RU2309403C2 - Method for determining fat availability in feces - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining the number of fat drops available in fecal emulsion preparation by means of microscope. Feces sample mass under study, mean fat drop radius, acetic acid volume as reagent, methylene blue or Nile blue volume placed under cover glass, amount of feces excreted per day or in single defecation, distilled water volume added to feces sample for preparing fecal emulsion, fecal emulsion volume placed under cover glass for carrying out microscopic analysis is additionally determined. Fat quantity is calculated in the feces under study on basis of data received.

EFFECT: high accuracy in determining total fat quantity, non-decomposed (neutral) fat quantity and fatty acid quantity in feces for setting differential diagnosis of exocrinic pancreatic insufficiency and substance absorption disorders in intestine.

Description

The invention relates to medicine, namely to the field of clinical laboratory diagnostics, and can be used in the determination of fats in feces.

Currently, fats in feces are determined by qualitative (microscopy) and quantitative methods (chemical). Quantitative methods include gravimetric, titrimetric methods and fecal lipidograms. Quantitative methods allow a total determination of the total amount of fat in feces, taking into account fat of exogenous (food) origin. The determination of the amount of fat in feces is necessary for the diagnosis of pancreatic insufficiency, acholia syndrome and impaired digestion in the intestines, in particular with the syndrome of accelerated evacuation of chyme and impaired absorption of substances by the intestines. However, with pancreatic insufficiency, the content of unsplit fat - neutral fat increases, and with the pathology of the small intestine associated with impaired absorption of substances - breakdown fat - fatty acids and salts of fatty acids. The gravimetric method allows you to determine the total fat content in feces without differentiation between split (fatty acids and salts of fatty acids) and unsplit fat (neutral fat). For differential laboratory diagnosis of pancreatic digestion insufficiency syndrome and digestive digestion syndrome in the small intestine (enteric syndrome), the titrimetric method and feces lipid profile are performed. However, limitations on the use of the titrimetric method include the loss of volatile fatty acids and differences in the titration factor depending on which fatty acid is used as the standard. Gravimetric, titrimetric methods require the collection of feces within 72 hours, intake of 50-150 g of fat / day with food at least 2 days before and during the collection of feces. The gravimetric method leads to interference in cases of taking large doses of aminosalicylic acid, azathioprine, bisacodyl (long-term use), cholestyramine (dose more than 15 g / day), kanamycin, neomycin, tetracycline.

However, the gravimetric, titrimetric methods and fecal lipidogram to date remain not accessible to every laboratory, rather laborious, time consuming, expensive. Gravimetry analysis takes a long time. Operations such as "aging" the precipitate, filtering, calcining, take several hours, so a lot of time passes from the beginning to the end of the analysis. However, it should be noted that during this time the constant work of a biochemist-analyst is not required, since the “aging” of the precipitate, the calcination of crucibles or the drying of the precipitate takes place practically without its participation. Therefore, it is not difficult to perform several (5-10) parallel operations at once. Thus, gravimetric analysis is not used in cases where a quick analysis of a large number of samples is required. Duration, laboriousness and high cost of determination practically exclude the use of these methods for analyzes put on stream in polyclinics and clinical hospitals. In addition, for these methods it is necessary to collect feces within 72 hours, which is almost not carried out in modern clinics, since the need for a complete quantitative collection of feces presents known difficulties, requires special dishes and appropriate instruction of patients and staff of medical institutions. When determining daily fat, it is not necessary to collect daily feces, it is enough after each bowel movement during the day to determine the fat. Daily fat loss is the sum of fat loss for each bowel movement during the day. However, due to the high cost, duration and complexity of gravimetry and titrimetry methods, fat is not determined in every portion of feces, but in daily stool. The proposed method for determining fats in feces is devoid of these disadvantages and allows, without collecting daily stool to determine fats after each bowel movement.

Closest to the proposed method is a coprological method for the study of steatorrhea, which allows you to obtain indicative information about the exocrine function of the pancreas and the absorption function of the intestine [V.T. Morozova, I.I. Mironova, R.L. Martshevskaya. Laboratory diagnosis of the pathology of the digestive system. M., 2001, p. 63-98]. Determination of split and unsplit fat in feces using a coprological study gives a qualitative result. Microscopy of feces determines neutral fats, fatty acids and soaps. However, this method is not reliable, since it does not give an idea of the amount of released fats. Quantitative methods are more accurate and allow you to select more effective doses of enzyme preparations for the correction of exocrine pancreatic insufficiency. These difficulties led to the search for an alternative method that quantifies steatorrhea.

