RU2640929C1 - Method for cholesterol content determination in blood serum by dynamic surface tension results - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: dynamic surface tension of a 1-3 ml blood serum sample is measured on a tensiometer operating by the principle of maximum pressure in the vial, the cholesterol content ([Cholesterol], mmol/l) is determined from the obtained dynamic surface tension values using the regression-correlation analysis formulas [Cholesterol]=0.25σ₁-0.22σ₂-0.1λ₀+0.12λ₁+0.71, where σ₁ [mN/m] is the surface tension at t=0.02 seconds, σ₂ [mN/m] is the surface tension at t=1 second, λ₀ [mN/m⋅s^1/2] is the angle of inclination of the initial section of the curve in the coordinates σ/(t^1/2). λ₁ [mN/m⋅s^-1/2] is the angle of inclination of the final section of the curve in the coordinates σ/(t^-1/2).

EFFECT: increased accuracy of determination.

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Description

The invention relates to the field of veterinary medicine, in particular to a method for determining the serum cholesterol content, which allows to determine the cholesterol content in it based on the measurement of the dynamic surface tension of the blood serum of cows using a regression method.

The development of veterinary science presents new requirements for the diagnosis, treatment and prevention of diseases in animals. Using methods of early, preclinical diagnosis can reduce treatment costs and prevent a decrease in the productivity of farm animals. The determination of the important lipid component of the blood, cholesterol, is currently carried out by costly biochemical methods, most often by the enzymatic colorimetric method. Principle of the method: during the hydrolysis of cholesterol esters, free cholesterol is formed. Cholesterol formed as a result of hydrolysis and present in the sample is oxidized by atmospheric oxygen under the action of cholesterol oxidase to form equimolar amounts of hydrogen peroxide. Under the action of peroxidase, hydrogen peroxide oxidizes chromogenic substrates with the formation of a colored compound, the color intensity of which is directly proportional to the concentration of cholesterol in the sample and is measured photometrically.

The regression method is actively used in foreign practice to model biological processes (Rossau CD., Mortensen R.V. Risk factors for suicide in patients with schizophrenia: tested case-control study. The British Journal of Psychiatry. October 1997, vol. 171 (4 ). P. 355-359; Abraham HD, Degli-Esposti S, Marino L. Seroprevalence of hepatitis C in a sample of middle class substance abusers. Journal of Addictive Disease 1999. Vol. 18 (4). P. 77-87 ; Watson P., Petrie A. Statistics for Veterinary and Animal Science. John Wiley & Sons, 2013. 408 p.), While in domestic literature it is much less common (Kurilovich S.A., Reshetnikov O.V. , Shakhmatov SG et al. Prevalence and risk factors for gallstone development diseases in the female population of Novosibirsk. // Therapeutic archive. - 2000. No. 2. P. 21-26). RF patent No. 245692, published on July 27, 2012; RF patent No. 2277243, publ. 05/27/2006). Also relatively new for veterinary medicine is the method for determining serum DPN. There is a method of determining the surface tension of human blood serum (VN Kazakov, OV Sinyachenko, GA Ignatenko and others. Interfacial tensiometry of biological fluids in therapy. - Donetsk: Donetsk, 2003, 584 S.) to determine the effectiveness ongoing treatment. In veterinary practice, the determination of dynamic surface tension is used to assess the readiness of horses for competition (RF Patent, No. 2336524, G01N 33/49, publ. 20.10.2008).

A known method of determining fat and protein in milk according to the results of dynamic surface tension using a regression model (RF Patent, No. 2600820, G01N 33/04, published October 27, 2016). However, the determination of serum cholesterol by measuring its dynamic surface tension is not known in the art.

Fats or triglycerides - natural organic compounds, complete esters of glycerol and monobasic fatty acids; enter the class of lipids. Cholesterol is a naturally occurring lipophilic alcohol found in the cell membranes of all animal organisms. It is a secondary monohydric aromatic alcohol, with a polycyclic core cyclopentane perhydrophenanthrene. The chemical structure of triglycerides and cholesterol are significantly different.

