Vitamin B6 and PLP/PA ratio
Vitamin B6 is a water soluble vitamin. The main circulating forms of vitamin B6 are pyridoxal (PL) and pyridoxal 5’- phosphate (PLP). These two forms are detectable in both plasma and red blood cells. The functional form is PLP. It acts as a coenzyme for many enzymes in the body and is involved in metabolism of proteins, carbohydrates, fats and homocysteine and synthesis of several neurotransmitters (e.g. serotonin, epinephrine and norepinephrine) and haem. These enzymes include decarboxylases, aminotransferases and glycogen phosphorylase. Especially diverse are the PLP-dependent enzymes involved in amino acid metabolism. Vitamin B6 also influences the immune system and may modulate action of some steroid hormones.
Deficiency and Toxicity
Body stores of vitamin B6 are low (20-30mg), so deficiency can develop fairly quickly when intake is inadequate or losses are excessive.
Specific vitamin B6 deficiency is rare; it usually occurs in combination with other water soluble vitamin deficiencies as a result of malabsorption states, increased losses (e.g. during renal dialysis) and as a result of inadequate nutrition. However, interaction with certain drugs (e.g. isoniazid, penicillamine and hydrocortisone) may induce specific vitamin B6 deficiency.
Deficiency is associated with various symptoms: irritability, depression, confusion, inflammation of the tongue, sores or ulcers of the mouth and epileptiform convulsions. Haematological manifestations may also occur and include a normocytic, microcytic or sideroblastic anaemia. Vitamin B6 deficiency is associated with hyperhomocysteinaemia which is an independent risk factor for cardiovascular disease.
Although no adverse effects have been linked to high dietary intake of vitamin B6, chronic high doses of vitamin B6 supplements have been linked to sensory neuropathy and dermatological lesions.
Assessment of status
Plasma vitamin B6 (PLP) is mainly bound to albumin and so concentrations can fall in the presence of systemic inflammatory response independent of vitamin B6 nutritional status. This is because plasma albumin concentrations fall as part of the systemic inflammatory response and also perhaps due to the redistribution of PLP and increased uptake by tissues. Hypoalbuminemia can also lower plasma PLP concentration independent of its nutritional status.
Work done by STEMDRL has shown that the red cell PLP concentration is unaffected by systemic inflammatory response in patients 48 hours after undergoing elective surgery for knee arthroplasty,76 whereas the plasma PLP concentration decreases significantly (~50%; see Table 1). This reduction is dependent on the magnitude of the systemic inflammatory response (see Table 2).28 Red cell PLP also reflects supplementation in patients with critical illness.79 We therefore recommend the measurement of PLP in red cells in patients that have evidence of systemic inflammatory response. Results are expressed as pmoles of PLP per g of haemoglobin.
Nutritional status: We routinely only report red cell PLP for assessment of vitamin B6 nutritional status. However, if required, plasma PLP, its metabolite pyridoxic acid (PA) and pyridoxal (PL) can be measured to assess vitamin B6 status in patients who are non inflamed and for research studies (please contact the laboratory before sending plasma for vitamin B6 nutritional status).
Investigation and monitoring of hypophosphatasia: Measurement of plasma PLP and PA is particulary useful to aid in the diagnosis of hypophosphatasia. Testing for this purpose is indicated only in patients with low alkaline phosphatase (ALP) activity and features present that are consistent with hypophosphatasia, such as recurrent fractures (e.g. metatarsal/femur) or on-going joint/muscle/bone pain. Markedly elevated concentrations of plasma PLP with a low plasma concentration of PA and very high plasma PLP:PA ratio are observed in cases of hypophosphatasia. The plasma PLP:PA ratio is more sensitive for hypophosphatasia than urine phosphoethanolamine. The plasma PLP:PA ratio is also useful for monitoring hypophosphatasia patients e.g. assessment of the efficacy of enzyme replacement therapy.
Recommended Daily Allowance
Adults: 1.3 mg/day but requirements depend on protein intake.