The invention solves the problem of determining the amount of total fat, unsplit (neutral) fat and fatty acids in the feces, which allows differential diagnosis between exocrine pancreatic insufficiency and impaired absorption of substances in the intestine.

A comparison was made of the values of fat, neutral fat and fatty acids obtained using titrimetry and gravimetry and indicators obtained by the claimed method in 9 samples. The results showed that the proposed method is not inferior in accuracy to gravimetric and titrimetric studies, and at the same time is more accurate than coprological research. The proposed method is simple and quick to perform, so it can be recommended for determining total fat, neutral fat, fatty acids for the diagnosis of lipolytic pancreatic insufficiency and intestinal diseases, in particular malabsorption syndrome.

In the proposed method for determining the amount of total fat, neutral fat and fatty acids, the amount of feces weighed is additionally measured, the amount of each substance in the preparation is determined, and the result is recounted for the total amount of excreted feces per day or for one bowel movement. When determining the daily fat by the proposed method does not require the collection of daily feces, it is enough after each bowel movement during the day to determine the fat. Daily fat loss is the sum of fat loss for each bowel movement during the day.

Where

Q - the amount of the test substance (total fat, neutral fat, fatty acids) in the studied feces M;

n is the number of fields of view in the preparation (with an increase of 7 × 40 4031 fields of vision; with an increase of 7 × 10 - 233 fields of view with a cover glass area of 324 mm ² );

N is the average number of drops (fat, neutral fat, fatty acids) in one field of view;

ρ is the density of fats, fatty acids 0.9 g / cm ³ ;

R is the average radius of one drop of fat;

S _p is the area of the integumentary glass;

v _k - the volume of the reagent (acetic acid, methylene or Nile blue), placed under a coverslip for microscopy;

M - the amount of feces per day or one bowel movement;

m is the mass of a sample of feces taken for the preparation of fecal emulsion;

V is the volume of distilled water added to the fecal sample of mass m for the preparation of fecal emulsion;

v is the volume of fecal emulsion placed under a coverslip for microscopy.

It should be noted that the amount of fecal emulsion, placed under a coverslip piece of glass for microscopy, is approximately equal to its mass, density is taken to be about 1 g / cm ^3, since a large part of the study drug, placed under a piece of glass coverslip, consists of water.

The obtained value is compared with the normal value of fat in feces. There are different opinions about how much fat is excreted in the feces normally. According to Korovina N.A., Zakharova I.N., Malova N.E. normally, no more than 10% of fat introduced with food is excreted in feces [Questions of Modern Pediatrics, 2003, vol. 2, No. 5, www.solvay-phanna.ru/creon/article.asp?id=2384]. According to Shulpekova Yu.O., Ivashkina V.T. - the norm is 7 g / day [Correction of digestive disorders by enzyme preparations of the pancreas (www.solvay-pharma.ru/creon/article.asp?id=3089)]. According to the data of V.T. Morozova, I.I. Mironova, and R.L. Martshevskaya, almost healthy people with feces emit 5% of fat in the form of salts of fatty acids (soaps) [Laboratory diagnosis of pathology of the digestive system. M., 2001, p. 76]. The study is informative only with normal intestinal evacuation function, which provides a fecal water content of 80-85%. With accelerated evacuation of chyme through the small intestine in the feces, neutral fat is determined, which does not indicate pancreatic lipolytic insufficiency.

The method is as follows. Feces for research are collected after spontaneous bowel movements in a specially designed dish: clean, dry, moisture-proof. You can not examine the material after an enema, taking medications (belladonna, pilocarpine, castor oil, paraffin oil), keeping candles.

For the study, from the total amount of feces M, a piece of feces is prepared with a mass of - m (a portion of feces). Dilute it in distilled water with a volume of V to the consistency of a thick syrup. A fecal emulsion volume is obtained from which a specific fecal emulsion volume (v) is taken and 3 preparations are prepared to determine total fat, neutral fat and fatty acids. 1 preparation - the volume of fecal emulsion (v) is applied to a glass slide, covered with a coverslip to determine the remains of fatty foods. If needles or clumps are found, the native preparation is heated on a flame of an alcohol lamp, without bringing to a boil, it is quickly microscopic. Needles and clumps of fatty acids melt easily, turning into drops, and crystallize again when cooled. 2 preparation - the volume of fecal emulsion (v) and the volume of acetic acid (v _k ) are mixed on a glass slide. Cover with a coverslip. Diagnose salts of fatty acids (soaps) by boiling. When the preparation is boiled with acetic acid, the salts of fatty acids melt, forming droplets that quickly crystallize when the preparation cools. 3 preparation - fecal emulsion with volume (v) and methylene or Nile blue volume (v _k ) are mixed on a glass slide. Cover with a coverslip. Drops of neutral fat from drops of fatty acids are differentiated. Drops of fatty acids turn blue or navy blue. Drops of neutral fat do not stain with methylene blue, and Nile blue stains them yellowish or pinkish or does not stain at all [V.T. Morozova, I.I. Mironova, R.L. Martshevskaya. Laboratory diagnosis of the pathology of the digestive system. M., 2001, p.77]. Cover the test preparations with a coverslip neatly so that the test mixture is evenly distributed and does not flow out from under the coverslip. The material is uniformly located under a 324 mm ² cover slip and does not spread when applying a 0.1 ml fecal emulsion and 0.1 ml reagent (acetic acid, methylene or nile blue).

