Objective: The mechanism by which evinacumab, a fully human monoclonal antibody directed against ANGPTL3 (angiopoietin-like 3 protein) lowers plasma LDL (low-density lipoprotein) cholesterol levels in patients with homozygous familial hypercholesterolemia is unknown. We investigated apoB (apolipoprotein B) containing lipoprotein kinetic parameters in patients with homozygous familial hypercholesterolemia, before and after treatment with evinacumab. Approach and Results: Four patients with homozygous familial hypercholesterolemia underwent apoB kinetic analyses in 2 centers as part of a substudy of a trial evaluating the efficacy and safety of evinacumab in patients with homozygous familial hypercholesterolemia. The enrichment of apoB with the stable isotope (5,5,5- 2 H 3 )-Leucine was measured in VLDL (very LDL), IDL (intermediate-density lipoprotein), and LDL at different time points before and after intravenous administration of 15 mg/kg evinacumab. Evinacumab lowered LDL-cholesterol by 59±2% and increased IDL apoB and LDL apoB fractional catabolic rate in all 4 homozygous familial hypercholesterolemia subjects, by 616±504% and 113±14%, respectively. VLDL-apoB production rate decreased in 2 of the 4 subjects. Conclusions: In this small study, ANGPTL3 inhibition with evinacumab is associated with an increase in the fractional catabolic rate of IDL apoB and LDL apoB, suggesting that evinacumab lowers LDL-cholesterol predominantly by increasing apoB-containing lipoprotein clearance from the circulation. Additional studies are needed to unravel which factors are determinants in this biological pathway. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04722068.
BackgroundInfants undergoing cardiac surgery are at risk of a negative protein balance, due to increased proteolysis in response to surgery and the cardiopulmonary bypass circuit, and limited intake. The aim of the study was to quantify the effect on protein kinetics of a short-term high-protein (HP) diet in infants following cardiac surgery.MethodsIn a prospective, double-blinded, randomized trial we compared the effects of a HP (5 g · kg−1 · d−1) versus normal protein (NP, 2 g · kg−1 · d−1) enteral diet on protein kinetics in children <24 months, on day 2 following surgical repair of congenital heart disease. Valine kinetics and fractional albumin synthesis rate (FSRalb) were measured with mass spectrometry using [1-13C]valine infusion. The Mann–Whitney U test was used to investigate differences between group medians. Additionally, the Hodges-Lehmann procedure was used to create a confidence interval with a point estimate of median differences between groups.ResultsTwenty-eight children (median age 9 months, median weight 7 kg) participated in the study, of whom in only 20 subjects isotopic data could be used for final calculations. Due to underpowering of our study, we could not draw conclusions on the primary outcome parameters. We observed valine synthesis rate of 2.73 (range: 0.94 to 3.36) and 2.26 (1.85 to 2.73) μmol · kg−1 · min−1 in the HP and NP diet, respectively. The net valine balance was 0.54 (−0.73 to 1.75) and 0.24 (−0.20 to 0.63) μmol · kg−1 · min−1 in the HP and NP group. Between groups, there was no difference in FSRalb. We observed increased oxidation and BUN in the HP diet, compared to the NP diet, as a plausible explanation of the metabolic fate of surplus protein.ConclusionsIt is plausible that the surplus protein in the HP group has caused the increase of valine oxidation and ureagenesis, compared to the NP group. Because too few patients had completed the study, we were unable to draw conclusions on the effect of a HP diet on protein synthesis and balance. We present our results as new hypothesis generating data.Trial registrationDutch Trial Register NTR2334.
Key points Placental structure and function can be modified as a result of maternal obesity affecting materno‐fetal fatty acids (FA) transport. We report for the first time, in humans and in vivo, the kinetics of placental FA transfer in normo‐weight and in normolipemic obese pregnant women using stable isotopes. The administration of different tracer FA with similar behaviour to the mother at different time points allows the collection of kinetic information on materno‐fetal transfer of FA despite only one sample of placenta and cord can be collected per subject. Computational modelling showed a good fit to the data when considering all maternal plasma lipid classes but not when based only on non‐esterified FA. The novel approach using multiple tracer FA administration combined with computational modelling shows a consistent time course of placental tracer FA and predicted total FA accumulation. Abstract We analyse for the first time the in vivo materno‐fetal kinetic transfer of fatty acids (FA) labelled with stable isotopes in control and obese (OB) pregnant women. Labelled FA with a similar metabolism (stearic acid: 13C‐SA; palmitic acid: 13C‐PA; oleic acid: 13C‐OA) were orally administered at −4 h, −8 h and −12 h, respectively prior to elective caesarean section to 10 pregnant women with a body mass index >30 (OB) and 10 with a body mass index in the range 20–25 (NW). Placenta, venous and arterial cord blood were collected obtaining a wide range of FA enrichments. A combined experimental and computational modelling analysis was applied. FA fractional synthesis rate (FSR) in placenta was 11–12% h–1. No differences were observed between NW and normo‐lipidemic OB. It was not possible to estimate FA FSR in cord blood with this oral bolus dose approach. Computational modelling demonstrated a good fit to the data when all maternal plasma lipid classes were included but not with modelling based only on the non‐esterified FA fraction. The estimated materno‐fetal 13C‐FA transfer was ∼1%. In conclusion, our approach using multiple 13C‐FA tracers allowed us to estimated FSR in placental/maternal plasma but not in fetal/maternal compartments. Computational modelling showed a consistent time course of placental 13C‐FA transfer and predicted total fetal FA accumulation during the experiment. We conclude that, in addition to non‐esterified FA fraction in the maternal circulation, maternal plasma very low‐density lipoprotein and other lipoproteins are important contributors to placental FA transfer to the fetus.
