Vitamin A and Carotenoids
Preparing the column on the high performance liquid chromatograph for vitamin A and E analysis
Vitamin A is well known for its role in vision but it is also a powerful antioxidant and required for gene expression, embryonic development, and for normal immune and reproductive function. The main functional forms of vitamin A are retinal and retinoic acid. As well as being derived from the diet these can also be produced by oxidation of retinol in the eyes, liver and skin. Retinal is required for vision, whereas retinoic acid promotes cell differentiation of epithelial cells to allow growth and development and has roles in embryonic development.
Carotenoids are chemically similar to vitamin A and exist in the circulation mainly as α-carotene, β-carotene, lutein, and lycopene. Like vitamin A they are fat soluble and are bound to lipoproteins and chylomicrons. Although not essential, β-carotene is a precursor for vitamin A and like lycopene and lutein is a strong lipophilic antioxidant.
Deficiency and Toxicity
Vitamin A deficiency may develop due to inadequate intake and is most likely to occur in patients receiving long term nutritional support. Deficiency may also occur secondary to some chronic disease states, such as cystic fibrosis and other severe defects in lipid absorption and severe intestinal and liver diseases. Preterm and very low birth weight babies are at risk of vitamin A deficiency which potentially may lead to broncho-pulmonary dysplasia. Vitamin A deficiency states take some time to develop because large body stores in liver and fat confer a half-life of about six months. Symptoms of deficiency are night blindness, dry eyes (leading eventually to keratomalacia and blindness), respiratory disease, and diarrhoea.
Pharmacological doses of vitamin A can produce acute toxic symptoms of nausea, headache, fatigue, loss of appetite, dizziness, and dry skin. Symptoms and signs of chronic toxicity include hypercalcaemia, anorexia, joint and bone pain, osteoporosis, alopecia, hyperkeratosis and haemorrhage. Prolonged exposure to high vitamin A doses will ultimately lead to liver damage, therefore liver function tests should be checked in patients when the plasma vitamin A concentration is above 4.5 μmol/L.67 Because of harmful effects on the foetus, vitamin A intake should not be increased during pregnancy. Synthetic retinoids are used in treating psoriasis and troublesome acne.
At high concentrations, β-carotene imparts yellow/orange skin colouration and so its measurement is useful in the differential diagnosis of some cases of jaundice, especially in children. The activity of the enzyme β-dioxygenase may be impaired in children leading to non-pathological accumulation of β-carotene, especially when consuming food rich in β-carotene.
Assessment of status
Vitamin A status is assessed by measurement of plasma retinol concentration. Retinol is bound in the circulation to retinol binding protein (RBP) and pre-albumin forming a complex that prevents renal loss of vitamin A. Low RBP levels found during the systemic inflammatory response may result in a low plasma vitamin A concentration which does not represent nutritional deficiency. Work done in STEMRDRL shows that the systemic inflammatory response can cause a reduction in plasma vitamin A concentration of up to 50% in patients 48 hours after undergoing elective surgery for knee arthroplasty (see Table 1).68 This reduction is dependent on the magnitude of the systemic inflammatory response (see Table 2).28 The interpretation of plasma levels may be complicated by co-existing liver disease and protein-calorie malnutrition resulting in reduced synthesis of RBP. Vitamin A concentration in plasma may increase in renal failure due to decreased excretion of RBP.69
Zinc is required for synthesis of RBP, therefore zinc deficiency can result in a reduction in plasma vitamin A concentration while hepatic vitamin A stores remain adequate. This reduced plasma vitamin A concentration can only be corrected by zinc administration.
A normal plasma vitamin A concentration does not necessarily reflect adequate hepatic vitamin A stores, as plasma vitamin A concentrations do not start to fall until hepatic vitamin A stores have been significantly depleted, by 30 to 50%. Once liver vitamin A stores are depleted, the plasma concentration is a good marker of status. Plasma vitamin A concentrations of 0.5 to 0.7 μmol/L are considered sub-optimal and symptoms of vitamin A deficiency have been associated with concentrations of less than 0.5 μmol/L.
Recommended Daily Allowance
Adults: 900 μg/day
Effect of Systemic Inflammatory Response on Plasma Retinol Concentrations
Table 1: Baseline, peak/ trough and day 7 concentrations of CRP and retinol (vitamin A) following elective surgery for knee arthroplasty (n = 20).68
|CRP (mg/L)||<6 (<6–16)||150 (69–255)||31 (<6–134)||<0.001|
|Retinol (μmol/L)||2.0 (1.2–3.8)||0.9 (0.4–1.9)||1.9 (1.0–3.8)||<0.001|
Table 2: Distribution of median plasma vitamin A concentrations according to increments of CRP concentrations (n = 2186).28
|n||Median plasma vitamin A concentration (μmol/L)||p|
Sample Requirements and Reference Ranges for Vitamin A and Carotenoids
|Sample Type||Plasma / serum or whole blood (fasting sample preferred*).|
|Container||Lithium heparin (non-gel), EDTA, or plain. SST and lithium heparin gel tubes are unsuitable.|
|Precautions||Light-sensitive; wrap in tin foil. Send by first class post within 72 hours of collection. If delivery to Glasgow is outwith 72 hours, separate sample and store plasma frozen until sending and then send by first class post.|
|Minimum volume*||300 µL** plasma or 600 µL whole blood (vitamin E can be measured simultaneously on same volume)|
Vitamin A: 1.0 to 3.0 µmol/L70 (adults)
0.8 to 0.9 µmol/L (sub-clinical state: at risk of
≤ 0.7 µmol/L (clinical deficiency likely)
> 4.5 µmol/L (risk of toxicity78)
0.5 to 1.5 µmol/L29 (< 1 year)
0.7 to 1.5 µmol/L29 (1 to 6 years)
0.9 to 1.7 µmol/L29 (7 to 12 years)
0.9 to 2.5 µmol/L29 (13 top 18 years)
α-carotene: 14 to 60 µg/L70
β-carotene: 90 to 310 µg/L70
|Mean turnaround time||4.0 days|
|Method||HPLC with UV detection70 (In-house simultaneous method for vits A and E)|
|Traceability||Traceable to National Institute of Standards and Technology (NIST) standard reference material.|
|Intermediate Precision (CV)||
Vitamin A: 5.1% at 1.4 µmol/L, 5.6 % at 2.3 µmol/L
α-carotene: 11.4% at 27.8 µg/L, 14.8% at 47.4 µg/L
β-carotene: 12.8% at 168 µg/L, 8.2% at 361 µg/L
|Measurement Uncertainty, U||
Vitamin A: 1.3 ± 0.17 µmol/L, 2.2 ± 0.28 µmol/L
α-carotene: 30.8 ± 11.18 µg/L, 35.0 ± 15.36 µg/L
β-carotene: 136 ± 39.6 µg/L, 193 ± 73.7 µg/L
Vitamin A: Acceptable 4.7%, Desirable 3.1 %***
α-carotene: Acceptable 18%, Desirable 12%***
β-carotene: Acceptable 13.5%, Desirable 9%***
|EQA Scheme||UK NEQAS, Birmingham (once per month) & INSTAND scheme Düsseldorf, Germany (four times per year).|
* Ideally a fasting sample should be collected, especially if the patient is receiving oral or parenteral vitamin A supplementation. If this is not possible, sample should be taken at least 8 hours post treatment for patients receiving oral supplementation or TPN.
** Absolute minimum volume; this volume is insufficient to carry out repeat analysis if analysis fails.
*** Goal origin: biological variation71