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 functional forms of vitamin A are retinol and retinal, but these can also be produced from dietary retinoic acid and some carotenoids.
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, respiratory disease, and diarrhoea.
Retinol is bound in the circulation to retinol binding protein (RBP) and, to a lesser extent, pre-albumin. As a result the low RBP levels found during the acute phase response results in a low vitamin A concentration which does not represent nutritional deficiency. The interpretation of plasma levels may be complicated by co-existing liver disease and protein-calorie malnutrition as well as the acute phase response.
Pharmacological doses of vitamin A can produce toxic symptoms of nausea, headache, fatigue, loss of appetite, dizziness, and dry skin, and ultimately in liver damage, osteoporosis, and haemorrhage. 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.
Carotenoids are chemically similar to vitamin A and exist in the circulation as α-carotene, β-carotene, lutein, and lycopene. Like vitamin A they are fat soluble and are bound to lipoproteins and chylomicrons. Although they are not essential, β-carotene is a precursor for vitamin A and like lycopene and lutein is a strong lipophilic antioxidant. At
high concentrations (over 100µg/L), β-carotene imparts a yellow skin colouration and so its measurement is occasionally useful in the differential diagnosis of some cases of jaundice.
Sample Requirements and Reference Ranges for Vitamin A and Carotenoids
| Sample Type | Plasma or serum |
|---|---|
| Container | Lithium heparin, EDTA, or plain. SST unsuitable. |
| Precautions | Highly light-sensitive; wrap in tin foil. Send first class. If delivery to Glasgow is outwith 24 hours, freeze until sending and then send on ice, or preferably dry ice. |
| Minimum volume* | 300 µL |
| Reference ranges | |
| - general | 1.0 to 3.0 µmol/L (adults) 0.5 to 1.2 µmol/L (sub-clinical state: at risk of developing clinical deficiency) < 0.5 µmol/L (clinical deficiency likely) > 5.0 µmol/L (risk of toxicity) |
| - age-related | 0.5 to 1.5 µmol/L (< 1 year) 0.7 to 1.5 µmol/L (1 to 6 years) 0.9 to 1.7 µmol/L (7 to 12 years) 0.9 to 2.5 µmol/L (13 top 18 years) |
| - carotenoids | alpha-carotene: 14 to 60 µg/L beta-carotene: 90 to 310 µg/L Lutein: 80 to 200 µg/L Lycopene: 100 to 300 µg/L |
| Method | HPLC with UV detection |
* This is the absolute minimum volume; these volumes are insufficient to carry out a repeat analysis in the event of an analytical problem.