(P26-017-23) Comparing Plasma Phylloquinone Concentrations Measured Using Commercial Enzyme-linked Immunoassays to High Performance Liquid Chromatography

Objectives: Enzyme-linked immunoassays to measure circulating phylloquinone (PK) have become commercially available but their validity is uncertain. High performance liquid chromatography (HPLC) is an established and validated method to quantify circulating PK. The objective of the study was to compare the plasma PK concentrations measured by two commercially available ELISAs to concentrations measured using HPLC.

Methods: Plasma PK concentrations were measured in 108 samples obtained from adults who participated in a vitamin K depletion (~10 mcg PK/day for 28 days) –vitamin K supplementation (~500 mcg PK/day for 28 days) cross-over study using the Abbexa and LSBio ELISAs and a validated HPLC assay. Pearson correlation coefficients between each ELISA and the HPLC measures were calculated. Inter-assay agreement between both ELISAs and the HPLC measurement was determined using Bland-Altman and regression analyses.  Plasma PK concentrations measured using each assay were compared between the vitamin K depletion and the vitamin K supplementation phases using repeated measures ANOVA.

Results: Plasma PK concentrations measured using the Abbexa and LSBio ELISAs did not correlate with the concentrations measured using HPLC (Pearson r=0.13 (p=0.17) and r=0.01 (p=0.94), respectively). Plasma PK concentrations were on average 1.0 nmol/L lower using the Abbexa assay (95% limits of agreement:  -0.76, 2.76) and 12.4 nmol/L higher using the LSBio assay (95% limits of agreement:  2.9, 27.7), compared to the HPLC measures. Plasma PK concentrations (mean±SEM) measured using HPLC were significantly lower during the vitamin K depletion phase (0.4±0.1 nmol/L) than in the vitamin K supplementation phase (1.2±0.2 nmol/L; p< 0.001), as expected. However, neither ELISA detected any significant difference in plasma PK between the vitamin K depletion and supplementation phases (Abbexa: vitamin K depletion phase 14.5±0.7 nmol/L, vitamin K supplementation phase 13.1±1.1 nmol/L; p=0.29).

Conclusions: These findings raise doubt about the validity of two commercially available plasma PK ELISAs.  Until such assays are validated, circulating PK should be measured using the valid HPLC assay.

Funding Sources: USDA ARS cooperative agreement 58-8050-9-004