Anemia, associated with folic acid deficiency

Whatever the reason folic acid deficiency anemia is characterized by the appearance in the bone marrow megaloblasts, intramedullary destruction erythrokaryocytes, patsitopeniey, macrocytosis and hyperchromia red blood cells and sometimes - mental disorders.

Folic (pteroilglutaminovaya) acid (vitamin B₉) It consists of three components:

• pteridine ring;
• P-aminobenzoic acid (together constitute pteroievuyu acid);
• glutamic acid.

The molecular weight of folic acid - 441. Folic acid is active in the form of tetrahydrofolic acid derivatives. Folic acid content in different forms in the human body is about 5-10 mg. Stocks in violation of its revenue in the body may be depleted over 4 of the month.

The daily requirement of folic acid is 100-200 micrograms, Pregnancy, hemolytic anemia, it increases many times.

The total amount of folic acid in the diet with the full power of 500-600 micrograms per day. Folate - folates contained in considerable amounts in the liver, yeast, spinel, meat. More 50 % folic acid, in food, It can break down when cooked.

Folic acid is absorbed in the upper small intestine. The ability of the intestines to absorb folic acid is much greater than the daily requirement.

Folic acid participates in the synthesis of pyrimidine and purine bases,, along with methylcobalamin - the synthesis of timidinm nofosfata uridinmonofosfata. Folate coenzymes More involved in a number of reactions. In particular, tetrahydrofolate participates in the synthesis of glutamic acid histidine.

The etiology and pathogenesis of anemia during folic acid deficiency

Folic acid deficiency, as well as deficit of cobalamin, It leads to the development of megaloblastic anemia.

Malabsorption of folate observed in individuals, resection of the small intestine, especially jejunum, as well as in long-term taking anticonvulsants such as diphenylhydantoin (difenina) and phenobarbital (luminal). The absorption of folic acid is broken when the sprue and celiac disease. Folic acid deficiency, as well as deficit of cobalamin, observed at blind loop syndrome. Described rare cases of hereditary disorders of folic acid transport across the gut wall and into the cerebrospinal fluid. Absorption of other substances without suffering.

Folic acid deficiency in newborns observed in prematurity, violation of intestinal absorption, fed goat's milk, containing a small amount of folic acid.

A significant malabsorption of folic acid may occur in patients, alcoholics.

Increased consumption of folic acid during pregnancy can lead to a deficiency of folic acid, However, in developed countries today it is rare. Folic acid deficiency in pregnant women occur when poor nutrition, the presence of hemolytic anemia, use a large amount of pre-pregnancy alcohol.

Clinical manifestations of folate deficiency

Folic acid deficiency in children and young women. At the same time, patients complain of general weakness, dizziness. Unlike cobalamin deficiency sore tongue rarely observed. Paresthesias, and other signs of funicular myelosis missing. Sclera often ikterichnost.

In contrast to the deficit of cobalamin deficiency of folic acid is rarely observed gistaminoupornaya Akhil, gastratrophia, although the decrease of gastric secretion is often.

For folate deficiency, as well as for cobalamin deficiency, characterized by the appearance in the peripheral blood macrocytosis, megalocytes and megaloblasts, the presence of hyperchromic anemia, reducing the number of reticulocytes, platelets and leukopenia with neutrophilic shift to the left and right. In the bone marrow - megaloblasts.

The changes in the nervous system are different from the manifestations at B₁₂-deficiency anemia.

Proved, that persons with epilepsy, Folic acid deficiency leads to frequent attacks, to their greater severity. This is particularly important due to the fact, that taking anticonvulsants can cause malabsorption of folate. According to the literature, in individuals with schizophrenia folic acid deficiency causes exacerbations, more severe symptoms and less effective treatment.

Differential diagnosis of cobalamin deficiency and folic acid

For the differential diagnosis of cobalamin deficiency and folic acid is used radioimmunoassay method for determination of folic acid in the serum and particularly in erythrocytes. The content of cobalamin is determined by microbiological method. As a test organism used as Streptococcus faecalis and Lactobacillis casei. Normally, the content of folic acid in the blood serum ranges, According to the authors, using different research methods, from 3 to 25 ng / ml, and erythrocyte - from 100 to 425 ng / ml.

