True digestibility of tryptophan in plant and animal protein.

BACKGROUND: Protein quality, evaluated using Digestible Indispensable Amino Acid Score (DIAAS) requires ileal digestibility values of individual indispensable amino acids (IAA) in each protein. However, true tryptophan (Trp) digestibility has rarely been quantified in humans. OBJECTIVE: To measure the true Trp digestibility and DIAAS of 2H-intrinsically labelled plant and animal protein sources in humans, using the dual isotope tracer technique. METHODS: The true Trp digestibility of 2H intrinsically labelled plant proteins such as whole mung bean (n=6) and dehulled mung bean (n=6), chickpea (n=5), and yellow pea (n=5), and protein from animal source foods such as egg white (n=6), whole egg (n=6), chicken meat (n=6) and goat milk (n=7) was determined against the known digestibility of U-13C spirulina whole cell protein as reference, except for goat milk protein which was measured against free crystalline 13C-Trp as reference. Banked samples from earlier studies conducted to determine true IAA digestibility of different protein sources were used for the analysis. DIAAS was calculated for each test protein using digestibility corrected IAA scores (mg IAA/g of protein) in comparison to the IAA requirement score for adults. RESULTS: The true Trp digestibility of whole mung bean, dehulled mung bean, chickpea, yellow pea, egg white, whole egg, chicken meat, and goat milk were 67.6±3.7%, 74.5±4.4%, 72.6±2.3%, 72.5±2.2%, 89.7±2.5%, 91.4±2.6%, 95.9±2.2%, and 92.8±2.9% respectively. The true Trp digestibility of plant protein sources was significantly lower than that of animal protein sources (p<0.05). Trp was not a limiting IAA in all the tested proteins. CONCLUSION: The true Trp digestibility determined in the present study ranged from 67.6±3.7% to 95.9 ± 2.2% for whole mung bean and chicken meat respectively, and adds to the database of individual true IAA digestibility of different protein sources. This study was registered in Clinical Trials Registry of India (CTRI) with registration number: CTRI/2017/11/010468, CTRI/2020/04/024512, CTRI/2018/03/012265.

Untargeted Maternal Plasma Metabolomics in Hirschsprung Disease: A Pilot Study.

Background and Aims: Hirschsprung disease (HSCR) is a congenital disorder of unknown etiology affecting the enteric nervous system (ENS). Since the early gestational development of the ENS is dependent on the prenatal maternal metabolic environment, the objective of this pilot study was to explore the role of specific maternal plasma metabolites in the etiology of HSCR. Methods: In this cross-sectional study, postnatal (as a surrogate for prenatal) plasma samples were obtained from mothers of children diagnosed with HSCR (n = 7) and age-matched mothers of normal children (n = 6). The plasma metabolome was analyzed by ultra-high-pressure liquid chromatography and mass spectrometry. Metabolites were identified by mzCloud using Compound Discoverer software. Using an untargeted metabolomics workflow, metabolites with case versus control group differences were identified. Results: A total of 268 unique plasma metabolites were identified and annotated in maternal plasma. Of these, 57 were significantly different between case and control groups (P < 0.05, t-test). Using a false discovery rate corrected cutoff of 10% to adjust for multiple comparisons, 19 metabolites were significantly different in HSCR cases, including carnitines, medium-chain fatty acids, and glutamic acid. Pathways affected were for amino acid and lipid metabolism. Conclusion: Disordered prenatal metabolic pathways may be involved in the etiopathogenesis of HSCR in the developing fetus. This is the first study to assess maternal plasma metabolomics in HSCR.

Bioavailability and daily requirement of vitamin B12 in adult humans: an observational study of its colonic absorption and daily excretion as measured by [13C]-cyanocobalamin kinetics.

BACKGROUND: Clinical and biochemical vitamin B12 (B12) deficiency is lower than anticipated in vegetarians. Extraileal absorption, such as from the colon, as well as reduced daily excretion, may be adaptive mechanisms to maintain B12 homeostasis with marginal intakes. OBJECTIVE: To measure the absorption of B12 from the small and large intestine, and its daily rate of excretion from the body, using a [13C]-cyanocobalamin tracer. METHODS: Oral B12 bioavailability was measured over 12 h after administration of [13C]-cyanocobalamin tracer (2.5 µg) in normal participants. The colonic B12 bioavailability was evaluated by direct instillation of [13C]-cyanocobalamin (5 µg) into the ascending colon. Bioavailability was calculated from 2-compartmental modeling of the tracer appearance in plasma. The excretion rate of B12 was measured from [13C]-cyanocobalamin elimination from the body over 4 wk after oral dosing (5 µg). RESULTS: The oral B12 bioavailability (n = 11) was 63% ± 10% measured over 12 h. A late absorption peak, accounting for 12% of the absorption, was observed after an average lag time of 8.7 h from dosing. The colonic B12 bioavailability (n = 10) was 7% ± 5% over 4 h. The daily B12 excretion rate (n = 4) was 0.7 ± 0.2 µg/d. The minimum daily requirement of B12 in these participants was derived at 1 µg /d. CONCLUSIONS: B12 is absorbed in the human colon. This observation confirms the potential contribution of the colon in daily B12 nutriture, and along with a possible lower requirement, could explain the absence of clinical deficiency in populations with marginal B12 intakes. TRIAL REGISTRATION NUMBER: This study was registered in Clinical Trials Registry of India (CTRI) with the registration number CTRI/2018/04/012957, available from https://ctri.nic.in/Clinicaltrials/showallp.php?mid1=49319&EncHid=&userName=029108.

