Vitamin A in the blood

The reference values (norm) of the concentration of vitamin A (retinol) in the blood serum: in children 1-6 years - 0.7-1.5 μmol / l, 7-12 years - 0.91-1.71 μmol / l, 13 -19 years - 0,91-2,51 μmol / l; in adults it is 1.05-2.09 μmol / l.

Vitamin A refers to fat-soluble and exists in two forms - actually vitamin A, or retinol (found only in animal products), and provitamin A, known as carotene (derived from animal and vegetable products) that can be converted into retinol in the walls the digestive tract. Approximately 50-90% of the retinol taken with food is absorbed in the small intestine and transported in the complex bound to the chylomicrons to the liver, where it is stored in the form of retinol palmitate. If necessary, it is released into the bloodstream in the form of retinol, which is in combination with vitamin A-binding protein. In the serum, the vitamin A-binding protein + retinol complex binds to transthyretin. From serum, retinol is captured by target cells, such as retinal photoreceptors and epithelium.

When vitamin A enters the body in excess of the requirements (180-430 μg of retinol per day depending on age, sex and physiological condition), its excess is deposited in the liver forming the depot of this vitamin. With a reduced intake of retinol from food, its liver stores are released into the bloodstream, maintaining the serum retinol concentration at a normal level (above 0.7 μmol / L). Other biologically active forms of vitamin A (retinal and retinoic acid) are present in the blood in very low concentrations (below 0.35 μmol / L); on the esters of retinol accounts for approximately 5% of total vitamin A (0.1-0.1 μmol / l).

Vitamin A has an important role in oxidation-reduction processes. Retinol promotes the formation of glycogen in the liver and muscles, promotes the increase of cholesterol in the blood, takes part in the synthesis of steroid and sex hormones. It is necessary for the growth and formation of the bone skeleton, the resynthesis of rhodopsin, and also contributes to the normal functioning of the mucous membranes and the integumentary epithelium of the skin, preventing its metaplasia, hyperkeratosis and excessive sloughing. Vitamin A helps strengthen hair, teeth and gums. In recent years, the multiple role of vitamin A in preventing cancer and regulating immunity has been shown (necessary for the completion of phagocytosis, increases Ig synthesis, stimulates the formation of T-killers, stimulates T helper type II, etc.). Vitamin A - an active antioxidant, mainly acting in the presence of vitamin E; it protects vitamin C from oxidation. Deficiency of vitamin A is regarded as a risk factor for malignant neoplasms. In experimental studies it was shown that an increase in the vitamin A content in the diet increases the median duration of life by 17.5%. Zinc is an essential cofactor of vitamin A metabolism (necessary for the synthesis of vitamin A-binding protein).

The average daily requirement for retinol for adults (20-50 years) is 1.2 mg (4000 IU, 1 IU is equivalent to 0.3 μg retinol), for pregnant women - 1.5 mg (5000 IU), for breast-feeding - 1, 8 mg (6000 IU), for people over 60 years - 2.5 mg (10,000 IU). At least a third of the daily requirement of retinol should be delivered to the body in ready-made form; the rest can be absorbed by the use of carotenoids, of which retinol forms in the body. It should be borne in mind that approximately 30% of retinol in foodstuffs is destroyed by their heat treatment. The activity of retinol is 2 times higher than carotene, in addition, the latter is only 30-40% absorbed into the intestine. Therefore, in assessing the dietary intake, it is considered that 1 mg of retinol approximately corresponds to 6 mg of carotenoids.

Determination of retinol (vitamin A) and carotenoids in serum according to Bessey in the modification of LA Anisimova

Principle of the method

The determination of vitamin A and carotenoids is based on their hydrolysis in an alkaline alcohol solution, followed by extraction with a mixture of organic solvents.

Reagents

• 11 M solution of potassium hydroxide (KOH).
• 96% ethyl alcohol.
• 1 M solution of potassium hydroxide (KOH) in 96% ethyl alcohol: 1 volume of 11 M KOH solution is mixed with 10 volumes of 96% ethyl alcohol. The reagent is prepared on the day of the study. If staining occurs during mixing, the alcohol must be cleaned by distillation before use.
• Xylene, h.c.
• Oktan, h.ch.
• Xylene-octane mixture: prepared by mixing equal volumes of xylene and octane.

The investigations are carried out on a spectrophotometer.

The course of the determination of vitamin A

Blood taken from the finger (about 1 ml) is added to the centrifuge label and placed in a glass cup with warm water (temperature 40-45 ° C) for 20-30 minutes. To separate the serum, a blood clot is carefully wound around the edge of the tube with a thin glass rod and centrifuged at 3000 rpm for 10 minutes.

Select 0.12 ml of serum and transfer it to an agglutination tube, then add 0.12 ml of a 1 M alcohol potassium hydroxide solution. The contents are shaken thoroughly.

Test tubes with probes are placed in a water bath for 20 minutes at a temperature of 60 ° C for hydrolysis.

The samples are cooled and 0.12 ml of a xylene-octane mixture is added to them, shaken vigorously for 10-15 seconds. Cool again and centrifuge.

Carefully remove the top layer containing vitamin A and carotenoids with a Pasteur pipette with a rubber can and transfer it to microcuvettes.

Samples are spectrophotometric at a wavelength of 328 nm - to determine vitamin A, and at a wavelength of 460 nm - to determine carotenoids.

After spectrophotometry, the samples under study are subjected to ultraviolet irradiation to destroy vitamin A. For these purposes, a quartz (bactericidal) lamp is installed at a distance of 15-20 cm from the microcubes in such a way that a part of the cell filled with liquid is exposed to irradiation; the irradiation time is 45-60 minutes.

The samples are repeatedly spectrophotometric at a wavelength of 328 nm. The content of vitamin A is determined from the difference in extinction values (optical density), taking into account the coefficient (factor) 637, calculated by Bessey for vitamin A.

The calculation is carried out according to the formula:

X = 637 × (E328 (1) -E328 (2)),

Where X is the vitamin A content, μg / dL; 637 is the coefficient calculated by Bessey for the determination of vitamin A; Е328 (1) - the optical density of the solution before irradiation; E328 (2) is the optical density of the solution after irradiation.

The coefficient for transferring the concentration of vitamin A from μg / dL in μmol / l is 0.035.

The content of carotenoids is calculated by the formula:

X = 480-E480,

Where X is the content of carotenoids, μg / dL; 480 is the coefficient calculated by Bessey to determine carotenoids; E480 is the optical density of the test solution.

Note

According to Bessey, larger or smaller serum volumes can be taken during the studies, but its ratio to the volume of the alcohol solution should be constant with any change in the volume (amount) of the xylene-octane mixture.

The norm in the content of vitamin A in the blood serum is: in newborns and infants - 160-270 μg / l; in adults, 1.05-2.45 μmol / l (300-700 μg / l). The content of carotenoids in the adult serum is 800-2300 μg / l.