Elecsys folate 3

A semiquantitative food frequency questionnaire was used to assess diet from the last menstrual period of the index pregnancy to the time of study recruitment.^{34 (link),35 (link)} An interview was conducted at recruitment to assess supplement intake for the 3-month period before the date of the index pregnancy detection. Pregnancy dietary folate equivalent (DFE) intake (micrograms per day) included both natural food folate and folic acid from fortification and supplement intake and was estimated by multiplying the frequency of intake of standard portion sizes of each food item or supplement times its folate or folic acid content, according to the Harvard nutrient composition database.^{35 (link)} To account for the variations in folate intake by total energy consumption, we adjusted the DFE intake for total energy intake via the residual method.^{36 (link)}Baseline plasma samples for a subpopulation were sent to Boston Children’s Hospital’s Clinical and Epidemiological Research Laboratory for folate quantification (to convert from nanograms per milliliter to millimoles per liter, multiply by 2.266) by an electrochemiluminescence binding assay (Elecsys Folate III, Roche Diagnostic). The assay is approved by the US Food and Drug Administration and has high reliability for plasma folate measurement (day-to-day imprecision <3%).

In order to collect measurements of Hcy concentration in plasma, a Hcy-specific ELISA assay (Cell Biolabs Inc., San Diego, CA, USA) was used [18 (link)]. Briefly, samples were incubated in the homocysteine-coated plates for ten minutes at RT, followed by incubation with the primary anti-homocysteine antibody and then the provided secondary antibody. Lastly, the substrate solution was added for 30 min, followed by the stop solutions. iMark Absorbance Microplate Reader (BioRad, Hercules, CA, USA) was used to read the absorbance of each sample at a wavelength of 450 nm. The Elecsys Folate III (Roche Diagnostics; Indianapolis, IN, USA) method was utilized to quantify folate and vitamin B12 concentrations using an intrinsic folate binding protein and vitamin B12 factor. Human Vitamin B6 (VB6) ELISA Kit (MyBioSource, San Diego, CA, USA) and Methionine Assay Kit (Fluorometric) (Abcam, Cambridge, MA, USA) were used to measure vitamin B6 and methionine, respectively, following the manufacturer’s protocols.