Variability of human fasted venous plasma metabolomic profiles with tourniquet induced hemostasis.

Venous plasma metabolomics is a potent and highly sensitive tool for identifying and measuring metabolites of interest in human health and disease. Accurate and reproducible insights from such metabolomic studies require extreme care in removing preanalytical confounders; one of these is the duration of tourniquet application when drawing the venous blood sample. Using an untargeted plasma metabolomics approach, we evaluated the effect of varying durations of tourniquet application on the variability in plasma metabolite concentrations in five healthy female subjects. Tourniquet application introduced appreciable variation in the metabolite abundances: 73% of the identified metabolites had higher temporal variation compared to interindividual variation [Intra-Class Correlation (ICC) > 0.50]. As such, we recommend tourniquet application for minimal duration and to wait for 5 min with the needle in situ after removing the tourniquet, to reduce hemostasis-induced variability and false flags in interpretation.

Measuring vitamin B-12 bioavailability with [13C]-cyanocobalamin in humans.

BACKGROUND: Vitamin B-12 deficiency is widespread in many parts of the world, affecting all age groups and increasing with age. It is primarily due to a low intake of animal source foods or malabsorption. The measurement of bioavailability of vitamin B-12 is etiologically important in deficiency but is limited due to the use of radioactive isotopes like [57Co]- or [14C]-cyanocobalamin. OBJECTIVES: The aim of this study was to measure the bioavailability of [13C]-cyanocobalamin in humans and to assess the effect of parenteral replenishment of vitamin B-12 on the bioavailability. METHODS: We synthesized a stable isotope-labeled vitamin B-12, [13C]-cyanocobalamin, using Salmonella enterica by providing [13C2]-ethanolamine as a sole carbon source. After purification and mass spectrometry-based characterization, its oral bioavailability was measured in the fasted state with high and low oral doses, before and after parenteral replenishment of vitamin B-12 stores, from the kinetics of its plasma appearance in a 2-compartment model. RESULTS: [13C]-cyanocobalamin was completely decyanated to [13C]-methylcobalamin describing metabolic utilization, and its plasma appearance showed early and late absorption phases. At a low dose of 2.3 µg, the mean bioavailability was 46.2 ± 12.8 (%, mean ± SD, n = 11). At a higher dose of 18.3 µg, the mean bioavailability was 7.6 ± 1.7 (%, mean ± SD, n = 4). Parenteral replenishment of the vitamin B-12 store in deficient individuals prior to the measurement resulted in a 1.9-fold increase in bioavailability. CONCLUSIONS: Vitamin B-12 bioavailability is dose dependent and at a low dose that approximates the normal daily requirement (46%). The stable isotope method described here could be used to define the etiology of deficiency and to inform the dietary requirement in different physiologic states as well as the dose required for supplementation and food fortification. This trial was registered at the Clinical Trials Registry of India as CTRI/2018/04/012957.

Acyl ethanolamides in Diabetes and Diabetic Nephropathy: Novel targets from untargeted plasma metabolomic profiles of South Asian Indian men.

The pathophysiology of diabetic nephropathy (DN) in type 2 diabetes (T2D) patients is minimally understood. We compared untargeted high-resolution accurate mass (HRAM) orbitrap-based plasma metabolomic profiles of 31 T2D-DN (with estimated glomerular filtration rate ≤80 mL/min/1.73 m2), 29 T2D and 30 normal glucose tolerance (NGT) Indian men. Of the 939 plasma metabolites that were differentially abundant amongst the NGT, T2D and T2D-DN (ANOVA, False Discovery Rate - FDR adjusted p-value < 0.05), 48 were associated with T2D irrespective of the renal function of the subjects. Acyl ethanolamides and acetylcholine were decreased while monoacylglycerols (MAGs) and cortisol were elevated in both T2D and T2D-DN. Sixteen metabolites, including amino acid metabolites Imidazolelactate and N-Acetylornithine, changed significantly between NGT, T2D and T2D-DN. 192 metabolites were specifically dysregulated in T2D-DN (ratio ≥2 or ≤0.5 between T2D-DN and T2D, similar abundance in NGT and T2D). These included increased levels of multiple acylcarnitine and amino acid metabolites. We observed a significant dysregulation of amino acid and fatty acid metabolism in South Asian Indian male T2D-DN subjects. Unique to this study, we report a reduction in acyl ethanolamide levels in both T2D and T2D-DN males. Those with dysregulation in acyl ethanolamides, which are endogenous agonists of GPR119, are likely to exhibit improved glycemic control with GPR119 agonists.