Determination of dermatan sulfate and chondroitin sulfate as related substances in heparin by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5cmx50mum i.d.) at 40 degrees C and -30kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, DeltaUA-->GalNAc-4S Na(2) and DeltaUA-->GalNAc-6S Na(2) were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.

Oxalate-degrading activity in Bifidobacterium animalis subsp. lactis: impact of acidic conditions on the transcriptional levels of the oxalyl coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes.

Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.

An EPR method for measuring the rate of distribution of organic substrates between cyclodextrin, micelles and water.

The combined use of selected nitroxides and EPR spectroscopy has been proved to be suitable for studying the partitioning rate of a given substrate in cyclodextrin-micelle systems.

Determination of oxalyl-coenzyme A decarboxylase activity in Oxalobacter formigenes and Lactobacillus acidophilus by capillary electrophoresis.

Oxalyl-coenzyme A decarboxylase (OXC) is a key enzyme in the catabolism of the highly toxic oxalate, catalysing the decarboxylation of oxalyl-coenzyme A (Ox-CoA) to formyl-coenzyme A (For-CoA). In the present study, a capillary electrophoretic (CE) method was proposed for the assessment of the activity of recombinant OXC from two bacteria, namely Oxalobacter formigenes DSM 4420 and Lactobacillus acidophilus LA 14. In particular, the degradation of the substrate Ox-CoA occurring in the enzymatic reaction could be monitored by the off-line CE method. A capillary permanently coated with polyethylenimine (PEI) was used and in the presence of a neutral background electrolyte (50 mM phosphate buffer at pH 7.0), a reversal of the electroosmotic flow was obtained. Under these conditions, the anodic migration of Ox-CoA (substrate) and For-CoA (reaction product) occurred and their separation was accomplished in less than 12 min. The CE method was validated for selectivity, linearity (range of Ox-CoA within 0.005-0.650 mM), sensitivity (LOD of 1.5 microM at the detection wavelength of 254 nm), precision and accuracy. Steady state kinetic constants (V(max), K(m) or k') of OXC were finally estimated for both the bacteria showing that although L. acidophilus LA 14 provided a lower oxalate breakdown than O. formigenes DSM 4420, it could be a potentially useful probiotic in the prevention of diseases related to oxalate.