A fast semi-quantitative LC-MS method for measurement of intact apolipoprotein A-I reveals novel proteoforms in serum.

BACKGROUND: Surrogate markers for reverse cholesterol transport (RCT) efficiency such as HDL cholesterol and immune methods for apolipoprotein A-I (ApoA-I) may not fully reflect the actual efficiency of the RCT pathway. Several genetic variants and different posttranslational proteoforms of ApoA-I may unevenly affect the functionality of the HDL particle to efflux cholesterol. A method employing top-down immunoaffinity LC-MS of ApoA-I in order to characterize the most prevalent ApoA-I proteoforms in human plasma is described. METHODS: Diluted plasma was directly injected into a 2D LC-MS system consisting of an affinity column and an analytical column. Enriched ApoA-I fractions were introduced into the MS and intact or fragmented ApoA-I was analyzed. RESULTS: ApoA-I as detected by the described LC-MS method distributes into at least 14 major potential proteoforms exceeding detection limit in human plasma. Substantial amounts of ApoA-I in plasma were found to occur as truncated, oxidized, glycated and glycosylated proteoforms. Levels of glycated ApoA-I distinguished significantly diabetic from non-diabetic samples. In addition novel truncated and glycosylated proteoforms were detected. CONCLUSIONS: ApoA-I proteoforms measured by LC-MS represent a useful approach to augment the clinical picture of ApoA-I and its function in health and disease.

Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7 alpha1,3-fucosyltransferase gene.

The alpha1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLe(x)) on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct produced a Fuc-TVII enzyme with impaired activity compared with the wildtype enzyme. Polymorphonuclear granulocytes from an individual carrying this mutation homozygously also showed a reduced expression of SLe(x). In the present study, we have established Epstein-Barr virus-transformed B-cell lines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B-cell origin by flow cytometry analysis. IWO cells interacted with E-selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell was transiently transfected with wildtype FUT7 cDNA, interaction with E-selectin could be restored. Cell surface expression of the SLe(x)-related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared with that on SIGN cells, consistent with a role of these antigens in E-selectin recognition. These cell lines will be useful in further characterization of E-selectin ligands and encourage further studies on the consequences of the FUT7-G329A mutation in vivo.

Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene.

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

The radiological exposure of man from radioactivity in the Baltic Sea.

A radiological assessment has been carried out considering discharges of radioactivity to the Baltic Sea marine environment since 1950. The sources of radioactivity that have been evaluated are atmospheric nuclear-weapons fallout, fallout from the Chernobyl accident in 1986, discharges of radionuclides from Sellafield and La Hague transported into the Baltic Sea, and discharges of radionuclides from nuclear installations located in the Baltic Sea area. Dose rates from man-made radioactivity to individual members of the public (critical groups) have been calculated based on annual intake of seafood and beach occupancy time. The dose rates to individuals from the regions of the Bothnian Sea and Gulf of Finland are predicted to be larger than from any other area in the Baltic Sea due to the pattern of Chernobyl fallout. The dose rates are predicted to have peaked in 1986 at a value of 0.2 mSv year-1. Collective committed doses to members of the public have been calculated based on fishery statistics and predicted concentrations of radionuclides in biota and coastal sediments. The total collective dose from man-made radioactivity in the Baltic Sea is estimated at 2600 manSv, of which approximately two-thirds originate from Chernobyl fallout, approximately one-quarter from atmospheric nuclear-weapons fallout, approximately 8% from European reprocessing facilities, and approximately 0.04% from nuclear installations bordering the Baltic Sea area. An assessment of small-scale dumping of low-level radioactive waste in the Baltic Sea in the 1960s by Sweden and the Soviet Union has showed that doses to man from these activities are negligible. Dose rates and doses from natural radioactivity dominate except for the year 1986 where dose rates to individuals from Chernobyl fallout in some regions of the Baltic Sea approached those from natural radioactivity.