BackgroundPrimary hyperoxaluria type 1 (PH1) is an inborn error of glyoxylate metabolism, characterized by increased endogenous oxalate production. The metabolic pathways underlying oxalate synthesis have not been fully elucidated, and upcoming therapies require more reliable outcome parameters than the currently used plasma oxalate levels and urinary oxalate excretion rates. We therefore developed a stable isotope infusion protocol to assess endogenous oxalate synthesis rate and the contribution of glycolate to both oxalate and glycine synthesis in vivo.MethodsEight healthy volunteers and eight patients with PH1 (stratified by pyridoxine responsiveness) underwent a combined primed continuous infusion of intravenous [1-13C]glycolate, [U-13C2]oxalate, and, in a subgroup, [D5]glycine. Isotopic enrichment of 13C-labeled oxalate and glycolate were measured using a new gas chromatography–tandem mass spectrometry (GC-MS/MS) method. Stable isotope dilution and incorporation calculations quantified rates of appearance and synthetic rates, respectively.ResultsTotal daily oxalate rates of appearance (mean [SD]) were 2.71 (0.54), 1.46 (0.23), and 0.79 (0.15) mmol/d in patients who were pyridoxine unresponsive, patients who were pyridoxine responsive, and controls, respectively (P=0.002). Mean (SD) contribution of glycolate to oxalate production was 47.3% (12.8) in patients and 1.3% (0.7) in controls. Using the incorporation of [1-13C]glycolate tracer in glycine revealed significant conversion of glycolate into glycine in pyridoxine responsive, but not in patients with PH1 who were pyridoxine unresponsive.ConclusionsThis stable isotope infusion protocol could evaluate efficacy of new therapies, investigate pyridoxine responsiveness, and serve as a tool to further explore glyoxylate metabolism in humans.
Primary hyperoxalurias (PH) are inborn errors of glyoxylate metabolism characterized by an increase in endogenous oxalate production. Oxalate overproduction may cause calcium-oxalate crystal formation leading to kidney stones, nephrocalcinosis, and ultimately kidney failure. Twenty-four hour urine oxalate excretion is an inaccurate measure for endogenous oxalate production in PH patients and not applicable in those with kidney failure. Treatment efficacy cannot be assessed with this measure during clinical trials. We describe the development and validation of a gas chromatography−tandem mass spectrometry method to analyze the samples obtained following a stable isotope infusion protocol of 13 C 2 -oxalate and 1-13 C-glycolate in both healthy individuals and PH patients. Isotopic enrichments of plasma oxalate, glycolate, and glyoxylate were measured on a gas chromatography−triple quadrupole mass spectrometry system using ethylhydroxylamine and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) for analyte derivatization. Method precision was good for oxalate and glycolate (coefficients of variation [CV] were <6.3% and <4.2% for inter-and intraday precision, respectively) and acceptable for glyoxylate (CV <18.3% and <6.7% for inter-and intraday precision, respectively). The enrichment curves were linear over the specified range. Sensitivity was sufficient to accurately analyze enrichments. This new method allowed calculation of kinetic features of these metabolites, thus enabling a detailed analysis of the various pathways involved in glyoxylate metabolism. The method will further enhance the investigation of the metabolic PH derangements, provides a tool to accurately assess the therapeutic efficacy of new promising therapeutic interventions for PH, and could serve as a clinical tool to improve personalized therapeutic strategies.
Background: High protein intake in early life is associated with an increased risk of childhood obesity. Dietary protein intake may be a key mechanistic modulator through alterations in endocrine and metabolic responses. Objective: We aimed to determine the impact of different protein intake of infants on blood metabolic and hormonal markers at the age of four months. We further aimed to investigate the association between these markers and anthropometric parameters and body composition until the age of two years. Design: Term infants received a modified low-protein formula (mLP) (1.7 g protein/100 kcal) or a specifically designed control formula (CTRL) (2.1 g protein/100 kcal) until 6 months of age in a double blinded RCT. The outcomes were compared with a breast-fed (BF) group. Glucose, insulin, leptin, IGF-1, IGF-BP1, -BP2, and -BP3 levels were measured at the age of 4 months. Anthropometric parameters and body composition were assessed until the age of 2 years. Groups were compared using linear regression analysis. Results: No significant differences were observed in any of the blood parameters between the formula groups (n = 53 mLP; n = 44 CTRL) despite a significant difference in protein intake. Insulin and HOMA-IR were higher in both formula groups compared to the BF group (n = 36) (p < 0.001). IGF-BP1 was lower in both formula groups compared to the BF group (p < 0.01). We found a lower IGF-BP2 level in the CTRL group compared to the BF group (p < 0.01) and a higher IGF-BP3 level in the mLP group compared to the BF group (p = 0.03). There were no significant differences in glucose, leptin, and IGF-1 between the three feeding groups. We found specific associations of all early-life metabolic and hormonal blood parameters with long-term growth and body composition except for IGF-1. Conclusions: Reducing protein intake by 20% did not result in a different metabolic profile in formula-fed infants at 4 months of age. Formula-fed infants had a lower insulin sensitivity compared to breast-fed infants. We found associations between all metabolic and hormonal markers (except for IGF-1) determined at age 4 months and growth and body composition up to two years of age.
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