Micronutrient Biomarker Selection and Assay Methods and Performance in Double-Blind, Randomized, Controlled Micronutrient Dose Response (MiNDR) Trials among Women of Reproductive Age and Pregnant Women in Rural Bangladesh.

BACKGROUND

Comprehensive documentation of micronutrient biomarker assessments, capturing status from deficiency to excess, remains limited, specifically in the context of multiple micronutrient supplementation (MMS) trials.

OBJECTIVES

We document biomarker selection, preanalytical and analytical methods, assay performance evaluation, and biomarker interpretation for modeling the dose-response effects of MMS in 2 parallel bioefficacy trials among women of reproductive age and pregnant women in rural Bangladesh.

METHODS

Blinded analysis of biomarker assays is being performed in the field and at 2 laboratories. Automated clinical chemistry analyzers are used to measure conventional serum and plasma biomarkers of vitamin D, B12, folate, iron, inflammation, iodine, and bone turnover. Plasma vitamers of A, E, B2, and B6, and urinary B1, B2, and B3 are measured by ultra-performance liquid chromatography (UPLC). A serum mineral panel is analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Urinary iodine and functional assays for vitamin B1, B2, and B12, iron, and selenium are measured using 96-well plate methods. Point-of-care tests are performed for hemoglobin in venous blood, whereas liver and kidney function, glucose, and a lipid panel are performed in plasma.

RESULTS

Limits of detection and quantitation for biomarker assays are reported. Interassay coefficient of variations of quality control (QC) materials for primary outcome biomarkers are 4%-10% for automated analyzers, ICP-MS, and 96-well plate, and 2%-11% for UPLC assays, where available. Measurements of two-thirds of the primary outcome biomarkers could be evaluated using established external QC materials to ensure assay performance.

CONCLUSIONS

The detailed account of micronutrient biomarker assays in the dose-response MMS trials provides a useful framework for designing future research involving comprehensive assessments of micronutrient status in vulnerable populations. External quality assurance tools are warranted for UPLC-based B1, B2, and B3 vitamers, and for kinetic assays of B1, B2, and selenium.