An average drop of neutral fat is selected, with the help of an ocular micrometer, its diameter is measured and the average number of drops of fat, neutral fat and fatty acids in one field of view is calculated. The determination of the average weight of one drop of fat depends on the thickness of the test drug. If the average diameter of the fat drop is less than or equal to the thickness of the test drug, then the average volume of the drop of fat is calculated as the volume of the ball, if the average diameter of the fat drop is larger than the thickness of the test drug located under the coverslip, the average volume is calculated as the volume of the cylinder. Knowing the number of drops of fat in one field of view (N), the number of fields of vision in the preparation (n) and the mass of one drop of fat, find the mass of fat contained in the test drug, located under a coverslip and containing the volume of fecal emulsion (v). Considering the feces dilution (mass of feces (m) diluted in the volume of distilled water (V)), the amount of fat contained in the feces allocated per day or for one bowel movement is found.

Where

Q - the amount of the test substance (total fat, neutral fat, fatty acids) in the studied feces M;

q is the amount of fat in the test preparation (v) under a glass cover with an area S _p equal to n · N · ρ-4/3 · π · R ³ (if 2R≤ (v + v _k ) / S _p ) or equal (n · N · ρ · π · R ² · (v + v _k ) / S _p (if 2R> (v + v _k ) / S _p ));

v _k - the volume of the reagent (acetic acid, methylene or Nile blue), placed under a coverslip for microscopy;

S _p - the area of the glass cover equal to 324 mm ²

M - the amount of feces per day or one bowel movement;

m is the mass of a sample of feces taken for the preparation of fecal emulsion;

V is the volume of distilled water added to the fecal sample of mass m for the preparation of fecal emulsion;

v is the volume of fecal emulsion placed under a coverslip for microscopy.

or

Where

Q - the amount of the test substance (total fat, neutral fat, fatty acids) in the studied feces M;

n is the number of fields of view in the preparation (with an increase of 7 × 40 4031 fields of vision; with an increase of 7 × 10 - 233 fields of view with a cover glass area of 324 mm ² );

N is the average number of drops (fat, neutral fat, fatty acids) in one field of view;

ρ is the density of fats, fatty acids 0.9 g / cm ³ ;

R is the average radius of one drop of fat;

A-4/3 · R, if 2R≤ (v + v _k ) / S _p ; (v + v _k ) / S _p if 2R> (v + v _k ) / S _p ;

Sp is the area of the integumentary glass;

v _k - the volume of the reagent (acetic acid, methylene or Nile blue), placed under a coverslip for microscopy;

M - the amount of feces per day or one bowel movement;

m is the mass of a sample of feces taken for the preparation of fecal emulsion;

V is the volume of distilled water added to the fecal sample of mass m for the preparation of fecal emulsion;

v is the volume of fecal emulsion placed under a coverslip for microscopy.

It should be noted that the volume of fecal emulsion placed under the coverslip for microscopy is approximately equal to its mass, the density is assumed to be approximately 1 g / cm ³ , since most of the test drug placed under the coverslip consists of water.

The density of fatty acids at a temperature of 37 ° C on average is approximately 906.57 kg / m ³ . The density of milk fat at 15 ° C is from 0.936 to 0.944 g / cm ³ ; animal fat at 18 ° C is from 0.915 to 0.953 g / cm ³ . Thus, you can use the average value of the density of fats, equal to 0.9 g / cm ³ .

According to the proposed method, 9 patients were examined who, for control, also carried out the determination of fats in feces using gravimetric and titrimetric methods. It was found that the calculated indices of neutral fat, fatty acids and total fat almost coincide with those obtained using gravimetric and titrimetric methods. The difference in indicators obtained by the proposed method of titrimetry and gravimetry, averaged 7.5% for total fat and neutral fat and 8% for fatty acids.

