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How to Calculate the Results of Metabolism Detection

Source: Elabscience®Published: Mar 08,2024

Metabolism detection is a method that uses chemical reactions to detect various products and enzymes in the process of biological metabolism. The main detection targets include carbohydrates, lipids, small organic molecules, inorganic ions, proteins and other substances, as well as various enzymes involved in metabolic reactions. Colorimetry (detection of absorbance) or fluorescence (detection of fluorescence intensity) are usually used for metabolism detection.

The standard curve method is used commonly in the calculation of metabolism detection, which involves preparing a standard series with known concentration of standard substances, determining the absorbance or fluorescence intensity of each standard sample under standard conditions, and a standard curve y=f(x) is established using the absorbance or fluorescence intensity values (Y) and the concentration of the standard substances (X). The absorbance or fluorescence intensity of the test sample is measured under the same conditions, and then the concentration is calculated through the standard curve.

The results of metabolism detection are presented in two main ways: concentration and activity.

01 concentration

The detection of various metabolites is usually indicated by their concentration. For example, when determining the concentration of hydrogen peroxide in serum, the standard curve established by the color development of the standard sample is y=0.0045x+0.02022 (as shown in the figure). The absorbance of the serum sample after color development is measured to be 0.359, then the concentration of hydrogen peroxide in the serum is calculated as (0.359-0.02022)/0.0045=75.28 mmol/L.

When using the standard curve method, the standard substance used can be the same substance as the detected metabolite, or the substance that is generated in equal proportion after a certain biochemical reaction of the detected metabolite. For example, when detecting the concentration of hydrogen peroxide (H2O2) in serum, H2O2 is prepared into a standard sample of known concentration for detection. While detecting the content of triglyceride in serum, glycerol, the hydrolysis product of triglyceride, is generally used as a standard for color development reaction. Glycerol is hydrolyzed to glycerol at a ratio of 1:1 during the detection experiment. Therefore, the glycerol concentration calculated through the detection result is the concentration of triglyceride in the sample.

02 activity

Besides the detection of the concentration of various substances, the results are usually expressed in activity units for the detection of enzymes in metabolic reactions. Enzyme activity detection is also one of the unique detection methods of metabolism detection.

Enzyme activity is usually expressed in the activity unit (U), and different enzyme activity units have different definitions that usually referring to the rate at which the enzyme consumes the reaction substrate and generates products within a unit time under normal physiological conditions. The detection of enzyme activity is also achieved by measuring the rate of change in the concentration of the relevant substrates or products. For example, sucrose catalyzes sucrose hydrolysis to produce glucose, so the rate of glucose concentration increase is used to measure the activity of sucrose. Catalase catalyzes the decomposition of hydrogen peroxide, so the decrease rate in hydrogen peroxide concentration is used to measure catalase activity. In addition to the standard curve method to detect the concentration of substrates or products, special attention should be paid to the reaction time and temperature when measuring enzyme activity, and the total protein content is usually introduced to correct the sample treatment level when detecting tissue sample.

To determine the content of sucrose in rat ileum tissue, its activity unit U is defined as the amount of sucrose hydrolyzed per milligram of tissue protein per minute at 37°C. Using glucose as the standard substance, the standard curve corresponding to the measured absorbance of glucose concentration (mmol/L) was y = 0.0306 x + 0.0025. Within 20 minutes of the reaction time, the sucrose in the sample hydrolyzed the substrate, and the absorbance of glucose detection increased from 0.079 to 0.204. Additionally, the total protein concentration in the sample is measured to be 6.48 mgprot/mL, therefore the activity of sucrose in the ileum sample is “(0.204 - 0.079 - 0.0025) / 0.0306 / 20 / 6.48 * 1000 = 30.89 U/mgprot”.

It should be noted that the detection of enzyme activity in metabolic testing is an assessment of its catalytic ability under simulated physiological conditions (include temperature, pH, ionic strength, cofactors, etc.), and there is no essential relationship with the amount of substances of the enzyme molecule. The possibility exists that an augmentation in the quantity of enzyme substance may result in a reduction of its activity.