Effect of Systemic Inflammatory Response on Plasma and Red Cell Vitamin B6 Concentrations
Table 1: Baseline, peak/ trough and day 7 concentrations of CRP, plasma and red cell pyridoxal 5 phosphate (PLP) and albumin following elective surgery for knee arthroplasty (n = 20).76 Median (range).
|
Baseline values
(0 h) |
Peak/trough
(48 h) |
Final values
(168 h) |
Friedman
(0–168 h) |
|
| C-reactive protein (mg/L) | <6 (<6–<6) | 170 (70–242) | 31 (<6–168) | <0.001 |
| Plasma PLP (nmol/L) | 25 (17-36) | 13 (10-18) | 23 (13-29) | <0.001 |
| Albumin (g/L) | 41 (38-45) | 36 (24-39) | 39 (34-43) | <0.001 |
|
Red cell PLP (pmol/g Hb) |
295 (210-550) | 320 (307-544) | 320 (240-580) | 0.409 |
Table 2: Distribution of median plasma vitamin B6 concentrations according to increments of CRP concentrations (n = 796).28
|
CRP concentration (mg/L) |
n |
Median plasma vitamin B6 concentration (nmol/L) |
P |
| ≤5 | 401 | 48 | - |
| >5-10 | 102 | 27 | <0.001 |
| >10-20 | 83 | 32 | <0.01 |
| >20-40 | 67 | 24 | <0.001 |
| >40-80 | 61 | 18 | <0.001 |
| >80 | 82 | 15 | <0.001 |
Sample Requirements and Reference Ranges for red cell vitamin B6 and plasma PLP/PA ratio
| Sample Type |
Red cell (fasting sample preferred*) - for assessment of vitamin B6 nutritional status. Whole blood or packed red blood cells (please label tube as red cells) required. If sending red cells please discard plasma and do not send (unless plasma is required for a different test). Please write 'vitamin B6' on request form.
|
|---|---|
| Container |
Red cell:Lithium heparin (non-gel) or EDTA. Lithium heparin gel tubes unsuitable. Plasma: EDTA. |
| Precautions |
Light-sensitive; wrap in tin foil (red cell and plasma). Red cell: Send whole blood by first class post within 72 hours (do not freeze whole blood). If delivery to Glasgow is outwith 72 hours of sample collection, prepare red cells (minimum volume 300 µL) by removing and discarding plasma and buffy layer (mark clearly on tube that they are red cells) and store frozen until sending and then send by first class post (ice or dry ice not required). Plasma: Send by first class post to arrive within 72 hours of collection. If delivery to Glasgow will be outwith 72 hours store plasma frozen until sending and then send by first class post(ice or dry ice not required). |
| Minimum volume** |
Red cell: 1 mL whole blood or 400 µL of red cells**** (Vitamin B2 can be analysed simultaneously on this volume). Plasma: 600 µL*** |
| Reference range |
Red cell PLP****: 250-680 pmol/g Hb80 <150 pmol/g Hb (At risk of deficiency) >2000 pmol/g Hb (over supplementation) >4000 pmol/g Hb (risk of toxicity) Plasma (for diagnosis/investigation of hypophosphatasia) PLP: 20 – 140 nmol/L80 PA: 9 - 60 nmol/L80 PLP:PA ratio: <5 (This reference range only applies when the plasma PLP is raised) |
| Mean turnaround time | Red cell: 4.5 days (see this page for STEMDRL TAT targets) |
| Method | UPLC with fluorimetric detection80 |
| Traceability | EQA. Please contact laboratory for current EQA performance. |
| Intermediate Precision (CV) | See this page for latest data |
| Measurement Uncertainty, U | See this page for latest data |
| Analytical Goals (CV) | See this page for latest data |
| EQA Scheme |
Red cell and plasma (PLP only): INSTAND whole blood/plasma EQA scheme, Düsseldorf, Germany (four times per year). |
| Included in UKAS scope? |
Red cell: Yes Plasma: No |
* Ideally a fasting sample should be collected, especially if the patient is receiving oral or parenteral vitamin B6 supplementation. If this is not possible, sample should be taken at least 8 hours post treatment for patients receiving oral supplementation or TPN.
** Patient must not be on vitamin B6 supplements for at least 2 weeks prior to sample collection.
*** Absolute minimum volume; this volume is insufficient to carry out repeat analysis if analysis fails.
**** Please contact the Laboratory if vitamin B6 in plasma for assessment of nutritional status is required.