To confirm the operability of the method, the following clinical examples are provided.

Example No. 1. A patient suffering from chronic pancreatitis for several years. In a gravimetric study, the amount of fat lost during the day was 18 g. When determining the daily steatorrhea using the proposed method, the amount of total fat was 20 g / day.

Where

Q - the amount of the test substance (total fat, neutral fat, fatty acids) in the studied feces M;

n is the number of fields of view in the preparation (with an increase of 7 × 40 4031 fields of vision; with an increase of 7 × 10 - 233 fields of view with a cover glass area of 324 mm ² );

N is the average number of drops (fat, neutral fat, fatty acids) in one field of view;

ρ is the density of fats, fatty acids 0.9 g / cm ³ ;

R is the average radius of one drop of fat;

S _p is the area of the integumentary glass;

v _k - the volume of the reagent (acetic acid, methylene or Nile blue), placed under a coverslip for microscopy;

M - the amount of feces per day or one bowel movement;

m is the mass of a sample of feces taken for the preparation of fecal emulsion;

V is the volume of distilled water added to the fecal sample of mass m for the preparation of fecal emulsion;

v is the volume of fecal emulsion placed under a coverslip for microscopy.

Since 2R = 2 · 0.05 mm;

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = 100/324 mm ² = 0.3 mm,

then A = 4/3 · R;

n = 4031 fields of view;

N = 4, i.e. an average of 4 drops of fat in one field of view;

ρ = 0.9 g / cm ³ ;

R = 0.05 mm;

A = 4/3 · R, since 2R = 2 · 0.05 mm;

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = 100/324 mm ² = 0.3 mm,

then A = 4/3 · R;

S _p - 324 mm ² ;

M = 250 g / day;

m = 5 g;

V = 0.3 ml of distilled water;

v = 0.1 ml.

Q = (4031 · 4 · 0.9 g / 1000 mm ³ · 3.14 · (0.05) ² · (4/3 · (0.05)) · 250 g / day · (5 + 0.3 )) / (5 · 0.1) = 20 g / day;

Q = 20 g / day.

Example No. 2. A patient with generalized pancreatic exocrine insufficiency, taking enzyme replacement drugs. With a titrimetric study, steatorrhea was 25 g / day, fatty acids 20 g / day. When determining using the proposed method, the total fat is 26 g / day, fatty acids - 19.6 g / day. Therefore, the dose of the drug that was prescribed to the patient is sufficient, and the steatorrhea that the patient has is associated with impaired absorption of substances in the intestine.

Calculation of the amount of total fat:

Since 2R = 2 · 0.04 mm;

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = ^100/2 = 324 mm 0.3 mm,

then A = 4/3 · R;

n = 4031 fields of view;

N = 10, i.e. an average of 4 drops of fat in one field of view;

ρ = 0.9 g / cm ³ ;

R = 0.05 mm;

A = 4/3 · R, since 2R = 2 · 0.04 mm

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = ^100/2 = 324 mm 0.3 mm,

then A = 4/3 · R;

S _p - 324 mm ² ;

M = 250 g / day;

m = 5 g;

V = 0.3 ml of distilled water;

v = 0.1 ml.

Q = (4031 · 4 · 0.9 g / 1000 mm ³ · 3.14 · (0.04) ² · (4/3 · (0.04)) · 250 g / d · (0.3 + 5 )) / (5 · 0.1) = 26 g / day;

Q = 26 total fat / day.

Calculation of the amount of fatty acids:

n = 4031 fields of view;

N = 18, i.e. an average of 18 drops of fatty acids in one field of view;

ρ = 0.9 g / cm ³ ;

R = 0.03 mm;

A = 4/3 · R, since 2R = 0.06 mm;

v = 0.1 ml;

v _k = 0,1 ml;

_{(v + v k) / S} p = 0,2 ml / 324 mm ² mm ³ = ^200/2 = 324 mm 0.6 mm,

then A = 4/3 · R;

S _p - 324 mm ² ;

M = 250 g / day;

m = 5 g;

V = 0.3 ml of distilled water;

v = 0.1 ml.

Q = (4031 · 18 · 0.9 g / 1000 mm ³ · 3.14 · (0.03) ² · (4/3 · (0.03)) · 250 g / day · (5 + 0.3 )) / (5 · 0.1) = 19.6 g / day;

Q = 19.6 g / day.

The amount of total fat in feces according to the proposed method is 26 g / day, the amount of fatty acids is 19.6 g / day.

