repeatedly. Encapsulation much improves the chiral discrimination by lipase and makes this enzyme even more useful in organic chemistry.

New selectivity in peptide hydrolysis by metal complexes. Platinum(II) complexes promote cleavage of peptides next to the tryptophan residue.

Tryptophan-containing N-acetylated peptides AcTrp-Gly, AcTrp-Ala, AcTrp-Val, and AcTrp-ValOMe bind to platinum(II) and undergo selective hydrolytic cleavage of the C-terminal amide bond; the N-terminal amide bond remains intact. In acetone solution, bidentate coordination of the tryptophanyl residue via the C(3) atom of indole and the amide oxygen atom produces complexes of spiro stereochemistry, which are characterized by (1)H, (13)C, and (195)Pt NMR spectroscopy, and also by UV-vis, IR, and mass spectroscopy. Upon addition of 1 molar equiv of water, these complexes undergo hydrolytic cleavage. This reaction is as much as 10(4)-10(5) times faster in the presence of platinum(II) complexes than in their absence. The hydrolysis is conveniently monitored by (1)H NMR spectroscopy. We report the kinetics and mechanism for this reaction between cis-[Pt(en)(sol)(2)](2+), in which the solvent ligand is water or acetone, and AcTrp-Ala. The platinum(II) ion as a Lewis acid activates the oxygen-bound amide group toward nucleophilic attack of solvent water. The reaction is unimolecular with respect to the metal-peptide complex. Because the tryptophanyl fragment AcTrp remains coordinated to platinum(II) after cleavage of the amide bond, the cleavage is not catalytic. Added ligand, such as DMSO and pyridine, displaces AcTrp from the platinum(II) complex and regenerates the promoter. This is the first report of cleavage of peptide bonds next to tryptophanyl residues by metal complexes and one of the very few reports of organometallic complexes involving metal ions and peptide ligands. Because these complexes form in nonaqueous solvents, a prospect for cleavage of membrane-bound and other hydrophobic proteins with new regioselectivity has emerged.

[Effect of autogenic training on glucose regulation and lipid status in non-insulin dependent diabetics]. / Uticaj autogenog treninga na glikoregulaciju i lipidni status kod insulinneza visnih dijabeticara.

INTRODUCTION: The objective of this study was to examine the benefits of autogenic training in patients with type 2 diabetes and 40 diabetics treated with oral antidiabetic agents were assigned to receive autogenic training. Treatment effects on GHb levels, glycemia, lipids and lipid peroxidases were evaluated after 12 weeks. RESULTS: Subjects demonstrated significant improvements of GHb level (8.94 +/- 2.21% vs. 7.9 +/- 2.395) (p < 0.005). Fasting glucose was significantly lower after treatment (11.6 +/- 6.1 mmol/l vs. 8.32 +/- 4.53 mmol/l) (p < 0.005). The serum level of HDL cholesterol was significantly higher after autogenic training (1.21 +/- 0.11 vs. 1.36 +/- 1.42) (p < 0.005). Cholesterol level was significantly lower after training (6.63 +/- 1.66 mmol/l vs. 6.10 +/- 1.12 mmol/l) (p < 0.05). Lipid peroxidase was significantly lower after treatment (4.05 +/- 0.58 vs. 3.69 +/- 1.26) (p < 0.005). CONCLUSION: Autogenic training in selected patients, especially those who are most responsive to stress would provide benefits for glucosE control and lipid metabolism that are not always achieved by conventional treatment.

[Effect of hyperlipoproteinemia on blood fibrinolytic activity and thrombocyte aggregation]. / Uticaj hiperlipoproteinemije na fibrinoliticku aktivnost krvi i agregaciju trombocita.