In milk, fat is in the form of so-called "fat globules", rather large formations (2.5-5.0 microns in diameter) of complex structure. The main component of milk fat globules that make up the core are triglycerides, other fat components are located in the shell of fat globules and are present in small amounts (1% are phospholipids, 0.012-0.014% are cholesterol and other sterols).

The blood cholesterol content is much higher. It is found in serum as part of complexes of different densities with proteins. Cholesterol in such complexes can be from 5% (in chylomicrons) to 40-50% (in LDL and HDL), which in total amounts to 5 mmol of cholesterol in a liter of blood serum. With high cholesterol in the blood, hypercholesterolemia occurs - this is a symptom of the following diseases: coronary heart disease, myocardial infarction, atherosclerosis, liver disease, kidney disease, chronic pancreatitis, pancreatic cancer, diabetes mellitus, hypothyroidism, obesity, growth hormone deficiency (STH) and etc. Therefore, the control of this biochemical parameter in the blood has important diagnostic value.

Typically, serum components are determined biochemically. The biochemical method of analysis is based on color reactions, during which the color of the reaction medium changes. In turn, the intensity of the color change is proportional to the content of the metabolite in the test material and is determined photometrically.

A prerequisite for such an analysis is the initial transparency of the blood serum and a slightly yellowish tint. Thus, the method has limitations in the presence of, for example, chiles in the test sample - in this case, clouding is due to an increased level of triglycerides in the blood serum, which gives it a turbidity, white color, creamy consistency. At the same time, it is impossible to isolate individual components, and therefore to conduct an analysis. Similar problems arise when there are destroyed red blood cells in the sample (hemolysis) or an excess of bile pigment concentration (ictericity).

Interfacial tensiometry allows measurements in colored media, which greatly expands the possibilities of biochemical analysis.

The proposed method is aimed at determining cholesterol depending on the dynamic surface tension of blood serum, measured by the method of maximum pressure in the bubble on the BPA-1P tensiometer using the formulas of regression-correlation analysis. The method will significantly reduce costs not only for expensive instruments for biochemical research, but also for chemical reagents used for analysis, as well as to accelerate the receipt of the result.

The technical result achieved by the implementation of the proposed method is a significant reduction in cost, acceleration, simplification of the determination of cholesterol in blood serum based on measurements of dynamic surface tension (DPN).

The specified technical result is achieved by the fact that a blood serum sample of any quality (including the presence of chylosis, hemolysis and ikterichnost) with a volume of 1-3 ml measure the dynamic surface tension on a tensiometer operating on the principle of maximum pressure in the bubble. Not only fresh blood serum can be used, but also after two or three times freezing and after storage in the refrigerator for up to 4 days; when using the biochemical method of analysis, only a single freezing is allowed. Based on the obtained values of dynamic surface tension, the serum cholesterol content is determined using regression-correlation analysis formulas that determine the relationship between the serum cholesterol content and the parameters of its dynamic surface tension. To measure dynamic surface tension, a blood serum sample is introduced into the measuring cell of the device.

DPN is determined on a BPA-1P tensiometer (Maximum Bubble Pressure Tensiometer) (Germany, Sinterface Technologies). Significant advantages of this technique are: ease of use of equipment and software of the device; lack of consumables, which affects the total cost of the study; setup and calibration of the device passes through water, which does not require additional costs for calibrators and norms; the possibility of using preservatives in work, which allows for delayed research; the ability to use already measured samples for other studies.

The principle of operation of the device is quite simple. Air from the compressor enters the capillary, which is lowered into the test fluid (blood serum). Using an electric converter, the excess pressure in the system (the maximum pressure in the bubble) is determined, which is used to calculate the surface tension. Electrical signals from the measuring systems are fed to an electronic unit, which is connected to a personal computer via an analog-to-digital converter. As a result of the measurements, the device allows you to obtain graphs of the dependence on the surface tension time in the boundary fluid layer. On the tensiograms, using the program, the points corresponding to t → 0 (σ ₀ ), t = 0.02 s (σ ₁ ), t = 1 s (σ ₂ ) are determined, and the equilibrium PN t → ∞ (σ ₃ ) is also calculated by extrapolation of the curve in the coordinates σ / (t ^-1/2 ). In the course of computer data processing (ASD program), the coordinate system changes: when determining the angle of inclination of the initial portion of the curve (λ ₀ ), the coordinates σ / (t ^1/2 ) are used, and for the final portion of the curve (λ ₁ ) the coordinates σ / (t ^{- 1/2} ).

