Trimetazidine does not alter metabolic substrate oxidation in cardiac mitochondria of target patient population.

BACKGROUND AND PURPOSE: Trimetazidine, known as a metabolic modulator, is an anti-anginal drug used for treatment of stable coronary artery disease (CAD). It is proposed to act via modulation of cardiac metabolism, shifting the mitochondrial substrate utilization towards carbohydrates, thus increasing the efficiency of ATP production. This mechanism was recently challenged; however, these studies used indirect approaches and animal models, which made their conclusions questionable. The goal of the current study was to assess the effect of trimetazidine on mitochondrial substrate oxidation directly in left ventricular myocardium from CAD patients. EXPERIMENTAL APPROACH: Mitochondrial fatty acid (palmitoylcarnitine) and carbohydrate (pyruvate) oxidation were measured in permeabilized left ventricular fibres obtained during coronary artery bypass grafting surgery from CAD patients, which either had trimetazidine included in their therapy (TMZ group) or not (Control). KEY RESULTS: There was no difference between the two groups in the oxidation of either palmitoylcarnitine or pyruvate, and in the ratio of carbohydrate to fatty acid oxidation. Activity and expression of pyruvate dehydrogenase, the key regulator of carbohydrate metabolism, were also not different. Lastly, acute in vitro exposure of myocardial tissue to different concentrations of trimetazidine did not affect myocardial oxidation of fatty acid. CONCLUSION AND IMPLICATIONS: Using myocardial tissue from CAD patients, we found that trimetazidine (applied chronically in vivo or acutely in vitro) had no effect on cardiac fatty acid and carbohydrate oxidation, suggesting that the clinical effects of trimetazidine are unlikely to be due to its metabolic effects, but rather to an as yet unidentified intracardiac mechanism.

Location of PRODAN in lipid layer of HDL particle: a Raman study.

FT Raman spectroscopy has been applied to determine the location of PRODAN within HDL and to investigate its influence on the structure of the particle. The complex spectra of HDL and HDL labeled with PRODAN were divided into three regions according to the wave numbers, and adherent spectra were compared separately. Additionally, recorded spectra of protein and lipid fractions of HDL were used as a support for the assignment of particular vibrations in intact particles. In high frequency region, the shift in vibrational frequencies of CH3 groups but almost negligible shift of CH2 groups suggests that PRODAN is situated at the water/lipid interface in the vicinity of the protein. The statement is supported by the observed influence of PRODAN on particular lipid vibrations of phospholipids head-groups. In the fingerprint region, the influence of PRODAN is observed as the slight change in beta-strand secondary structure of apolipoprotein and strongly reduced vibrations of the acyl chain in lipids. That additionally confirms that PRODAN mainly interacts with the lipid domain of the particle. In the low frequency region, the lack of change in Tyr Fermi resonance doublet and only slight differences in the pattern of CS and SS stretching vibrations in labeled HDL confirms that PRODAN has no influence on structure of apolipoprotein embedded in lipid domain. The main conclusions drawn from the vibrational spectra of HDL with and without PRODAN clearly confirm that PRODAN induces negligible changes in HDL structure and hence is reliable fluorescent label for the structural analysis.

Investigation of spermatozoa and seminal plasma by fourier transform infrared spectroscopy.

Fourier transform infrared (FT-IR) spectra of human spermatozoa and seminal plasma were recorded and analyzed. The procedure that was established for sample preparation enabled acquisition of reproducible spectra. The parameter I(1087)/I(966) for controlling spectra reproducibility was defined. The assignment of bands was carried out using an empirical approach and the origin of the "sperm specific doublet", the bands at 968 cm(-1) and 981 cm(-1), was determined. The principal component regression (PCR) algorithm was used to define the specific spectral regions correlating to characteristics of spermatozoa, such as concentration, straight-line velocity (VSL), and beat cross frequency (BCF). Then, simple spectral parameters, such as band intensities and band ratios, were tested to determine which one best correlates to characteristics of spermatozoa. The region of the amide I band, between 1700 cm(-1) and 1590 cm(-1), was defined as a specific spectral region that correlates to the concentration of spermatozoa. The parameter that gave the linear dependence to the concentration of spermatozoa was the intensity of the amide I band. For VSL, the bands between 1119 cm(-1) and 943 cm(-1) were defined as the specific spectral region. The relative amount of nucleic acids with respect to proteins showed linear dependence on the straight-line velocity of spermatozoa. BCF showed the best correlation to the bands between 3678 cm(-1) and 2749 cm(-1), which largely represent lipids and proteins. These results suggest that FT-IR spectroscopy can serve as an adjunct to conventional histopathology studies.

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.

EPR evidence for the oxidation-induced formation of negatively charged species on the low-density lipoprotein surface.

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electrophoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.

Causal relationship between the transitions in the core and the surface in porcine low-density lipoproteins.