BACKGROUND AND AIM: An impaired lipid metabolism and macrovascular disease are often associated with diabetes mellitus. Effects of hyperlipoproteinemia on fibrinolytic blood activity and platelet aggregation were investigated in noninsulin-dependent diabetes mellitus. MATERIAL AND METHODS: The study sample consisted of 50 patients with noninsulin-dependent diabetes mellitus, only on dietetic regime. The following was studied: lipids (total cholesterol, HDL cholesterol and total triglycerides) fibrinolytic blood activity and platelet aggregation in patients with increased levels of cholesterol and triglycerides. We used enzymatic methods for lipids, Buckell's method for fibrinolysis (> 180 min) and Crean's method for platelet aggregation (6-8 s). RESULTS: Statistical analysis of the obtained results shows significant changes in lipids. Triglycerides were significantly higher in diabetics with poor control (p < 0.01). Total cholesterol was significantly higher in patients with coronary disease, hypertension and diabetics with occlusive disease of low extremities (p < 0.01). HDL cholesterol was significantly lower in diabetics with coronary heart disease (p < 0.05). Statistical analysis showed significant changes in blood coagulability. Decreased fibrinolytic blood activity (158.10 + 31.3 min v.s. 169.82 + 21.05 min) (p < 0.05), as well as faster platelet aggregation (6.7 + 0.48 sec v.s. 6.8 + 0.93 sec) (p < 0.1) in patients with hypertriglyceridemia compared with the diabetics with normal triglycerides. Other lipids did not influence significantly the fibrinolysis and platelet aggregation in noninsulin-dependent diabetics. CONCLUSION: All of this indicates the importance of the studied factors in the onset of atherosclerosis with patients with noninsulin-dependent diabetes; the latter is relevant to the importance of planned diabetic regime and moderate physical activity in preventing vascular complications of diabetes.

Spectroscopic, kinetic, and mechanistic study of a new mode of coordination of indole derivatives to platinum(II) and palladium(II) ions in complexes.

Binding of tryptophan residue to intrinsic metal ions in proteins is unknown, and very little is known about the coordinating abilities of indole. Indole-3-acetamide displaces the solvent ligands from cis-[Pt(en)(sol)2]2+, in which sol is acetone or H2O, in acetone solution and forms the complex cis-[Pt(en)(indole-3-acetamide)]2+ (3) of spiro structure, in which the new bidentate ligand coordinates to the Pt(II) atom via the C(3) atom of the indolyl group and the amide oxygen atom. This structure is supported by 1H, 13C, 15N, and 195Pt NMR spectra and by UV, IR, and mass spectra. Molecular mechanical simulations by Hyperchem and CHARMM methods give consistent structural models; the latter is optimized by density-functional quantum chemical calculations. Dipeptide-like molecules N-(3-indolylacetyl)-L-amino acid in which amino acid is alanine, leucine, isoleucine, valine, aspartic acid, or phenylalanine also displace the solvent ligands in acetone solution and form complexes cis-[Pt(en) N-(3-indolylacetyl)-L-amino acid)]2+ (6), which structurally resemble 3 but exist as two diastereomers, detected by 1H NMR spectroscopy. The bulkier the amino acid moiety, the slower the coordination of these dipeptide-like ligands to the Pt(II) atom. The indolyl group does not coordinate as a unidentate ligand; a second donor atom is necessary for bidentate coordination of this atom and the indolyl C(3) atom. The solvent-displacement reaction is of first and zeroth orders with respect to indole-3-acetamide and cis-[Pt(en)(sol)2]2+, respectively. A mechanism consisting of initial unidentate coordination of the ligand via the amide oxygen atom followed by closing of the spiro ring is supported by 1H NMR data, the kinetic effects of acid and water, and the activation parameters for the displacement reaction. In the case of N-(3-indolylacetyl)-L-phenylalanine, the bulkiest of the entering ligands, the reaction is of first order with respect to both reactants. The bidentate indole-3-acetamide ligand in 3 is readily displaced by (CH3)2SO and 2-methylimidazole, but not by CNO-, CH3COO-, and CH3CN. Complexes cis-[Pd(en)(sol)2]2+ and cis-[Pd(dtco)(sol)2]2+ react with indole-3-acetamide more rapidly than their Pt(II) analogues do and yield complexes similar to 3. This study augments our recent discovery of selective, hydrolytic cleavage of tryptophan-containing peptides by Pd(II) and Pt(II) complexes.

Redox reactivity and reorganization energy of zinc cytochrome c cation radical.

