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How to calculate the results of metabolic tests

Time：

2023-05-16

Metabolic testing is a method that uses chemical reactions to detect various products and enzymes in the biological metabolic process. Its main detection targets include substances such as sugars, lipids, organic small molecules, inorganic ions, and proteins, as well as various types of enzymes involved in the metabolic reaction process. The assay is usually performed using a colorimetric method (measuring absorbance) or a fluorometric method (measuring fluorescence intensity).

　　Metabolic testing is a method that uses chemical reactions to detect various products and enzymes in the biological metabolic process. Its main detection targets include substances such as sugars, lipids, organic small molecules, inorganic ions, and proteins, as well as various types of enzymes involved in the metabolic reaction process. The assays are usually performed using colorimetric methods (measuring absorbance) or fluorometric methods (measuring fluorescence intensity).

　　In the calculation of metabolic assays, the standard curve method is usually used, i.e., a standard series with known concentration is prepared from standard substances, and the absorbance (or fluorescence intensity) of each standard sample is measured under standard conditions, and a standard curve y=f(x) is established with the absorbance (fluorescence intensity) value (Y) to the concentration (X) of the standard substance under test, and the absorbance (fluorescence intensity) of the sample to be tested is measured under the same conditions, and then The concentration is calculated from the standard curve.

　　The results of the metabolic assay are expressed in two main ways: concentration and activity.

　　01 Concentration

　　For the detection of various metabolites, the concentrations are usually shown. For example, when determining the concentration of hydrogen peroxide in serum, the standard curve established by color development of a standard sample with the equation y=0.0045x+0.02022 (as shown in the figure), the absorbance of the serum sample after color development was measured to be 0.359, and the concentration of hydrogen peroxide in the serum sample was calculated to be: (0.359-0.02022)÷ 0.0045=75.28 mmol/L.

　　When using the standard curve method, the standard substance used can be the same as the metabolite being tested, or it can be a substance generated in equal proportions from the metabolite being tested after a certain biochemical reaction. For example, in the detection of hydrogen peroxide (H2O2) concentration in serum, the H2O2 reagent is used to prepare a standard sample of known concentration for testing. In the case of triglycerides in serum, glycerol, a hydrolysis product of triglycerides, is generally used as a standard for the color development reaction. Triglycerides are hydrolyzed to produce glycerol at a ratio of 1:1 during the assay experiment, so the concentration of glycerol calculated from the assay results is the concentration of triglycerides in the sample.

　　02 Activity

　　In addition to the detection of the concentration of various substances, for the detection of various enzymes in the process of metabolic reactions, the results are usually expressed in activity units. The detection of enzyme activity is also one of the tests unique to metabolic testing.

　　Enzyme activity is usually expressed as activity units (U), and different enzyme activity units have their own definitions, which usually refer to the rate at which the enzyme consumes the reaction substrate and produces the product per unit time under normal physiological conditions. The detection of enzyme activity is also achieved by detecting the rate of change of the relevant substrate or product concentration. For example, sucrase catalyzes the hydrolysis of sucrose to produce glucose, so the rate of increase in glucose concentration is used to determine sucrase activity; catalyze the decomposition of hydrogen peroxide, so the rate of decrease in hydrogen peroxide concentration is used to determine peroxidase activity. In addition to the same standard curve method for the concentration of substrate or product, special attention should be paid to the reaction time and temperature when measuring enzyme activity, and total protein content is usually introduced to correct for the sample treatment level when performing tissue sample assays.

　　For example, for the determination of sucrase content in rat ileal tissue, the activity unit U is defined as 1 nmol of sucrose per minute hydrolyzed per mg of tissue protein at 37°С. Using glucose as a standard substance, the standard curve of absorbance corresponding to the glucose concentration (mmol/L) was measured as y = 0.0306 x + 0.0025. Within the reaction time of 20 min, sucrase in the sample hydrolyzed the substrate, causing an increase in absorbance of the glucose assay from 0.079 to 0.204. In addition, the total protein concentration in this sample was additionally measured as 6.48 mgprot /mL, so the sucrase activity in this ileal sample was (0.204-0.079-0.0025)÷0.0306÷20÷6.48×1000=30.89 U/mgprot.

　　It should be noted that the assay of enzyme activity in the metabolic assay is a test of its catalytic capacity under conditions that simulate its physiological conditions (including temperature, pH, ionic strength, cofactors, etc.) and is not essentially related to the amount of the substance that constitutes the molecule of the enzyme, so it is possible that the amount of the substance of the enzyme increases, but the activity decreases instead.