The Childhood Acute Illness and Nutrition (CHAIN) network nested case-cohort study protocol: a multi-omics approach to understanding mortality among children in sub-Saharan Africa and South Asia

Introduction: Many acutely ill children in low- and middle-income settings have a high risk of mortality both during and after hospitalisation despite guideline-based care. Understanding the biological mechanisms underpinning mortality may suggest optimal pathways to target for interventions to further reduce mortality. The Childhood Acute Illness and Nutrition (CHAIN) Network ( www.chainnnetwork.org) Nested Case-Cohort Study (CNCC) aims to investigate biological mechanisms leading to inpatient and post-discharge mortality through an integrated multi-omic approach. Methods and analysis; The CNCC comprises a subset of participants from the CHAIN cohort (1278/3101 hospitalised participants, including 350 children who died and 658 survivors, and 270/1140 well community children of similar age and household location) from nine sites in six countries across sub-Saharan Africa and South Asia. Systemic proteome, metabolome, lipidome, lipopolysaccharides, haemoglobin variants, toxins, pathogens, intestinal microbiome and biomarkers of enteropathy will be determined. Computational systems biology analysis will include machine learning and multivariate predictive modelling with stacked generalization approaches accounting for the different characteristics of each biological modality. This systems approach is anticipated to yield mechanistic insights, show interactions and behaviours of the components of biological entities, and help develop interventions to reduce mortality among acutely ill children. Ethics and dissemination. The CHAIN Network cohort and CNCC was approved by institutional review boards of all partner sites. Results will be published in open access, peer reviewed scientific journals and presented to academic and policy stakeholders. Data will be made publicly available, including uploading to recognised omics databases. Trial registration NCT03208725

Adiponectin levels correlate with the severity of hypertriglyceridaemia in glycogen storage disease Ia

Glycogen storage disease type Ia (GSD Ia) is characterized by severe hypercholesterolaemia and hypertriglyceridaemia. Little is known about the aetiology of the hyperlipidaemia in GSD Ia. Adipokines play an important regulatory role in lipid metabolism. We investigated whether adipokine concentrations were correlated with the degree of hyperlipidaemia in GSD Ia patients. Six patients with GSD Ia were studied in semi-fasted conditions. Adiponectin, but not leptin, correlated (r(2) = -0.79, p = 0.02) with plasma triglyceride concentrations in the GSD Ia patients. Leptin correlated well with BMI (r(2) = 0.59, p <0.01). However, neither body mass index (BMI) nor homeostasis model assessment (HOMA), as a marker of insulin sensitivity, correlated with triglyceride concentrations. Although a small number of patients were studied, these results indicate that adiponectin concentrations are correlated with the degree of hypertriglyceridaemia in GSD Ia. Pharmacological treatment aimed at increasing adiponectin levels might improve the metabolic status of these patients

Contribution of newly synthesized cholesterol to rat plasma and bile determined by mass isotopomer distribution analysis: bile-salt flux promotes secretion of newly synthesized cholesterol into bile.

To quantify the contribution of newly synthesized cholesterol to total plasma and biliary cholesterol under physiological conditions, unrestrained rats were infused intravenously with [1-13C]acetate (0. 6mmol/h per kg) from 12:00 to 24:00 h, and fractional and absolute cholesterol-synthesis rates were determined by mass isotopomer distribution analysis (MIDA). As bile diversion leads to changes in cholesterol metabolism, rats were equipped with permanent catheters in the bile duct and duodenum, allowing sampling of small amounts of bile from an intact enterohepatic circulation. For comparison, rats with chronic bile diversion were also studied. Fractional synthesis of plasma cholesterol was 10.8+/-1.7% (mean+/-S.D.) after 12 h in rats with intact circulation. Fractional synthesis of biliary cholesterol was significantly higher than that of plasma cholesterol, i.e. 16.5+/-2.0% (P<0.05) after 12 h. In contrast, no differences between fractional synthesis of cholesterol in plasma and bile were found in bile-diverted animals (31.8+/-2.1 and 33.1+/-3.3% respectively after 12 h). The calculated absolute rate of cholesterol biosynthesis increased from 53+/-10 to 221+/-19 micromol/day per kg after bile diversion. A comparison of MIDA results with those obtained from balance studies indicated that MIDA does not assess total body synthesis in rats, presumably because of incomplete equilibration of newly synthesized molecules with cholesterol in the plasma compartment. These studies demonstrate that the contribution of newly synthesized cholesterol to biliary cholesterol is higher than to plasma cholesterol under physiological conditions, probably reflecting bile-salt-induced secretion of newly formed cholesterol by the periportal hepatocytes