Little is known about transient intermediates in photoinduced electron-transfer reactions of metalloproteins. Oxidative quenching of the triplet state of zinc cytochrome c, 3Zncyt, is done at 20 degrees C, pH 7.00, and ionic strength of 1.00 M, conditions that suppress the thermal back-reaction and prolong the lifetime of the cation radical, Zncyt+. This species is reduced by [Fe(CN)6]4-, [W(CN)8]4-, [Os(CN)6]4-, [Mo(CN)8]4-, and [Ru(CN)6]4- complexes of similar structures and the same charge. The rate constants and thermodynamic driving forces for these five similar electron-transfer reactions were fitted to Marcus theory. The reorganization energy of Zncyt+ is lambda = 0.38(5) eV, lower than that of native cytochrome c, because the redox orbital of the porphyrin cation radical is delocalized and possibly because Met80 is not an axial ligand to the zinc(II) ion in the reconstituted cytochrome c. The rate constant for electron self-exchange between Zncyt+ and Zncyt, k11 = 1.0(5) x 10(7) M(-1) s(-1), is large owing to the extended electron delocalization and relatively low reorganization energy. These results may be relevant to zinc(II) derivatives of other heme proteins, which are often used in studies of photoinduced electron-transfer reactions.

Electron transfer to photosystem 1 from spinach plastocyanin mutated in the small acidic patch: ionic strength dependence of kinetics and comparison of mechanistic models.

A set of plastocyanin (Pc) mutants, probing the small acidic patch (Glu59, Glu60, and Asp61) and a nearby residue, Gln88, has been constructed to provide further insight into the electron transfer process between Pc and photosystem 1. The negatively charged residues were changed into their neutral counterparts or to a positive lysine. All mutant proteins exhibited electron transfer kinetics qualitatively similar to those of the wild type protein over a wide range of Pc concentrations. The kinetics were slightly faster for the Gln88Lys mutant, while they were significantly slower for the Glu59Lys mutant. The data were analyzed with two different models: one involving a conformational change of the Pc-photosystem 1 complex that precedes the electron transfer step (assumed to be irreversible) [Bottin, H., and Mathis, P. (1985) Biochemistry 24, 6453-6460] and another where no conformational change occurs, the electron transfer step is reversible, and dissociation of products is explicitly taken into account [Drepper, F., Hippler, M., Nitschke, W., and Haehnel, W. (1996) Biochemistry 35, 1282-1295]. Both models can account for the observed kinetics in the limits of low and high Pc concentrations. To discriminate between the models, the effects of added magnesium ions on the kinetics were investigated. At a high Pc concentration (0.7 mM), the ionic strength dependence was found to be consistent with the model involving a conformational change but not with the model where the electron transfer is reversible. One residue in the small acidic patch, Glu60, seems to be responsible for the major part of the ionic strength dependence of the kinetics.

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin.

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction 3Zncyt + az(II)-->Zncyt+ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrostatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.

Comparing the rates and the activation parameters for the forward reaction between the triplet state of zinc cytochrome c and cupriplastocyanin and the back reaction between the zinc cytochrome c cation radical and cuproplastocyanin.

This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.

[Sodium excretion in children with lithogenic disorders]. / Izlucivanje natrijuma kod dece s litogenim poremecajima.

