Method(s): LC-MS/MS (1).
What is the HKr?
The HKr (HK-ratio) is composed of HK and the four kynurenines that are products of the pyridoxal 5´-phosphate (PLP)-dependent enzymes, kynurenine transaminase (KAT) and kynureninase (KYNU). Specifically, HKr is HK:(KA+AA+XA+HAA). This is an extension, or further development, of the HK:XA index as a functional marker of B6 status (2). The principle behind HKr is the same as for other ratio-based functional markers. When vitamin deficiency affects the catalytic capacity (kcat) of an enzyme, the reponse, which will also serve to maintain flux through the enzyme(s), is an increase in upstream metabolites concomitant with a decrease in downstream metabolites. Thus, an index, like the HKr, for capturing such information is most efficient when it includes both upstream and downstream metabolites. The HKr shows a particularly steep inverse association with PLP at concentrations less than 20 nmol/L, serving as a concrete metabolic confirmation of a cutoff of 20 nmol/L for vitamin B6 deficiency. However, a notable increase in HKr in the interval from 40 to 20 nmol/L PLP also supports a concept of marginal vitamin deficiency in this interval. Compared to the index that only included XA as the downstream metabolite (HK:XA), HKr demonstrate better specificity, sensitivity and reproducibility across cohorts (3).
Performance of the assay (LC-MS/MS)
Lower limit of detection (LOD) (HK/KA/AA/XA/HAA): 2/0.4/0.7/0.5/2 nmol/L.
Within-day CV: na; between-day CV: na.
Assessment of vitamin B6 status.
Specimen, collection and processing
Matrix: EDTA plasma and serum.
Volume: Minimum volume is 60 µL, but 200 µL is optimal and allows reanalysis.
Preparation and stability: HK and HAA decrease, AA increases whereas KA and XA are stable during storage of serum/plasma samples. The blood sample must be centrifuged and the plasma/serum fraction put on ice, and frozen.
Transportation; for general instruction on transportation, click here.
Frozen, on dry ice.
Reported values, interpretation
Reported values (HKr x 100): 20-40.
Intraclass correlation coefficients (ICC): na.
1. Midttun, O., Hustad, S., and Ueland, P.M. (2009). Quantitative profiling of biomarkers related to B-vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Sp 23, 1371-79.
2. Ueland, P.M., Ulvik, A., Rios-Avila, L., Midttun, Ø., and Gregory, J.F. (2015). Direct and Functional Biomarkers of Vitamin B6 Status. Annu Rev Nutr 35, 33-70.
3. Ulvik, A., Midttun, Ø., McCann, A., Meyder, K., Tell, G., Nygård, O.and Ueland, P.M. (2019) Tryptophan catabolites as metabolic markers of vitamin B6 status evaluated in cohorts of healthy adults and cardiovascular patients. Am J Clin Nutr, in press.