Spectral-luminescent manifestation of the damaging action of brain ischemia on blood serum components

With the aid of spectral-luminescent analysis, our hypothesis on the determining role of free-radical oxidation in damage of blood serum components (low-density lipoproteins) in ischemia has been confirmed. An increase in the luminescence intensity of the blood serum of animals that suffered from brain ischemia as against that of healthy animals is registered. Lipoperoxide free-radical damage of phospholipids of the amphipathic layer of low-density lipoproteins after an ischemic procedure has also been confirmed by fluorescent probes (rhodamine 6G and Nile Blue). __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 72, No. 6, pp. 827–831, November–December, 2005.     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

The application of coulometry for total antioxidant capacity determination of human blood

New coulometric method for estimation of blood and plasma total antioxidant capacity (TAC) based on using electrogenerated bromine was proposed. TAC of blood from patients with chronic renal disease undergoing long-term hemodialysis was investigated. Statistical significant changes in TAC level of venous and arterial blood were found. Catalase activity and low density lipoproteins (LDL) concentrations were determined. Linear correlation between TAC and parameters mentioned was found. Contribution from some individual antioxidants was investigated. The developed method for TAC assay is expressive, simple, stable and reliable, and successfully could be used for TAC determination of some biological fluids. This method could be applied in clinic for estimation of blood TAC from patients.     

Anion-exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl-ligated gel with a perchlorate-containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion‐exchange high‐performance liquid chromatography using a column filled with nonporous, diethylaminoethyl‐ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high‐density lipoprotein, low‐density lipoprotein, intermediate‐density lipoprotein, very‐low‐density lipoprotein and chylomicrons) were eluted with step‐wise increases in a concentration of NaClO₄. The post‐column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein‐cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within‐day and between‐day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high‐fat diet increased lipoprotein‐cholesterol concentrations by 6‐ to 77‐fold. The method described here was nevertheless able to quantitate levels of lipoprotein‐cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous Quantitative Analysis for Phospholipids in Human HDL and LDL Using Two Internal Standards by Liquid Chromatography/Mass Spectrometry

Abstract

A liquid chromatography/mass spectrometry method using two internal standards was developed for the simultaneous quantitative determination of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (LPC) in human high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL). We evaluated this method by examining precision, accuracy, and recovery of phospholipid concentrations in several matrixes. We obtained the time course of phospholipid content in human HDL and LDL treated with sPLA₂‐X and quantitatively observed the decrease of PC, PI, PE, and the increase of LPC. This method should be useful for examination of simultaneous change of endogenous phospholipids in various enzymatic assays.