Lack of consensus between measurements of plasma phylloquinone by enzyme-linked immunoassays and a well-validated high performance liquid chromatographic method

Authors: RUDITSER, REBECCA - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; FU, XUEYAN - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; BOOTH, SARAH - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; LIU, MINYING - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; SHEN, XIAOHUA - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; SHEA, KYLA - Jean Mayer Human Nutrition Research Center On Aging At Tufts University

Submitted to: Current Developments in Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2023
Publication Date: 6/26/2023
Citation: Ruditser, R., Fu, X., Booth, S.L., Liu, M., Shen, X., Shea, K. 2023. Lack of consensus between measurements of plasma phylloquinone by enzyme-linked immunoassays and a well-validated high performance liquid chromatographic method. Current Developments in Nutrition. https://doi.org/10.1016/j.cdnut.2023.101959.
DOI: https://doi.org/10.1016/j.cdnut.2023.101959

Interpretive Summary: Vitamin K status is assessed clinically and for research purposes by measuring circulating phylloquinone. New assays that measure circulating phylloquinone have become commercially available. However, their validity was uncertain. To address this, we compared plasma phylloquinone concentrations measured using two new immunoassays to plasma phylloquinone concentrations measured using an established and validated high-performance liquid chromatography (HPLC) assay. Samples were obtained from participants in a vitamin K depletion - supplementation study. The plasma phylloquinone concentrations measured using HPLC increased in response to the phylloquinone supplementation, as expected. However, neither immunoassay detected any change in plasma phylloquinone between the depletion and supplementation phases. In addition, the plasma phylloquinone measured with one immunoassay was 37% lower, on average, than the validated HPLC assay, while the average plasma phylloquinone measured using the other immunoassay was >700% higher than the HPLC assay. These findings reinforce the need to validate circulating phylloquinone assays as they become available.

Technical Abstract: Enzyme-linked immunoassays (ELISAs) that measure circulating phylloquinone have become commercially available, but their validity is uncertain. The objective of the study was to compare the plasma phylloquinone concentrations measured by two commercially available ELISAs to concentrations measured using a validated HPLC assay in 108 samples obtained from participants in a depletion (~10 mcg phylloquinone/day)-supplementation (~500 mcg phylloquinone/day) study. The geometric mean of plasma phylloquinone measured with ELISA A was 0.70 nmol/L (37%) lower compared to the HPLC measures. The mean of the ELISA B measures was 12.4 nmol/L (>700%) higher compared to the HPLC measures. Plasma phylloquinone measured using HPLC was significantly lower during phylloquinone depletion than during supplementation (0.4 +/- 0.1 vs 1.2 +/- 0.2 nmol/L; p<0.001). Neither ELISA detected any significant difference in plasma phylloquinone between depletion and supplementation (ELISA A p=0.76; ELISA B p=0.29). These findings reinforce the need to validate plasma phylloquinone assays as they become available.