The absorption of gut-microbe-produced-B-vitamins-in humans by oral administration of 13C cellulose

Indians in general are thought to have monotonous and predominantly cereal-based vegetarian diets with very little milk intake. In consequence, their intake of micronutrients, particularly the B vitamins, is low. Taking B12 as an example, the prevalence of its deficiency should be extraordinarily high, considering that the median daily population intake is only 1.2 µg/day1 compared to the stated average requirement (AR) of 2 µg/day.2 Yet, a recent national survey of 1-19y Indian children found just ~20-30% to be biochemically deficient, with no observed clinical manifestations. This has been replicated in a recent DBT-funded national survey of Indian adolescents and adults in 8 states, where <30% of the population were found to be deficient.4

A possible explanation of this apparent paradox is that in vivo intestinal microbial nutrient synthesis contributes to human micronutrient homeostasis. If this were indeed true – two important criteria need to be met. i) Is the human gut microbiome capable of producing B vitamins and nutrients ii) can these nutrients be adequately absorbed from the colon, where maximal synthesis may be expected to take place

Objective: To determine whether colonic microbiota can synthesize vitamins for human host absorption by providing 13C labelled cellulose as substrate

Methodology: Subject will report to the 3rd floor division of nutrition at 7 am. Sociodemographic details and relevant medical history will be documented. The bodyweight will be measured to the nearest 0.01 kg using calibrated digital weighing scale (Goldtech, AE038, New Delhi, India). Height will be measured using Seca stadiometer to the nearest 0.1 cm. All measurements will be in triplicates and mean of 3 readings will be considered. In the preceding week before the study, the subject will be instructed not to make any drastic changes in his/her diet.Baseline blood sample (8 ml) will be collected in a fasting state. 0.5 g – 4g of U- 13C Cellulose (U-10508, 97% purity, Isolife, Netherlands) will be administered as a suspension in 100 ml water or within a sandwich of white bread and butter. The subject will be allowed to eat his/her regular diet and resume regular activity after dose administration and report again after 24 hours and subsequently as per blood sampling protocol

 Serial blood samples will be processed for respective vitamin measurements on a high-resolution analytical platform consisting of a Vanquish Flex Binary UHPLC coupled to a mass spectrometer (Q Exactive, LC-HRAM-MS; Thermo Scientific) with a heated electrospray ionization (HESI-II) probe. Separation of the metabolites will be achieved by using a Hypersil Gold aQ column (100 × 2.1 × 1.9 μm; Thermo Scientific) in a reverse-phase gradient. The pooled samples will also be injected periodically throughout the runs for QC check.

The Q Exactive mass spectrometer will be operated under electrospray ionization (HESI-II) positive and negative polarity mode in full scan (m/z 67–1000) and resolution set to 70,000 (FWHM) at m/z 200, with automatic gain control (AGC) target of 1×106 ions and a maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS profiles will be acquired on a “Top5” data-dependent mode using the following parameters: resolution 35,000; AGC 1×105 ions; maximum IT 50 ms; 1.0 amu isolation window; combined NCE 25%, 35% and 50% and dynamic exclusion time set at 10s; Spectrum data type, Centroid.

Compound Discoverer (CD) will be used to detect the labelled features and isotope incorporation analysis. The stable isotope labelling layout, using an unlabelled reference sample, detects unknown compounds above a specified minimum intensity threshold, determines their elemental composition and identity, and then determines the labelled counterparts (isotopologues) of these compounds in the samples marked as labelled. The output provides the Isotopologues Distribution Chart, Trend Chart, and Metabolic pathways associated with the labelled features including the relative exchange rate (RER) for each compound to identify features with 13C incorporation. Each sampling time point will be manually evaluated for potential isotopic shift of upto m+n isotopomers for 13C incorporation based on the precursor labelling and their metabolites.

Fecal samples will be obtained at baseline to characterize the gut microbiota for B vitamin production capacity and identify specific strains associated with the measured plasma appearance of different B vitamins. Fecal samples will also be obtained at end of study  for untargeted metabolomics of the fecal sample