Table 1.

Overview of the stable isotope techniques used to support sustainable food systems

Method	Measures	Methodology
Dual tracer stable isotope technique(36)	Protein and amino acid digestibility of foods	Involves the simultaneous ingestion of two stable isotopically labelled proteins – intrinsically labelled test (2H or 15N) and standard (13C) proteins – added to a standardised meal The test protein is generated by incorporating 2H or 15N into plant, microbial or animal-based proteins during biosynthesis. Isotopically labelled plant proteins are produced by growing plants in the presence of deuterated water (²H2O) or 15N-labeled fertilisers, ensuring uniform protein labelling The standard protein should be a highly digestible protein whose true indispensable amino acid (IAA) digestibility is known. A standard protein commonly used is 13C-spirulina whole cells In a plateau-feeding protocol, postprandial blood samples are collected, and the true IAA digestibility is determined by comparing the steady-state plasma enrichment ratio of IAA from the test protein to that of the standard protein using mass spectrometry
Iron isotope dilution technique(84)	Iron absorption and loss	A stable isotope tracer of iron (57Fe) is administered orally and becomes incorporated into erythrocytes. As these labelled erythrocytes reach the end of their life span, they break down, releasing the tracer into the circulating body iron pool, where it gradually distributes across all tissues After approximately 12 months in adults, or 8 months in infants and children, the tracer is uniformly enriched throughout the body’s iron stores. At this point, any iron with a natural isotopic composition (56Fe) that is consumed via diet or supplements will dilute the isotopically enriched body iron, so that the 57Fe concentration decreases. The rate at which 57Fe decreases in concentration is proportional to iron absorption Any iron that is lost has the same isotopic composition as the total body iron; thus, iron lost from the body will reduce the amount of tracer in the body. Blood samples are collected over a duration of time to capture the change in the slope – depending on the study design – and are analysed using ICP-MS or TIMS
Deuterium oxide dose-to-mother technique(71)	Volume or quantity of breast milk consumed	Before dosing, the mother should be weighed in light clothing to the nearest 0·1 kg, and the baby should be weighed naked to the nearest 0·01 kg. Baseline saliva samples are then collected from both the mother and baby Each mother receives a single oral dose of deuterated water (²H2O). The standard dose for assessing breast milk intake is 30 g at 99·8 at.% ²H, regardless of a mother’s body weight. After dosing, the mother should continue feeding her baby as usual, ensuring that the baby ingests ²H2O exclusively through breast milk Saliva samples are then collected from both the mother and baby at 1, 2, 3, 4, 13 and 14 days after the mother’s initial dose. Deuterium enrichment in the saliva samples is measured using FTIR, enabling the calculation of breast milk intake and the intake of water from sources other than breast milk

²H₂O, deuterated water or deuterium oxide; IAA, indispensable amino acid; ICP-MS, inductively coupled plasma mass spectrometry; TIMS, thermal ionisation mass spectrometry; FTIR, Fourier-transform infrared spectroscopy.