biochemistry profile and thyroid (T4) concentrations. Immediately after collection, heparinized samples were centrifuged, and the plasma was harvested. Plasma not required for biochemical analyses was frozen at −20°C until analysis, within 30 min of sample collection. Samples remained frozen until they were shipped on dry ice to the laboratories chosen for analyte measurement (see below).

Choline and betaine metabolite measurements
-------------------------------------------

Plasma concentrations of free choline and betaine, were quantified, by the Caudill Laboratory, Cornell University, USA, using liquid chromatography-tandem mass spectrometry according to methods of Holm and Wang \[[@B27],[@B28]\] with modifications based on the instrumentation \[[@B29]\]. The intra- and inter-assay precision (coefficient of variation) of the method is \<3%, based on in-house control materials.

Plasma amino acid assessment
----------------------------

Amino acids were analyzed with a commercial amino acid analyser^g^ by the Amino Acid Laboratory, University of California Davis, USA. Methionine was not measured because previous studies have demonstrated that this amino acid is susceptible to oxidation during storage \[[@B30]\]. Cysteine was also not measured because this amino acid is susceptible to breakdown if not treated with sulfosalicylic acid immediately after sampling \[[@B31]\].

Urinary selenium assessment
---------------------------

Urinary selenium measurements were made by the Food and Environmental Research Agency, York, UK. Aliquots (1 mL) of test sample plus certified reference materials were digested in nitric acid using quartz high pressure closed vessels and microwave heating. The digests were diluted with water containing propan-2-ol and internal standard (Rhodium standard, "Aristar" grade, 1000 ppm. VWR International Ltd.), prior to quantification by inductively coupled plasma-mass spectrometry, using collision cell run in hydrogen mode (7500ce ICPMS, Agilient Technologies). Reagent blanks and samp