INTRODUCTION: The causes of nephrolithisis are multifactorial and have not yet been enough investigated [1]. Hypercalciuria is the most common cause of metabolic nephrolithiasis [2-4]. Close relationship between urinary calcium and urinary sodium has been a subject of reported observations in the past, showing that high urinary sodium is associated with high urinary calcium [5-7]. Hyperoxaluria, hyperuricosuria and cystinuria are also metabolic disorders that can lead to nephrolithiasis. Recent studies have indicated that urinary elimination of cystine is influenced by urinary sodium excretion. Based on these observations it has been hypothesised that patients with high urinary sodium excretion are at high risk of urinary stone disease. The purpose of the study was to investigate sodium excretion in a 24-hour urine and first morning urine collected from children with lithogenic metabolic abnormalities (hypercalciuria, hyperoxaluria, hyperuricosuria, cystinuria), both with nephrolithiasis and without it, in order to determine its significance in urinary calculi formation. PATIENTS AND METHODS: Urinary sodium excretion was investigated in 2 groups of children: patients with lithogenic metabolic abnormalities, but without urinary stone disease (L group) and patients with nephrolithiasis (C group). Both groups were divided into 2 subgroups: patients with hypercalciuria and without it. There were 22 patients in group L (mean age 11.97 +/- 4.13 years), of whom 17 formed a hypercalciuric subgroup and 5 formed a non-hypercalciuric subgroup (3 patients with hyperuricosuria and 2 patients with hyperoxaluria). Group C consisted of 21 patients with nephrolithiasis (mean age 12.67 +/- 3.44 years), of whom 6 formed a hypercalciuric subgroup and 15 formed a non-hypercalciuric group (2 patients with cystinuria and 13 patients without lithogenic metabolic abnormalities). Control group consisted of 42 healthy age-matched children. All subjects had a normal renal function. A detailed history and clinical examination were done, and ultrasonography was performed in all patients. A 24-hour urine, first morning urine and serum specimen were analysed for sodium, potassium, calcium, uric acid, urea and creatinine. Fractional excretion of sodium, as well as urinary sodium to creatinin ratio and urinary sodium to potassium ratio, were calculated from the findings. Sodium and potassium levels were determined by flame photometry, calcium was measured by atomic absorption technique (Beckman Atomic Spectrophotometer, Synchron CX-5 model, USA), uric acid by carbonate method and creatinine by Jaffe technique. Cystine and dibasic amino acids were quantified by ion chromatography. Urinary oxalate excretion was determined by enzyme spectrophotometry. Hypercalciuria was defined by 24-hour calcium excretion greater than 3.5 mg/kg per day and/or calcium to creatinine ratio greater than 0.20 [8]. Uric acid excretion was expressed as uric acid excretion factored for glomerular filtration, according to Stapleton's and Nash's formula [9]. Normal values were lower than 0.57 mg/dl of glomerular filtration rate in 24-hour samples. Mean values were statistically analyzed by Pearson's linear correlation and analysis of variance (ANOVA). RESULTS: Urinary sodium concentration values including urinary sodium to potassium ratios, are shown in Table 1. We found that urinary sodium excretion was significantly increased in patients of both L and C groups when compared with controls (p < 0.05). Further analysis of the subgroups showed that urinary sodium excretion was significantly higher only in patients with hypercalciuria of both L and C groups in comparison to controls (p < 0.05) (Table 2). A significant positive correlation was found between 24-hour urinary sodium to creatinine ratio and urinary calcium to creatinine ratio (r = 0.31; p < 0.001) (Graph 1), as well as between urinary sodium to potassium ratio in 24-hour and first morning urine (r = 0.69; p < 0.001) (Graph 2). (A

Effects of single and double mutations in plastocyanin on the rate constant and activation parameters for the rearrangement gating the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin.

The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.

Effects of viscosity and temperature on the kinetics of the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin.

This is a study of the effects of viscosity (in the range of 0.8-790 cP), of temperature (in the range of 260.7-307.7 K), and of ionic strength (in the range of 2.5-20.0 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. The unimolecular rate constant is kF. The apparent activation parameters DeltaH*, DeltaS*, and DeltaG* for this reaction were obtained in experiments with aqueous glycerol solutions having a constant composition. The interpolation of kF values obtained at the constant composition into the dependence of kF on temperature at constant viscosity gave the proper activation parameters, which agree with those obtained in experiments with solutions having a constant viscosity. This agreement validates the latter method, which is more efficient than the former, for determining activation parameters of processes that are modulated by viscosity. The smooth change in kF is governed by the change in viscosity, not in other properties of the solvent, and it does not depend on the choice of the viscosigen. Donor/acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, are consistent with true electron transfer and with electron transfer that is coupled to, or gated by, a preceding structural rearrangement of the diprotein complex 3Zncyt/pc(II). The fact that at very high viscosity kF approaches zero shows that the reaction is probably gated throughout the investigated range of viscosity. Kinetic effects and noneffects of ionic strength, viscosity, and thermodynamic driving force indicate, but do not prove, that the reaction under consideration is gated. The kinetic effect of viscosity is analyzed in terms of two models. Because ln kF is a nonlinear function of ln eta, protein friction has to be considered in the analysis of viscosity effects on kinetics.