Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin.

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.

Automated measurement of lipoprotein(a) by immunoturbidimetric analysis.

Immunoturbidimetric analysis of lipoprotein(a) in plasma or serum was developed for use on the Roche COBAS FARA II and COBAS MIRA clinical chemistry analyzers. The components of the assay are: (1) buffer consisting of 2.25% polyethylene glycol in phosphate-buffered saline, 0.2% gelatin, and a surfactant; (2) fractionated goat anti-human lipoprotein(a) IgG; (3) five standards with lipoprotein(a) concentrations ranging from 0.05 to 1.0 g/l; (4) two controls with concentrations of approximately 0.2 and 0.5 g/l. The analyzer delivers sample and buffer, incubates the reaction mixture at 37 degrees C for 5 min, delivers neat lipoprotein(a) antibody, and incubates for an additional 10 min. The lipoprotein(a) concentration of samples is calculated by the COBAS DENS (Data Evaluation for Non-linear Standard Curves) option by fitting the standard curve values to a four-parameter logit-log curve model. Total imprecision results (CV%) for the FARA II and MIRA were under 11% (NCCLS protocol EP5-T). The assay is linear beyond the highest calibrator to 2.6 g/l. No interference was observed for plasminogen up to 2.3 g/l, apolipoprotein B up to 4.36 g/l, hemoglobin up to 10 g/l, bilirubin up to 4.0 g/l, and triglycerides up to 4.36 g/l. Comparison with a double monoclonal ELISA used at the Northwest Lipid Research Laboratories yielded: R = 0.970, slope = 1.013, and y-intercept = 0.00009 (n = 37). Comparison with a commercially available ELISA kit for lipoprotein(a) yielded: r = 0.987, slope = 1.243, and y-intercept = 0.024 (n = 40). This assay provides rapid, accurate, and precise screening of lipoprotein(a) in serum or plasma.

Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer.

We report here the first radioimmunoassay for a vitamin D metabolite utilizing a radioiodinated tracer. Antibodies were generated in a goat immunized with the vitamin D analog 23, 24, 25, 26, 27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. The 125I-labeled tracer was prepared by reacting a 3-amino-propyl derivative of vitamin D-C(22)-amide with Bolton-Hunter reagent. The primary antiserum, used at a 15,000-fold final dilution, cross-reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols; the antiserum did not cross-react with dihydrotachysterol. Calibrators were prepared in vitamin D-stripped human serum. 25-Hydroxycholecalciferol was quantitatively extracted from serum or plasma (50 microL) with acetonitrile. The assay consists of a 90-min incubation at room temperature with primary antiserum, followed by a 20-min incubation with a second antiserum and separation of bound from free fractions by centrifugation. The detection limit of the assay was 2.8 micrograms/L for 25-hydroxycholecalciferol. Results with the present assay compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.