Figure 1. Hypertriglyceridemia in fasted Col18a1^−/− mice.

(A) Retro-orbital sinus blood was taken from wild-type and Col18a1^−/− mice after fasting the animals for 4 hrs in the morning. Total plasma triglycerides were compared between mutant and wild-type (n = 10 mice, unpaired t-test, P<0.0001) and results were repeated. (B) Plasma cholesterol was analyzed in the same samples. (C) Triglyceride distribution across plasma lipoprotein subclasses. Plasma was pooled from 4 mice of each genotype and triglycerides in lipoprotein size classes were determined by LipoSEARCH FPLC. Control mice, filled bars; Col18a1^−/− mice, open bars. Chylomicron (CM) >80 nm particle diameter; VLDL, 30–80 nm; LDL, 16–30 nm; and HDL, 8–16 nm. The experiment was performed twice and the error bars represent the range in the recovery of triglycerides in the two experiments. (D) Equal volumes of lipoproteins of δ<1.006 g/ml (pooled from n = 3 mice of each genotype) were analyzed by negative staining transmission electron microscopy (inset, 20,000× magnification, bar = 50 nm). The diameters of particles present in eight fields were analyzed in a blinded fashion. The experiment was repeated twice with comparable results. (E). The fraction of fasting lipoproteins of δ<1.006 g/ml from Col18a1 mutants contained >4-fold more triglyceride than in wild type. The experiment was performed on pooled plasma samples from n = 4 mice and repeated three times. (F) Equal volumes of δ<1.006 g/ml lipoproteins were concentrated by membrane filtration and apolipoproteins were resolved by gradient SDS-PAGE, then visualized by Western blotting with rabbit anti-mouse apolipoprotein B-48/B-100 and apolipoprotein E (apoE) polyclonal antibodies.