In folic acid deficiency decreases as the level of serum, and in erythrocytes. At deficiency kolabaminov folic acid content in the blood serum often rises, and erythrocytes can sometimes be reduced, although the decline, usually, a small.

For the diagnosis of folic acid deficiency is also used Load method histidine (15 g), then define the content of acid in the urine formiminglutaminovoy. Normally, the bulk of the forms of glutamic acid histidine. With urine output 1-18 mg formiminglutaminovoy acid. At deficiency of folic acid for 8 hours after its introduction into the body is released from the urine 20 to 1500 mg formiminglutaminovoy acid. A significant number of formiminglutaminovoy acid excreted in the urine after administration of histidine in persons, treated with methotrexate. At deficiency of cobalamin, cirrhosis of the liver the urinary excretion of acid increases formiminglutaminovoy, to a lesser extent, than with a deficit of folic acid. Normal levels of urinary methylmalonic acid content increases formiminglutaminovoy acid histidine after receiving evidence of deficiency of folic acid, instead of cobalamin.

It helps to differentiate these two states and radionuclide study. At deficiency of cobalamin, It involves a violation of secretion of intrinsic factor, impaired absorption of labeled cyanocobalamin, a deficiency of folic acid, it is absorbed properly.

At deficiency of folic acid, its introduction into the body leads to a higher content of reticulocytes, whereas injection of cyanocobalamin (derivatives) ineffective. Folic acid deficiency methylcobalamin increases the level of reticulocytes.

Defining index segmentation neutrophils

Count the number of neutrophils, with 4 segment and more (Total 200 cell). Determine the percentage of cells, with 5 segments and more relative to the amount of cells, with 4 segment. The index is determined by the formula:

IS =(KS (5 and more))/KS(4);

where IC - index segmentation;

KS (5 and more) - The number of segmented neutrophils (on 200 cell), in which the number of segments is equal to 5 or more;

KS (4) - The number of segmented neutrophils, in which the number of segments is equal to 4.

Normally, this index ranges 2,5 to 25 %. Where, when it exceeds 30 % (vibration 120%), You can talk about hypersegmentation neutrophils. It observed deficiency of folic acid, metilkoʙalamina, and uremia.

Determination of methylmalonic acid in urine by Giordio and Plaud

Reagents.

1. The ion exchange resin Dowex-Zx4 (chloride form, 200-400 Mesh). The resin was washed with five volumes of distilled water 3 times, was suspended in two volumes of distilled water and stored in a dark dish at 4 ° C.

2. A solution of para-nitroaniline 0,075 % prepared by dissolving 37,5 mg reagent 500 ml 0,2 M solution of hydrochloric acid. Store in a dark pot, It remained stable for several months at room temperature.

3.Acetate buffer (pH 4,3) made from 1 M sodium acetate and 1 M solution of acetic acid.

4.Diazoreaktiv: 4 ml 0,5 % sodium nitrate solution was mixed with 15 ml para-nitroaniline. The mixture was placed in a refrigerator at 4 ° C and the cooled solution was added 4 ml 0,2 M sodium acetate. Reagent stable at temperatures 4 ° C for a day.

5.Standard 0,05 M solution of methylmalonic acid: 295 mg methylmalonic acid are dissolved in 50 ml of water.

Method

Urine was collected into a glass bottle and frozen until the study. To study the charge portion of urine (20- 25 ml), thawed, centrifuged, and the supernatant was collected using a weak solution of sodium hydroxide or hydrochloric acid to adjust pH 6,5.

Chromatographic column height 20 cm and a diameter 1 cm was filled with resin Dowex-Zx4. The resin layer should be within 3 cm. Is passed through the resin 5 ml of urine, washed with distilled water (2 × 50 ml at a rate of not more than 15 ml 1 from). Methylmalonic acid may be eluted from the resin 20 ml 0,1 M solution of hydrochloric acid. Further 2 ml of eluate were mixed with 3 ml 1 M acetate buffer, pH 4,3 in a test tube with a glass stopper (15×2cm). To the mixture is added 3 ml cold diazoreaktiva, wherein the pH is raised to 4,0. The mixture was heated in a water bath at a temperature 94 ° C. 3 min and 2 ml 3 M sodium hydroxide solution. The tube was then removed from the water bath, stoppered and the contents stirred. The mixture was cooled at room temperature for 10 min and at a photometric 620 nm in a cuvette with a pathlength 1 cm.

