title: Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.
abstract: This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 μg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation>90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages.
authors.affiliation: Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Xueting
authors.initials: X
authors.lastname: Jin
authors.affiliation: Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.affiliation: BioDtech Inc., Birmingham, AL 35209, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Champion
authors.affiliation: Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: kruthh@nhlbi.nih.gov.
authors.forename: Howard S
authors.initials: HS
authors.lastname: Kruth
mesh_terms: D016632:Apolipoprotein A-I / Q000032:analysis / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004340:Drug Contamination; D004731:Endotoxins / Q000032:analysis / Q000494:pharmacology*; D006801:Humans; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects*
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D004731:Endotoxins
keywords: Apolipoproteins; Atherosclerosis; Cell proliferation; Endotoxin; Macrophages