Two independent parameters, two characteristic temperatures, one indicating the change in the molecular organization of the core, Tc, and the other in the surface layer, Ts, were measured for a number of natural and triglyceride-enriched porcine low-density lipoprotein (LDL1 (buoyant density 1.020--1.063 g/ml) and LDL2 (buoyant density 1.063--1.080 g/ml) samples. Tc was determined by differential scanning calorimetry (DSC), whereas Ts was measured by Mn(II) binding to the lipoprotein surface followed by electron spin resonance (ESR) spectroscopy. A significant causal relationship between Tc and Ts in both LDL subfractions demonstrates the surface-core interaction in LDL. The significance of that interaction is emphasized as a possible link in the chain diet----lipoprotein changes----atherosclerosis.

Competitive ion binding to low density lipoproteins: an electron spin resonance study.

The electron spin resonance (ESR) technique was used to evaluate binding constants for Ca(II) and Mg(II) in interaction with low density lipoprotein (LDL). The Ca(II) or Mg(II) ions competed with the paramagnetic Mn(II) ions for the same binding sites of two different classes on the LDL surface. For each ion competing with Mn(II), the solutions of eight non-linear competition equations were fit to the experimental titration curves, with two adjustable parameters, the two binding constants. The derived "intrinsic" values (the values corrected for the electrolyte-induced change of the surface potential) for "strong" binding sites for Ca(II) (170 +/- 85 M-1) and Mg(II) (60 +/- 30 M-1) differ significantly from the respective value for Mn(II) (760 M-1). The values for the "weak" binding sites (18 M-1, 15 M-1 and 10 M-1 for Mn(II), Ca(II) and Mg(II), respectively are in the range of the binding constants for these ions in interaction with model membranes.

An ESR study of the effect of an electrostatic field on binding of divalent cations to the surface of serum low-density lipoproteins.

The ESR technique has been used to study binding of Mn(II) ions to low-density lipoprotein (LDL) in solutions of various electrolyte ionic strengths. A model of the binding has been proposed which describes all the observations in electrolytes of ten different concentrations in terms of two types of binding sites and two corresponding sets of intrinsic binding parameters (n1 = 8, Kd1 = 1.31 X 10(-3) mol X l-1 and n2 = 170, Kd2 = 5.71 X 10(-2) mol X l-1). These parameters, together with the values of the potential (phi 0) responsible for binding of the ions to specific charged sites on the surface, reproduce the observed binding curves well in all the systems studied. The phi 0 values are obtained as an appropriate solution of the Poisson-Boltzmann equation.

Surface-core correlations in serum lipoproteins.

It has been found that the capacity of lipoproteins for binding Mn(II) ions is dependent on the arrangement of the lipoprotein core. A change in the molecular organization of the LDL core (thermotropic transition) is associated with the change on the lipoprotein surface. In HDL there is no thermotropic transition and no abrupt change of the binding capacity. The observed surface-core correlation might be an important link in understanding how dietary fat influences the lipoprotein metabolism.

Adsorption of Mn(II) ions to human low density lipoproteins. Magnetic resonance studies.

Mn(II) ions were used to study ion-binding properties of human low density lipoproteins (LDL). From the intensity of the EPR lines corresponding to the unbound Mn(II) ions the percentage of the ions bound to LDL is determined. By the titration of LDL with Mn(II) the binding parameters, dissociation constant, Kd, and the number of binding sites, n, could be derived. It has been found that there are at least two types of binding site on the LDL surface: 'strong' sites characterized by n = 6, Kd =1 x 5 x 10(-5)M x 1(-1), and 'weak' sites characterized by n = 145 and Kd = 6.6 x 10(-3)M x l(-1) for the sample in 0.01 M Tris-HCl buffer at 10 degrees C. At very low Mn(II) concentrations binding to the 'strong' sites exhibits a cooperative behaviour. In the 0.1 M buffer the 'strong' sites are almost completely occupied or blocked by the monovalent buffer cations. The number of the 'weak' sites remains unaltered and Kd is decreased slightly (Kd = 4.9 x 10(-3)M x (-1)). The location, chemical nature and the structural and functional relevance of the binding sites are discussed.

Quaternary structure, hydration, and self-association of hemoglobin. A proton magnetic relaxation study.

The hydration of oxyhemoglobin, carbonylhemoglobin, and deoxyhemoglobin does not depend on the quaternary state of the hemoglobin molecule as revealed through the concentration dependence of proton magnetic relaxation rates. The biphasic relaxation behavior of isotopically diluted solutions of hemoglobin confirms that the self-association of hemoglobin molecules at higher concentration is independent of the quaternary structure. In the case of aquomethemoglobin, a biphasic relaxation and a diminished heme accessibility at higher concentrations are observed, caused by interaction of the associating hemoglobin molecules.

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions.

The proton and deuterium longitudinal relaxation rates were studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H2O/D2O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D2O/H2O) composition. The proton relaxation rates show two different linear dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem). The ratio of the slopes is 1.6 (high-to-low HB-conc). Only in the higher concentration range two T1's were observed if the solvent contained more than half of D2O. The slow relaxation phase of protons has T1's similar to those measured in solutions with less than half of D2O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.