Hepatic lipase (HL) Kit za ispitivanje aktivnosti

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar

Mačka ne: BC2380

Veličina:50T/24s

Komponente:

Reagens i: 100 ml × 1. Skladištenje na 4 ℃.

Reagens II: 3 ml × 1. Skladištenje na 4 ℃.

Reagens iii: Prah × 1. Skladištenje na 4 ℃. Prije upotrebe, dodati 30 mL of distilled water, fully dissolve.

Reagens IV: Powder×2. Skladištenje na -20 ℃. Prije upotrebe, dodati 2 mL of distilled water to the one, fully dissolve. The dissolved reagent can be stored at -20 °C after repacking. Avoid repeated freeze-thaw cycles;

Standard: Prah × 1. Prije upotrebe, 6.94 mL of aceton is added to prepare a 10 μmol/mL α-naphthol

standard solution, which was fully dissolved before use.

Opis proizvoda:

Hepatic lipase (HL) is a lipolytic enzyme synthesized in liver parenchymal cells. It is present on the surface of the liver sinusoidal endothelial cells and the surface of the hepatocyte microvilli in the sinusoidal space, and can hydrolyze various lipoproteins. The triglycerides (Tg) and phospholipids (PL) in the medium change the size and density of various lipoprotein particles. When the HL and its activity in the plasma increasing, it can lead to low density lipoprotein (LDL) levels in the plasma, increase and accelerate the occurrence and development of atherosclerosis.

HL hydrolyzes α-naphthyl acetate to produce α-naphthol, which can form a purple-red azo compound with fast blue B salt. It has a characteristic absorption peak at 595 NM, and its color depth is positively correlated with liver esterase activity within a certain range.

Potrebni reagensi i oprema, ali nisu pruženi:

Spektrofotometar, vodena kupelji, uravnotežiti, centrifuga, adjustable transferpettor, 1 ML staklena kiveta, minobacač/homogenizator, ultrasonic crusher, led i destilirana voda.

Postupak:

Ja. Enzim izvlačenje

1. Tkivo

According to the tissue mass (g): the volume of Reagent I (ml) je 1:5~10 to extract. It is recommended to add 1 mL of Reagent I to 0.1 g tkiva, and fully homogenize on ice bath. Centrifuge at 10000g for 10 minuta u 4 ℃ za uklanjanje netopljivih materijala, i prije testiranja uzmite supernatant na ledu.

2. Bakterije ili stanice

According to the bacteria or cells (104): the volume of Reagent I (ml) is 500~1000:1. It is recommended to add 1 mL of Reagent I to 5 million of bacteria or cells. Use ultrasonication to splitting bacteria and cells (stavljen na led, ultrasonic power 300W, Radno vrijeme 3s, Interval 7s, ukupno vrijeme 3 min). Centrifuga na, 10000g for 10 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

3. Culture medium or other liquid: Otkrijte izravno.

Ii. Otkrivanje

• Predgrijan spektrofotometar za 30 minuta, Podesite valnu duljinu na 595 NM, Postavite nulu s destiliranom vodom.
• Preheat reagent III at 30℃ for more than 20 minuta.
• Standard: Dilute the 10μmol/mL standard solution to 1.25, 0.625, 0.3125, 0.15625, 0.078μmol/mL with reagent I.
• Add the following reagents in 1.5 mL EP tubes:

	Contrast tube (C)	Epruveta (T)	Standard (S)	Prazna cijev (B)
Uzorak (μL)	100	100	–	–
Standard solution (μL)	–	–	100	–
Reagens i (μL)	450	400	400	500
Reagens II (μL)	–	50	50	50
Mix and react for 10 min at 30℃			–	–
Reagens iii (μL)	400	400	400	400
Reagens IV (μL)	50	50	50	50
Mix thoroughly and detect the absorbance at 595 NM, record as AC, NA, AS and AB respectively. ΔAT=(AT-AC), ΔAS=(AS-AB). A contrast tube is required for each test tube, and the standard curve need only be tested once or twice.

Iii. Izračunavanje:

1.Standard curve

The concentration of standard solution as x-axis, ΔAS as y-axis, obtain the equation y=kx+b. Take ΔAT to the equation to acquire x (μmol/ml) value.

2. Izračunavanje

• Tissue protein concentration

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every mg of protein in the reaction system per minute at 40℃.

HL Activity (U/Mg Prot)=x×Vs÷（Vs×Cpr）÷T =0.1x÷ Cpr

• Tissue weight

Definicija jedinice: One unit of enzyme activity is defined as the amount enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every gram of tissue in the reaction system per minute at 40℃.

HL Activity (U/g težina) = x×Vs÷(W×Vs÷Ve)÷T =0.0333x÷ W

• Liquid

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every milliliter of liquid sample in the reaction system per minute at 40℃.

HL Activity (U/ml) =x× Vs÷Vs÷T=0.1x

• Bacteria or cultured cells

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every 104 cells or bacteria in the reaction system per minute at 40℃.

HL Activity (U/104 cell) =x× Ve÷ cell amount÷ T= 0.1x÷ cell amount

Vs: Volumen uzorka (ml), 0.1 ml; VE: Extract solution volume, 1 ml;

CPR: Supernatant sample protein concentration (mg/ml); T: Vrijeme reakcije (min), 10 minuta;

W: Težina uzorka, g;

Količina ćelije: 10 thousand as unit.

Bilješka:

1. If the sample is animal liver, it is recommended to dilute the sample with reagent I more than 25 times before testing, and multiply the dilution factor in the calculation
2. If the sample is serum or plasma from obese animals, it is recommended to dilute the sample with reagent I more than 5 times before testing, and multiply the dilution factor in the calculation
3. When ΔA is greater than 0.8, it is recommended to measure the sample after diluting it with the reagent, and multiply it by the dilution factor in the calculation

Eksperimentalni primjer:

1. 1g rat liver was taken for sample processing, and the supernatant is diluted 24 vrijeme, then the operation is carried out according to the operation steps. Measured and calculated by 96 bunar ploča: ΔA = AT-AB = 0.713-0.001=0.712, and the standard curve: y = 0.6381x – 0.0005, calculate x =1.1166

HL activity (U/g masa) = x×VS ÷ (W×VS ÷ VST)÷ T ×48 = 53.597 U/g masa.

2. After the turkey serum was diluted 6 vrijeme, the operation was carried out according to the operation steps. Measured and calculated by 96 bunar ploča: ΔA = AT-AB =0.572-0.003=0.569, and the standard curve: y = 0.6381x – 0.0005, calculate x =892

HL activity (U/g masa) = x×VS ÷ (W×VS ÷ VST)÷ T ×12 = 1.071 U/g masa

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