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis

A new stable isotope procedure has been developed and validated in rats, applying [1-C-13]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-C-13]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t = 14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes. (C) 2000 Elsevier Science B.V. All rights reserved

Acute inhibition of glucose-6-phosphate translocator activity leads to increased de novo lipogenesis and development of hepatic steatosis without affecting VLDL production in rats

Glucose-6-phosphatase (G6Pase) is a key enzyme in hepatic glucose metabolism. Altered G6Pase activity in glycogen storage disease and diabetic states is associated with disturbances in lipid metabolism. We studied the effects of acute inhibition of G6Pase activity on hepatic lipid metabolism in nonanesthetized rats. Rats were infused with an inhibitor of the glucose-6-phosphate (G6P) translocator (S4048, 30 mg . kg(-1) . h(-1)) for 8 h. Simultaneously, [1-C-13]acetate was administered for determination of de novo lipogenesis and fractional cholesterol synthesis rates by mass isotopomer distribution analysis. In a separate group of rats, Triton WR 1339 was injected for determination of hepatic VLDL-triglyceride production. S4048 infusion significantly decreased plasma glucose (-11%) and insulin (-48%) levels and increased hepatic G6P (201%) and glycogen (182%) contents. Hepatic triglyceride contents increased from 5.8 +/- 1.4 mu mol/g liver in controls to 20.6 +/- 5.5 mu mol/g liver in S4048-treated animals. De novo lipogenesis was increased > 10-fold in S4048-treated rats, without changes in cholesterol synthesis rates. Hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were markedly induced. Plasma triglyceride levels increased fourfold, but no differences in plasma cholesterol levels were seen. Surprisingly, hepatic VLDL-triglyceride secretion was not increased in S4048-treated rats. These studies demonstrate that inhibition of the G6Pase system leads to acute stimulation of fat synthesis and development of hepatic steatosis, without affecting hepatic cholesterol synthesis and VLDL secretion. The results emphasize the strong interactions that exist between hepatic carbohydrate and fat metabolism

Acute inhibition of glucose-6-phosphate translocator activity leads to increased de novo lipogenesis and development of hepatic steatosis without affecting VLDL production in rats

Glucose-6-phosphatase (G6Pase) is a key enzyme in hepatic glucose metabolism. Altered G6Pase activity in glycogen storage disease and diabetic states is associated with disturbances in lipid metabolism. We studied the effects of acute inhibition of G6Pase activity on hepatic lipid metabolism in nonanesthetized rats. Rats were infused with an inhibitor of the glucose-6-phosphate (G6P) translocator (S4048, 30 mg . kg(-1) . h(-1)) for 8 h. Simultaneously, [1-C-13]acetate was administered for determination of de novo lipogenesis and fractional cholesterol synthesis rates by mass isotopomer distribution analysis. In a separate group of rats, Triton WR 1339 was injected for determination of hepatic VLDL-triglyceride production. S4048 infusion significantly decreased plasma glucose (-11%) and insulin (-48%) levels and increased hepatic G6P (201%) and glycogen (182%) contents. Hepatic triglyceride contents increased from 5.8 +/- 1.4 mu mol/g liver in controls to 20.6 +/- 5.5 mu mol/g liver in S4048-treated animals. De novo lipogenesis was increased > 10-fold in S4048-treated rats, without changes in cholesterol synthesis rates. Hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were markedly induced. Plasma triglyceride levels increased fourfold, but no differences in plasma cholesterol levels were seen. Surprisingly, hepatic VLDL-triglyceride secretion was not increased in S4048-treated rats. These studies demonstrate that inhibition of the G6Pase system leads to acute stimulation of fat synthesis and development of hepatic steatosis, without affecting hepatic cholesterol synthesis and VLDL secretion. The results emphasize the strong interactions that exist between hepatic carbohydrate and fat metabolism

Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique:application to rodents and humans

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in trace, rats, and humans. Decay of administered [2,2,4,4-H-2(4)]CA enrichment was measured in time in 50-mul plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 mu mol/ 100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 mu mol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics