Instability of Acylcarnitines in Stored Dried Blood Spots: The Impact on Retrospective Analysis of Biomarkers for Inborn Errors of Metabolism.

Stored dried blood spots (DBS) can provide valuable samples for the retrospective diagnosis of inborn errors of metabolism, and for validation studies for newborn blood spot screening programs. Acylcarnitine species are subject to degradation upon long-term storage at room temperature, but limited data are available on the stability in original samples and the impact on acylcarnitine ratios. We analysed complete acylcarnitine profiles by flow-injection tandem mass spectrometry in 598 anonymous DBS stored from 2013 to 2017, at +4 °C during the first year and thereafter at room temperature. The concentrations of C2-, C3-, C4-, C5-, C6-, C8-, C10:1-, C10-, C12:1-, C12-, C14:1-, C14-, C16:1-, C16-, C18:2-, C18:1-, C18-, C5OH+C4DC-, C18:1OH-, and C16DC-carnitine decreased significantly, whereas a positive trend was found for free carnitine. Only the C4/C8-, C8/C10-, C14:1/C10- and C14:1/C16-carnitine ratios appeared robust for the metabolite instability. The metabolite instability may provoke the wrong interpretation of test results in the case of retrospective studies and risk the inaccurate estimation of cut-off targets in validation studies when only stored control DBS are used. We recommend including control DBS in diagnostic, retrospective cohort studies, and, for validation studies, we recommend using fresh samples and repeatedly re-evaluating cut-off targets.

Regulation of bile acid synthesis by the nuclear receptor Rev-erbalpha.

BACKGROUND & AIMS: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbalpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbalpha in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. METHODS: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbalpha. RESULTS: Rev-erbalpha-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbalpha-deficient mice, whereas adenovirus-mediated hepatic Rev-erbalpha overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbalpha-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbalpha-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbalpha target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbalpha-deficient mice. CONCLUSIONS: These data identify a role for Rev-erbalpha in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression.

Secretory phospholipase A2 increases SR-BI-mediated selective uptake from HDL but not biliary cholesterol secretion.

High density lipoprotein cholesterol represents a major source of biliary cholesterol. Secretory phospholipase A2 (sPLA2) is an acute phase enzyme mediating decreased plasma HDL cholesterol levels. Clinical studies reported a link between increased sPLA2 expression and the presence of cholesterol gallstones. The aim of our study was to investigate whether the overexpression of human sPLA2 in transgenic mice affects biliary cholesterol secretion and gallstone formation. Liver weight (P < 0.01) and hepatic cholesterol content (P < 0.01) were significantly increased in sPLA2 transgenic mice compared with controls as a result of increased scavenger receptor class B type I (SR-BI)-mediated hepatic selective uptake of HDL cholesterol (P < 0.01), whereas hepatic SR-BI expression remained unchanged. However, biliary cholesterol secretion as well as fecal neutral sterol and fecal bile salt excretion remained unchanged in sPLA2 transgenic mice. Furthermore, gallstone prevalence in response to a lithogenic diet was identical in both groups. These data demonstrate that i) increased flux of cholesterol from HDL into the liver via SR-BI as a result of phospholipase modification of the HDL particle translates neither into increased biliary and fecal sterol output nor into increased gallstone formation, and ii) increased sPLA2 expression in patients with cholesterol gallstones might be a consequence rather than the underlying cause of the disease.

Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice.

Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 +/- 7.2 vs. 12.1 +/- 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 +/- 13 vs. 176 +/- 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 +/- 1.0 vs. 14.3 +/- 1.4 ml x kg(-1) x min(-1), P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.

Ventricular fibrillation without overt cardiomyopathy as first presentation of organic cation transporter 2-deficiency in adolescence.

This case report describes ventricular fibrillation without overt cardiomyopathy as the presenting symptom of primary carnitine deficiency due to organic cation transporter 2 (OCTN2)-deficiency in a 15-year-old girl. Normally this disease presents early in life with hypoketotic hypoglycemia, muscle weakness, and/or cardiomyopathy. The patient fully recovered after carnitine supplementation. Recognition of this disease is important because its treatment is easy and effective.

Hepatic VLDL production in ob/ob mice is not stimulated by massive de novo lipogenesis but is less sensitive to the suppressive effects of insulin.

Type 2 diabetes in humans is associated with increased de novo lipogenesis (DNL), increased fatty acid (FA) fluxes, decreased FA oxidation, and hepatic steatosis. In this condition, VLDL production is increased and resistant to suppressive effects of insulin. The relationships between hepatic FA metabolism, steatosis, and VLDL production are incompletely understood. We investigated VLDL-triglyceride and -apolipoprotein (apo)-B production in relation to DNL and insulin sensitivity in female ob/ob mice. Hepatic triglyceride (5-fold) and cholesteryl ester (15-fold) contents were increased in ob/ob mice compared with lean controls. Hepatic DNL was increased approximately 10-fold in ob/ob mice, whereas hepatic cholesterol synthesis was not affected. Basal rates of hepatic VLDL-triglyceride and -apoB100 production were similar between the groups. Hyperinsulinemic clamping reduced VLDL-triglyceride and -apoB100 production rates by approximately 60% and approximately 75%, respectively, in lean mice but only by approximately 20% and approximately 20%, respectively, in ob/ob mice. No differences in hepatic expression of genes encoding apoB and microsomal triglyceride transfer protein were found. Hepatic expression and protein phosphorylation of insulin receptor and insulin receptor substrate isoforms were reduced in ob/ob mice. Thus, strongly induced hepatic DNL is not associated with increased VLDL production in ob/ob mice, possibly related to differential hepatic zonation of apoB synthesis (periportal) and lipid accumulation (perivenous) and/or relatively low rates of cholesterogenesis. Insulin is unable to effectively suppress VLDL-triglyceride production in ob/ob mice, presumably because of impaired insulin signaling.

Stimulation of lipogenesis by pharmacological activation of the liver X receptor leads to production of large, triglyceride-rich very low density lipoprotein particles.

The oxysterol-activated liver X receptor (LXR) provides a link between sterol and fatty acid metabolism; activation of LXR induces transcription of lipogenic genes. This study shows that induction of the lipogenic genes Srebp-1c, Fas, and Acc1 upon administration of the synthetic LXR agonist T0901317 to C57BL/6J mice (10 mg/kg/day, 4 days) is associated with massive hepatic steatosis along the entire liver lobule and a 2.5-fold increase in very low density lipoprotein-triglyceride (VLDL-TG) secretion. The increased VLDL-TG secretion was fully accounted for by formation of larger (129 +/- 9 nm versus 94 +/- 12 nm, a 2.5-fold increase of particle volume) TG-rich particles. Stimulation of VLDL-TG secretion did not lead to elevated plasma TG levels in C57BL/6J mice, indicating efficient particle metabolism and clearance. However, T0901317 treatment did lead to severe hypertriglyceridemia in mouse models of defective TG-rich lipoprotein clearance, i.e. APOE*3-Leiden transgenic mice (3.2-fold increase) and apoE-/- LDLr-/- double knockouts (12-fold increase). Incubation of rat hepatoma McA-RH7777 cells with T0901317 also resulted in intracellular TG accumulation and enhanced TG secretion. We conclude that, in addition to raising high density lipoprotein cholesterol concentrations, pharmacological LXR activation in mice leads to development of hepatic steatosis and secretion of atherogenic, large TG-rich VLDL particles.