Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Enzyme distributions in subcellular fractions of BHK cells infected with Semliki forest virus: evidence for a major fraction of sphingomyelin synthase in the trans-golgi network.

BHK cells either untreated or infected with Semliki Forest virus have been fractionated on sucrose density gradients. Virus infection caused an increase in density of a membrane fraction enriched in sphingomyelin (SM), cholesterol, SM synthase and sialyltransferase activity. This increase in density was related to incorporation of viral proteins into this fraction, which is likely to contain trans-Golgi network (TGN) membranes. In contrast, glucosylceramide synthase and galactosyltransferase activities (markers for cis/medial and trans-Golgi respectively) underwent no density shift and alkaline phosphodiesterase, a plasma membrane marker, was only slightly density-shifted in infected cells. When cells were incubated with NBD-ceramide to enable them to synthesise NBD-SM and then washed with albumin to remove surface label, fluorescence in untreated cells was concentrated in a single juxtanuclear spot but in infected cells this region of bright fluorescence was larger and extended around the nucleus. After fractionation of these cells, NBD-SM (but only a small proportion of the NBD-ceramide) was found to be shifted into the higher density fraction in infected cells. This work provides further evidence that SM synthase is not mainly localised in the early Golgi cisternae as previously thought, but is associated more with a cholesterol-rich compartment which could be the TGN.     

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.     

Im-trityl protection of histidine.

A rational attempt to prepare FmocHis(piTrt)OH regiospecifically gave in fact the well-known tau-trityl isomer, and experiments with model systems indicate that the prospects for access to pi-trityl histidine derivatives, which would be of great value for the racemization-free synthesis of histidine-containing peptides, are poor.     

Pharmakogenetik der oralen Antikoagulation mit Cumarinen

The recent identification of VKORC1 has made important contributions to our understanding of the vitamin K cycle. The VKORC1 enzyme was shown to be the molecular target of coumarin drugs. Mutations and polymorphisms in coding and noncoding regions of the VKORC1 gene have been shown to cause both a partial to total coumarin resistance and coumarin sensitivity. Availability of molecular diagnostics (VKORC1, CYP2C9) and drug monitoring by HCPLC (determination of coumarin, vitamin K, and vitamin K epoxide levels) is helpful for detecting hereditary and acquired factors influencing coumarin therapy. In the future, these tools may be instrumental in designing individualized oral anticoagulation therapy regimens.