Vitamin A in the blood

Reference values (norm) for the concentration of vitamin A (retinol) in the blood serum: in children 1-6 years old - 0.7-1.5 μmol/l, 7-12 years old - 0.91-1.71 μmol/l, 13-19 years old - 0.91-2.51 μmol/l; in adults - 1.05-2.09 μmol/l.

Vitamin A is a fat-soluble vitamin and exists in two forms: vitamin A itself, or retinol (found only in animal products), and provitamin A, known as carotene (obtained from animal and plant products), which can be converted to retinol in the walls of the digestive tract. Approximately 50-90% of dietary retinol is absorbed in the small intestine and transported in a chylomicron-bound complex to the liver, where it is stored as retinol palmitate. When needed, it is released into the bloodstream as retinol complexed with vitamin A-binding protein. In the blood serum, the vitamin A-binding protein + retinol complex binds to transthyretin. From the blood serum, retinol is taken up by target cells, such as the photoreceptors of the retina and the epithelium.

When the body receives vitamin A in quantities exceeding the requirements (180-430 mcg of retinol per day depending on age, gender and physiological state), its excess is deposited in the liver, forming a depot of this vitamin. When the intake of retinol with food is reduced, its reserves from the liver are released into the bloodstream, maintaining the concentration of retinol in the blood serum 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); retinol esters account for approximately 5% of the total vitamin A (0.1-0.17 μmol/l).

Vitamin A plays an important role in oxidation-reduction processes. Retinol promotes the formation of glycogen in the liver and muscles, helps to increase the cholesterol content in the blood, and participates in the synthesis of steroid and sex hormones. It is necessary for the growth and formation of the skeletal system, rhodopsin resynthesis, and also promotes the normal functioning of the mucous membranes and the integumentary epithelium of the skin, preventing its metaplasia, hyperkeratosis, and excessive sloughing. Vitamin A helps to strengthen hair, teeth, and gums. In recent years, the diverse role of vitamin A in preventing cancer and regulating immunity has been shown (it is necessary for the completion of phagocytosis, increases Ig synthesis, stimulates the formation of T-killers, stimulates type II T-helpers, etc.). Vitamin A is an active antioxidant, mainly acting in the presence of vitamin E; it protects vitamin C from oxidation. Vitamin A deficiency is considered a risk factor for malignant neoplasms. Experimental studies have shown that increasing the content of vitamin A in the diet increases the median life expectancy by 17.5%. Zinc is an essential cofactor in the metabolism of vitamin A (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 mcg of retinol), for pregnant women - 1.5 mg (5000 IU), for breastfeeding women - 1.8 mg (6000 IU), for people over 60 years - 2.5 mg (10,000 IU). At least a third of the daily requirement for retinol should be supplied to the body in finished form; the rest can be covered by consuming carotenoids, from which retinol is formed in the body. It should be taken into account that approximately 30% of retinol in food products is destroyed during heat treatment. The activity of retinol is 2 times higher than that of carotene, in addition, only 30-40% of the latter is absorbed in the intestine. Therefore, when assessing the diet, it is believed that 1 mg of retinol is approximately equivalent to 6 mg of carotenoids.

Determination of retinol (vitamin A) and carotenoids in blood serum according to Bessey as modified by L. A. 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 potassium hydroxide (KOH) solution.
• 96% ethyl alcohol.
• 1 M potassium hydroxide (KOH) solution 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 coloration occurs during mixing, the alcohol should be purified by distillation before use.
• Xylene, chemically pure
• Octane, chemically pure
• Xylene-octane mixture: prepared by mixing equal volumes of xylene and octane.

The studies are carried out using a spectrophotometer.

The process of determining vitamin A

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

0.12 ml of serum is collected and transferred to an agglutination tube, where 0.12 ml of 1 M potassium hydroxide alcohol solution is then added. The contents are shaken thoroughly.

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

The samples are cooled and 0.12 ml of xylene-octane mixture is added to them, and they are shaken vigorously for 10-15 s. They are cooled again and centrifuged.

The upper layer containing vitamin A and carotenoids is carefully removed using a Pasteur pipette with a rubber bulb and transferred into microcuvettes.

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

After spectrophotometry, the samples are exposed to ultraviolet radiation to destroy vitamin A. For this purpose, a quartz (bactericidal) lamp is installed at a distance of 15-20 cm from the microcuvettes so that the part of the cuvette filled with liquid is exposed to radiation; the irradiation time is 45-60 min.

The samples are re-spectrophotometered at a wavelength of 328 nm. The vitamin A content is determined by 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 content of vitamin A, μg/dl; 637 is the coefficient calculated by Bessey for determining vitamin A; E328(1) is the optical density of the solution before irradiation; E328(2) is the optical density of the solution after irradiation.

The coefficient for converting vitamin A concentration from µg/dL to µmol/L is 0.035.

The content of carotenoids is calculated using the formula:

X = 480-E480,

Where X is the carotenoid content, μg/dl; 480 is the coefficient calculated by Bessey for determining carotenoids; E480 is the optical density of the test solution.

Note

According to Bessey, a larger or smaller volume of serum can be used when conducting research, but its ratio to the volume of the alcohol solution must be constant with any change in the volume (quantity) of the xylene-octane mixture.

The normal 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 blood serum of adults is 800-2300 μg/l.