Example No. 3. Patient with malabsorption syndrome. During titrimetric study, the fat in the daily feces amounted to 56 g, of which 50 g / day fatty acids. When studying the ratio of fats using the proposed method, fats were 54 g / day, fatty acids - 48.3 g / day.

Calculation of the amount of total fat:

Since 2R = 2 · 0.04 mm = 0.08 mm;

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = ^100/2 = 324 mm 0.3 mm,

then A = 4/3 · R;

n = 4031 fields of view;

N = 13, i.e. an average of 6 drops of fatty acids in one field of view;

ρ = 0.9 g / cm ³ ;

R = 0.04 mm;

A = 4/3 · R, since 2R = 2 · 0.04 mm;

_{(v + v k) / S} p = 0,1 ml / 324 mm ² mm ³ = ^100/2 = 324 mm 0.6 mm,

then A = 4/3 · R;

S _p - 324 mm ² ;

M = 400 g / day;

m = 5 g;

V = 0.3 ml of distilled water;

v = 0.1 ml.

Q = (4031 · 13 · 0.9 g / 1000 mm ³ · 3.14 · (0.04) ² · (4/3 · (0.04)) · 400 g / day · (5 + 0.3 )) / (5 · 0.1) = 54 g / day;

Q = 54 g / day.

Calculation of the amount of fatty acids:

n = 4031 fields of view;

N = 6, i.e. an average of 6 drops of fatty acids in one field of view;

ρ = 0.9 g / cm ³ ;

R = 0.05 mm;

A = 4/3 · R, since 2R = 0.1 mm;

v = 0.1 ml;

v _k = 0.1 ml;

_{(v + v k) / S} p = 0,2 ml / 324 mm ² mm ³ = ^200/2 = 324 mm 0.6 mm,

then A = 4/3 · R;

S _p - 324 mm ² ;

M = 400 g / day;

m = 5 g;

V = 0.3 ml of distilled water;

v = 0.1 ml.

Q = (4031 · 6 · 0.9 g / 1000 mm ³ · 3.14 · (0.05) ² · (4/3 · (0.05)) · 400 g / day · (5 + 0.3 )) / (5 · 0.1) = 48.3 g / day;

Q = 48.3 g / day.

The proposed method can be used not only to quantify steatorrhea, but also amylorrhea. The volume of starch grains, which have an elongated shape, must be calculated as the volume of two balls. To verify the proposed method for the quantitative determination of amylorrhea, the starch grains contained in 0.1 ml of the test solution, consisting of 2.5 g of starch dissolved in 1000 ml of water, were counted. The number of starch grains in 0.1 ml was 166710. When recalculating the mass of starch grains in 1000 ml using the proposed formula, the mass of starch grains in 1000 ml was 2.8 g.

Thus, due to the lack of methods for titrimetry, gravimetry and fecal lipidograms in clinical laboratories, the high cost of these methods, and the duration and complexity of these methods, the proposed method is important as an alternative method for determining neutral fat and fatty acids in order to diagnose enzymatic insufficiency and impaired absorption of substances in the intestine. At the same time, the proposed method is more accurate and objective than coprological research.

Claims (1)

A method for determining fats in feces, including determining the number of drops of fat in the preparation of fecal emulsion using microscopy, characterized in that it further determines the mass of the test sample of feces (m), the average radius of one drop of fat (R), the volume of the reagent is acetic acid, methylene or Nile blue, coverslip was placed under a piece of glass for microscopy (v _k), the number of feces per day or one defecation (M), the volume of distilled water was added to the sample of fecal mass m for the preparation of fecal emulsion (V), the volume of fecal emus sii, placed under a coverslip piece of glass for microscopy (v), then the calculated amount of fat in the feces of the test (Q) of formula Q = (n · N · ρ · π · R ² · A · M · (m + V)) / (m · V), where Q is the amount of the test substance (total fat, neutral fat, fatty acids) in the studied feces M; n is the number of visual fields in the preparation (with an increase of 7 × 40 4031 visual fields; with an increase of 7 · 10 - 233 fields of view when the area of the glass cover 324 mm ² ); N is the average number of drops (fat, neutral fat, fatty acids) in one field of view; ρ is the density of fats, fatty acids 0.9 g / cm ³ ; R is the average radius of one drop of fat;

S _p is the area of the glass cover (324 mm ² ); V _k - the volume of the reagent (acetic acid, methylene or Nile blue), placed under a coverslip for microscopy; M - the amount of feces per day or one bowel movement; m is the mass of a sample of feces taken for the preparation of fecal emulsion; V is the volume of distilled water added to the fecal sample of mass m for the preparation of fecal emulsion; v is the volume of fecal emulsion placed under a coverslip for microscopy.