In this way,

σ ₀ [mN / m] surface tension at the start of measurement (t → 0),

σ ₁ [mN / m] - surface tension at a time t = 0.02 seconds,

σ ₂ [mN / m] - surface tension at a time t = l second,

σ ₃ [mN / m] - surface tension at a time t = 10 seconds,

λ ₀ - [mN / m⋅s ^1/2 ] the slope of the initial portion of the curve in the coordinates σ / (t ^1/2 )

λ ₁ - [mN / m⋅s ^-1/2 ] the slope of the end of the curve in the coordinates σ / (t ^-1/2 )

To determine the effect of the amount of cholesterol in blood serum on the parameters of its DNA, a correlation analysis is performed between them in Microsoft Excel.

Based on the regression model, the following formula for calculating

[Cholesterol] = 0.25σ ₁ -0.222 ₂ -0.1λ ₀ + 0.12λ ₁ +0.71,

where σ ₁ [mN / m] is the surface tension at a time t = 0.02 seconds,

σ ₂ [mN / m] - surface tension at time t = 1 second

λ ₀ [mN / m⋅s ^1/2 ] is the slope of the initial portion of the curve in the coordinates σ / (t ^1/2 ),

λ ₁ [mN / m⋅s ^-1/2 ] is the angle of inclination of the final section of the curve in the coordinates σ / (t ^-1/2 ).

To simplify the calculation process, a statistical data processing program R (version 3.1.2.) Is used.

Obtained according to the proposed method, the data can be used to determine the cholesterol content in blood serum, which allows you to establish the state of the animal organism.

The measurement procedure for DPN is as follows: the test sample of blood serum (1-3 ml) is poured into a glass cup with a diameter of 1 cm, a height of 2 cm, placed under the capillary of the device (capillary radius 0.13 mm), the software starts and the measurement procedure begins.

The invention is illustrated by the following examples.

Example 1

According to the procedure described above, the dynamic surface tension of the blood serum of a black-motley cow was determined, sample 1. The parameters of its DPN were obtained: σ ₁ - 72.22 mN / m, σ ₂ - 64.18 mN / m; tilt angles: λ ₀ - 8.56 mN⋅m ^-1 s ^-1/2 and λ ₁ - 13.84 mN⋅m ^-1 s ^1/2 . In parallel, for comparison, the cholesterol content was measured at 5.5 mmol / L using the well-known biochemical method on a URIT-8030 biochemical analyzer (Urit Medical Electronic Co., Ltd, China).

According to the above formula, the calculation of cholesterol values (mmol / l)

[Cholesterol] = 0.25⋅72.22-0.22,264.18-0.1⋅8.56 + 0.12⋅13.84 + 0.71 = 5.45 mmol / L.

Thus, the cholesterol content determined by the comparative biochemical method and by the proposed method is practically the same (the difference is 0.9%).

The determination of dynamic surface tension allows a more complete description of the physicochemical properties of blood serum, which can be used in measures to control the physiological state and health of animals.

The study was carried out at the expense of the Russian Science Foundation (project No. 14-16-00046).

Claims (6)

A method for determining the cholesterol content in the blood serum of cows, characterized in that a 1-3 ml blood serum sample measures the dynamic surface tension with a tensiometer operating on the principle of maximum pressure in the bubble, and the cholesterol content is determined from the obtained values of the dynamic surface tension ([Cholesterol] , mmol / l) in blood serum using the formulas of regression-correlation analysis [Cholesterol] = 0.25σ ₁ -0.222 ₂ -0.1λ ₀ + 0.12λ ₁ +0.71, where σ ₁ [mN / m] is the surface tension at a time t = 0.02 seconds, σ ₂ [mN / m] - surface tension at a time t = 1 second, λ ₀ [mN / m⋅s ^1/2 ] is the slope of the initial portion of the curve in the coordinates σ / (t ^1/2 ), λ ₁ [mN / m⋅s ^-1/2 ] is the angle of inclination of the final section of the curve in the coordinates σ / (t ^-1/2 ).