-
Categories
-
Pharmaceutical Intermediates
-
Active Pharmaceutical Ingredients
-
Food Additives
- Industrial Coatings
- Agrochemicals
- Dyes and Pigments
- Surfactant
- Flavors and Fragrances
- Chemical Reagents
- Catalyst and Auxiliary
- Natural Products
- Inorganic Chemistry
-
Organic Chemistry
-
Biochemical Engineering
- Analytical Chemistry
- Cosmetic Ingredient
-
Pharmaceutical Intermediates
Promotion
ECHEMI Mall
Wholesale
Weekly Price
Exhibition
News
-
Trade Service
Experimental Purpose:1. To
the basic
of the determination of the
activity of
serum and alanine amino.2. To understand the determination method and clinical significance of serum alanine amino transfer enzyme.Principles ALT catalytic alanine and α-ketone diacids produce acetone and glutamate. The yield of acetone acid, i.e. the size of the reaction enzyme activity.acetone can react with 2,4-dinitrobenzene in an acidic solution to form a corresponding 2,4-dinitrobenzene , yellow, the latter in alkaline conditions red-brown. By measuring its light absorption at 520nm wavelength to understand the production of acetone acid, so as to determine the vitality of serum ALT, so this method is also known as color dating. Although the method also has disadvantages, but the operation is simple, does not need special
and
reagents, so it is widely used in clinical.the unit definition of />/>/>method is that each lml serum functions with the substrate for 30 minutes at pH7.4, 37 degrees C/> insulation and one unit for each 2.5 μg of acetone acid produced. The normal value of alanine amino transferase in human serum is 2 to 40U.Equipment and Reagents Equipment
Thermostat
Water Bath, 721, 722 phosphorescometer, liquid mixer, scale straw, dropper, test tube, etc. 2.Reagents(1) 0.1mol/L phosphate buffer (pH 7.1) 4): Kh2PO4 2.69 g/> and hydrophosphate potassium dioxide K2HPO4.3H2O13.97g/> distilled water dissolved and moved to a 1,000 ml capacity bottle, corrected pH to 7.4, and then added distilled water to the scale. Store in the refrigerator.(2) Alanine amino transferase substrate fluid (pH7.4): accurately named DL-alanine 1.78g/> and α-ketone diacid 29.2g />, first dissolved in 0.1mol/L phosphate buffer about 50 ml, then with 1mol/L NaOH correction pH to 7.4, and then diluted with phosphate buffer to 100 ml. Mix well and store in the fridge for one week.(3) 0.02% 2,4-dinitrobenzene solution (1mM/>): accurately named 2,4-dinitrobenzene 20mg dissolved in 1mo1/L hydrochloric acid, to be dissolved with 1mo1/L hydrochloric acid diluted to 100 ml
used.(4) 0.4M/>/L sodium hydroxide solution.(5) Acetone acid standard liquid (500 sg/ml): precisely called sodium acetone 62.5 mg dissolved in 0.1mol/LH2SO4100ml, this reagent needs to be ready-made.Step 1.1. Production of the standard curve take
dying
clean test tube 9, number, add reagents according to the table, mix well:take another dry clean test tube 11, number, in order to add in the 1 to 9 test tube The above diluted 1 to 9 acetone acid standard liquid 0.1 ml, in the 10th tube to add undiluted acetone acid standard liquid (500 sg/ml) 0.1 ml, and then follow the table operation.mix each tube, set at room temperature for 10 minutes, 520nm wavelength, with the 11th tube as the blank tube color, read each tube light density will each tube acetone acid content (5-50 mg) Pressed conversion into glutamate
transaminase
vitality unit: at every 100 ml of serum alanine amino transferase activity units as horizontal coordinates, light density for ordinates to plot the standard curve. 2. The determination of enzyme activity took 2 dry clean test tubes, indicated the determination tube and control tube, and operated according to the table. Mixing, static 10min, with 10min, at 520nm wavelength, with distilled water to adjust the zero point, read the measurement tube and control tube light density, to determine the tube light density value minus the control tube light density value, and then from the standard curve to find out the vitality unit of its enzyme. Note: 1. The specimen should take blood on an empty stomach and at that time be measured or the separated serum stored in the refrigerator. 2. The results of enzymes are related to enzyme action time, temperature, PH and reagent addition, etc. 3. When measuring reagent replacement, re-create the standard curve. ALT is widely found in
tissue
cells, with the highest level of this enzyme in liver cells. Hepatitis, toxic liver cell necrosis and other liver diseases, the activity of this enzyme in the serum increased, other diseases such as myocardial infarction, myocarditis, etc. also increased. Therefore, the determination of the activity of serum alanine amino transfer glutamate transaminase is of great significance in clinical diagnosis. Note: 1. The significance and measurement method of enzyme vitality When studying the influence of enzyme-promoting reaction speed and various factors on the speed of enzymatic reaction, the energy unit of enzyme is generally observed as an indicator. Enzyme vitality refers to the ability of enzymes to catalyz chemical reactions; the size of enzyme activity is expressed in enzyme vitality units, where an international unit of enzyme is the amount of enzyme that catalyses a 1 mol/L substrate within 25 degrees C/>, or the amount of enzyme that converts 1 micromolar (1 smol) substrate. When determining enzyme vitality, it is often calculated by the production of the product or the consumption of the substrate. Generally to determine the increase in the product is good, because the product from nothing, as long as the method is sensitive enough to accurately determine.
2. Introduction to the method of determining the activity of serum alanine amino transferase One is traphphosphonism and the other is coloration. The former is a method for determining enzyme rate and requires UV tern dph photometrics and enzyme reagents. The latter are King, Mohum and Reitman Frankel.
These three methods are the main methods of measuring ALT in China, their determination principle, reagent and operating steps and temperature are identical, the main differences are the action time of enzymes and substrates, standard curve drawing methods and unit definitions. In the action time, Kim's is 60min, the remaining two methods are 30min. The units of the Kim method are: 100 ml serum with sufficient amount of alanine, α-ketone diacid at pH7.437 degrees C/> insulation 1h, each produced by 1 mol acetone, called an amino alanine transferase vitality unit. The unit definition of the Muss method is: 30min per milliliter of serum insulation with the substrate at pH7.4, 37c/>, and one unit for each 2.5 mg of acetone acid produced. Lai's unit definition is: serum 1 ml, the total liquid response volume of 3 ml, at 25 degrees C />, 340nm light wave, light diameter 1cm/>, 1 min to reduce the light density of 0.001 as a unit. Because different methods measure the normal range of dynamic units of alanine amino transferase, so clinical evaluation of its diagnostic significance, it is necessary to first clarify what the dynamic unit and the normal value of the method, and then to make an accurate conclusion to the test results. the clinical significance 1 and ALT?
2, the normal value of ALT is 7
3, what is the meaning of the control tube? 4, the substrate liquid should be 37 degrees C / > temperature 5min?
.