Biotin
World-wide, millions of women enter preterm labor or have small newborns. Effective biomarkers are needed to identify women at risk for these adverse outcomes. A time and cost effective way to examine any potentially new biomarkers in samples collected during prior studies or trials that had been assayed for other metabolites would be highly useful. Thus, the current study aimed to determine if samples that had been previously thawed and re-frozen could be re-assayed for novel biomarkers, those being lipoprotein composition (sizing, proteome, lipids) and combined cholesterol and cytokine concentrations. Fasting blood was collected from 51 young non-pregnant women and plasma was analyzed for lipoprotein composition and cytokine concentrations after multiple freeze/thaw cycles in the cold or at room temperature and after being stored for 18 months. Plasma LDL-C, HDL-C, total cholesterol, and triglyceride concentrations decreased <6-7% (cholesterols) or <20% (triglyceride) after 7 thaws in the cold, 3 thaws at room temperature, and after 18 months of storage. As these decreases were less than day-to-day reported variation of lipids, they do not appear to be physiologically significant. Cytokine (IL-6, TNF a, IL-8, IL-1ß) and hsCRP concentrations decreased by 22%, 8%, 8%, 22%, and 35%, respectively; only IL-6, IL-1ß and hsCRP concentrations showed significant decreases greater than day-to-day variations of 20%. For measured triglyceride and cytokine, but not cholesterol concentrations, decreases with freeze/thaw cycles were greater when concentrations were elevated. Multiple thaws also led to changes in lipoprotein sizing, specifically to a shift from medium- and large-sized HDL particles to small-sized HDL particles and from large LDL to IDL. No changes occurred for VLDL particle numbers. Though particle sizes changed, the HDL proteome did not change with multiple thaw cycles or after long term storage. Overall, the results demonstrate that it is possible to use previously obtained frozen samples for plasma cholesterol and triglyceride levels and the lipoprotein proteome, and lipoprotein sizing and cytokine concentrations if one knows the history of the sample as changes should be relative to one another.
Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a 'humanized' serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic.