As a control, a mixture of, consisting of 2 ml 0,1 M solution of hydrochloric acid, 3 ml and diazoreaktiva 3 ml acetate buffer. Methylmalonic acid standard solution consists of the same components and 0,02 ml 0,05 M solution of methylmalonic acid. The absorbance of these solutions were measured under the same conditions.

Contents methylmalonic acid (mg) per unit volume of urine (1 l) determined by the formula:

(IS_Sample-IS_control)/(IS_standard-IS_control)*A*((1000*T)/M)

where E - absorbance of various samples;

A - the amount of methylmalonic acid in a standard test tube, ml;

1000 - The amount of urine, taken for research, ml;

T - the amount of hydrochloric acid, taken for elution, ml;

M - the number of the eluate, taken for research, ml.

Under the above conditions, the formula can be simplified as follows::

(IS_Sample-IS_control)/(IS_standard-IS_control)*236

Where, when the amount of methylmalonic acid in urine is greater than 300 mg / l, should be re-mixed with acetate buffer eluate and diazoreaktivom, but instead 2 ml of eluate is advisable to take 4- 10 times lower and bring the volume to 2 ml 0,1 M hydrochloric acid solution. When calculating the dilution should be considered.

Normally per day with urine output 0,6-4,7 mg of methylmalonic acid. At deficiency of cobalamin daily excretion of methylmalonic acid reaches 294 mg. Normally, reception 10 g valine not increase clearance of methylmalonic acid, cobalamin deficiency increases the excretion of methylmalonic acid.

Determination of methylmalonic acid in urine by Green

Reagents.

1. Ether.
2. Ethanol.
3. A solution of hydrochloric acid 0,1 M.
4. Ammonium sulfate.
5. Сильноосновная смола деацидит FF (200 mesh, chloride form).
6. A solution of sodium nitrate 0,5 % (weight / volume).
7. A solution of para-nitroaniline (750 mg dissolved in 1 l 0,2 M solution of hydrochloric acid).
8. Sodium acetate 0,2 M.
9. Acetate buffer 1 M, pH 4,3: mixed 1 M sodium acetate solution and 1 M solution of acetic acid in the ratio 16,35:33,9.
10. An aqueous solution of sodium hydroxide 3 M.

A. Standard solution methylmalonic acid: 100 mg dissolved in 100 ml of water, stored in the refrigerator.

B. Working standard methylmalonic acid: standard solution diluted in 20 time 0,1 M hydrochloric acid solution (1 mL of the standard and 19 ml of hydrochloric acid). This amount of methylmalonic acid equivalent 100 mg methylmalonic acid 1 l urine test conditions.

For the preparation of mixed diazoreaktiva 4 ml 0,5 % sodium nitrate solution and 15 ml para-nitroaniline. The mixture was placed in an ice bath and add 4 ml 0,2 M sodium acetate.

Method.

Urine was collected for one day, measured its volume and 10 ml of acidified 1 M sulfuric acid solution to pH 2,0 or slightly below. Ammonium sulfate was added to saturation. The mixture was placed in a separatory funnel and extracted three times with ether and ethanol in a ratio of 3:1. To prevent the formation of the emulsion mixture extracted used in excess.

The extract was passed through a small column chromatography (1 – 1,5 cm diameter), completed 5 ml resin. The column was washed 50 ml distilled water. Methylmalonic acid eluted 0,1 M hydrochloric acid solution. After elution selected 2 ml eluate, W is added ml of acetate buffer and 3 ml diazoreaktiva.

After mixing, the tube was placed in a water bath at a temperature 95 ° C 3 m. Then added 2 ml 3 M sodium hydroxide and once closed tube stopper to minimize contact with air. After cooling, the absorbance was measured at wavelength 620 them. For the blank instead of using the eluate 2 ml 0,1 M solution of hydrochloric acid, and for the standard solution - 2 ml of working standard.