Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA.

Major acute phase proteins (APPs) have proven diagnostically useful in dogs, cats and horses with routine use facilitated by commercially available automated heterologous assays. An automated assay applicable across all three species would highly facilitate further dissemination of routine use, and the aim of this study was to validate an automated latex agglutination turbidimetric immunoassay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline and equine SAA. Serum samples from 60 dogs, 40 cats and 40 horses were included. Intra- and inter-assay imprecision, linearity and detection limit (DL) were determined to assess analytical performance. To assess clinical performance, equine and feline SAA measurements were compared with parallel measurements using a previously validated automated SAA assay in a method comparison setting, and by assessing overlap performance of canine SAA in healthy dogs and diseased dogs with and without systemic inflammation. Intra- and inter-assay CVs ranged between 1.9-4.6% and between 3.0-14.5%, respectively. Acceptable linearity within a clinically relevant range of SAA concentrations was observed for all three species. The DL was 1.06 mg/L. Method comparison revealed acceptable agreement of the two assays measuring feline and equine SAA, and the overlap performance of canine SAA was acceptable. The tested assay measured SAA in canine, feline and equine serum with analytical and overlap performance acceptable for clinical purposes so improving practical aspects of clinical APP application. The monoclonal nature of the antibodies suggests strong, long-term inter-batch performance stability.

Isolation of mesenchymal stem cells from bone marrow wastes of spinal fusion procedure (TLIF) for low back pain patients and preparation of bone dusts for transplantable autologous bone graft with a serum glue.

Low back pain and subsequent disabilities are common. A lumbar spinal fusion procedure is an effective treatment with autologous bone grafts, but harvesting the bone from the iliac crest is associated with risks of complications. New treatments using stem cells together with osteoconductive and otesoinductive materials have made the procedure safer, but the inconsistency of the amount of stem cells harvested from bone marrow aspirate still remains to be solved. This study reports that the bone dusts, usually discarded as surgical wastes during transforaminal lumbar interbody fusion procedure (TLIF procedure), yielded cells which had the characteristics of mesenchymal stem cells (MSCs) in vitro. The cells were positive for the MSC markers and were able to differentiate in osteogenic and adipogenic directions. The cells grew robustly in an osteoconductive material, Bolheal (serum glue), and also proliferated well in culture medium supplemented with autologous serum. Therefore, the bone dust is a good candidate for the alternative source of stem cells other than bone marrow aspirate to increase the safety of the TLIF procedure.

Acid mediated hydrolysis of blueberry anthocyanins.

Acid mediated hydrolysis of anthocyanins was studied using capillary zone electrophoresis (CZE). A commercially available wild blueberry (Bilberry) extract was dissolved in different concentrations of TFA (0.1, 1, 3, 9%), then was subjected to thermodecomposition reaction at 95 degrees C. After the reaction, the samples were analyzed by CZE. The hydrolysis rate of each anthocyanin and the formation of the aglycon were determined by the change in the peak pattern of the anthocyanins in the electropherogram. Each anthocyanin peak decreased time dependently in a first order kinetic fashion. It was revealed that the hydrolysis rate of each anthocyanin was determined primarily by the type of conjugated sugar and not by the aglycon structure. The rate constant of anthocyanin hydrolysis was in the following order, arabinoside>galactoside>glucoside without regard to the aglycon structure. The kinetic behavior of this anthocyanin hydrolysis together with the CZE mobility allowed us to identify an unknown CZE peak as delphinidin 3-O-beta-arabinoside. At low TFA concentration, significant decomposition of the anthocyanidin nucleus occurred, but the glycoside hydrolysis predominated at high TFA concentration. It was further revealed that the aglycon released reacted successively to form polymeric products at higher TFA conditions.

Comparison of anthocyanin distribution in different blueberry sources by capillary zone electrophoresis.

Capillary zone electrophoretic separation of blueberry anthocyanins was studied using a Na-borate buffer containing trans-1,2-diaminocyclohexane-N,N,N',N'-tetra acetic acid monohydrate (CyDTA) as the carrier buffer. The separation conditions were precisely examined using an aqueous extract of bilberry (wild type blueberry) as the separation sample which is rich in this type and amount of anthocyanins. Each separated peak was identified by comparing the mobility with that of anthocyanin standards after normalization against the mobility of malvidin 3-o-glucoside (Mv 3-Glc) added as an internal standard. As salt concentrations of the running buffer increased, the peak resolution was markedly improved over the whole range of separation, especially, among the fast moving components (petunidin 3-glucoside, cyanidin 3-glucoside and malvidin 3-galactoside). Inversely, the peak separation both between petunidin 3-glucoside and peonidin 3-glucoside, and between delphinidin 3-glucoside and petunidin 3-galactoside, respectively, were decreased. The anthocyanins were, however, successfully separated by decreasing the buffer pH. Good separation of anthocyanins was finally achieved by 30 mM Na-borate (pH 8.78) containing 7.5 mM CyDTA within 10 min. Under this separation condition, anthocyanins from different blueberry sources were analyzed. The results revealed that different blueberry sources had their own patterns of anthocyanin distribution and amounts in the extracts, thus the present method is suitable for the quality control of anthocyanin-containing food materials.

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: effect of the hydrophobic chain length of the surfactants on the structural changes.

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion of alpha-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, the alpha-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.