High performance liquid chromatography method for the determination of pyridoxal-5-phosphate in human plasma: How appropriate are cut-off values for vitamin B6 deficiency?

Abstract

Objectives: Application of a HPLC (high performance liquid chromatography) method, using cyanide derivatisation, to the determination of plasma pyridoxal-5-phosphate (PLP) concentrations as an indicator of vitamin B 6 adequacy. Setting: The study was performed at the Institute of Food Research, Norwich, UK. Blood samples were taken at the Institute, at Health Centres, or in the volunteer's home. Subjects: 51 adolescent, 131 adult, 68 non-institutionalized elderly and 44 aged ( b 73 y) volunteers were recruited from local authority schools, local Health Centres and General Practitioners. Results: The mean PLP recovery was 92.8%. The intra-and inter-assay coef®cients of variation were 2.8% and 5.2% respectively. Mean PLP concentrations for males and females, respectively, were: adolescents (13 ± 14 y), 36.4 and 43.5 nM; adults (20 ± 64 y), 39.2 and 40.0 nM; elderly (68 ± 73 y), 34.8 and 35.3 nM; aged ( b 73 y), 57.8 and 49.0 nM. Percentages of subjects with PLP concentrations`34.4 nM were over 26% in all population groups. Mean vitamin B 6 intakes (mgag protein intake), as assessed by weighed dietary records, were all above reference nutrient intakes (15 mgag protein). Conclusions: An HPLC method, using cyanide derivitisation, has been applied to the determination of plasma PLP. Comparisons of results for local population groups with current cut-off values for plasma PLP, show large numbers of volunteers at risk of vitamin B 6 de®ciency although this is not re¯ected by vitamin B 6 intakes calculated from food tables. The 34.4 nM cut-off value for value for plasma PLP, indicating de®ciency, is questioned.

Pyridoxal Phosphate Decreases in Plasma but not Erythrocytes during Systemic Inflammatory Response

“…The reference intervals for PLP, PA, and PL in plasma were in good agreement with data reported by others (15)(16)(17)(18). PLP concentrations in red cells were adjusted to Hb rather than to volume of packed red cells because accurate pipetting of packed red cells is difficult and affects precision.…”

Use of chlorite to improve HPLC detection of pyridoxal 5′-phosphate

“…In the cyanide derivatization method [2,3], pyridoxal (PL) is oxidized to 4-pyridoxolactone; unfortunately, the lactone's fluorescence is maximal at pH 9-10 and markedly decreases at lower pH [7]. This might explain why acidic, isocratic cyanide methods fail to detect PL [2,3].…”

“…Derivatization of PLP is required for fluorescent detection by HPLC separation methods (cationexchange or reversed phase); generally utilized methods for derivatization include bisulfite [1], cyanide methods [2,3] and semicarbazide [4]. The chlorite oxidation technique of Lindgren and Nilsson [5] offers the potential to convert PLP to the more fluorescent 4-pyridoxic acid 5 -phosphate while avoiding the heating and toxicity of the cyanide procedure.…”

A comparison of the effect of advice to eat either ‘5-a-day’ fruit and vegetables or folic acid-fortified foods on plasma folate and homocysteine

“…Plasma pyridoxal phosphate (PLP) was measured as previously described (Bailey et al, 1999), with the following modifications: 100 ml of the cyanide derivatives were injected onto the HPLC column (APEX ODS 3 mm (25 cmÂ4 mm). PLP was eluted isocratically at a flow rate of 1 ml=minute using a 2 M acetate buffer, containing 1 mM heptane -sulphonic acid, adjusted to pH 3.75 with potassium hydroxide, and detected fluorometrically at an excitation and emission wavelengths of 325 and 418 nm (between batch CV 5.1%).…”