Hepatic lipase (HL) Kit di analisi dell'attività
Nota: Prendi due o tre campioni diversi per la previsione prima del test.
Attrezzatura operativa: Spettrofotometro
Gatto n: BC2380
Misurare:50T/24S
Componenti:
Reagente I: 100 ml×1. Conservazione a 4 ℃.
Reagente II: 3 ml×1. Conservazione a 4 ℃.
Reagente III: Polvere×1. Conservazione a 4 ℃. Prima dell'uso, aggiungere 30 mL of distilled water, fully dissolve.
Reagente IV: Polvere×2. Storage at -20℃. Prima dell'uso, aggiungere 2 mL of distilled water to the one, fully dissolve. The dissolved reagent can be stored at -20 °C after repacking. Evitare ripetuti cicli di congelamento-scongelamento;
Standard: Polvere×1. Prima dell'uso, 6.94 mL of acetone is added to prepare a 10 μmol/mL α-naphthol
standard solution, which was fully dissolved before use.
Descrizione del prodotto:
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 (T.G) 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.
Reagenti e attrezzature necessari ma non forniti:
Spettrofotometro, bagnomaria, bilancia, centrifuga, adjustable transferpettor, 1 Cuvetta di vetro da ml, mortaio/omogeneizzatore, ultrasonic crusher, ghiaccio e acqua distillata.
Procedura:
IO. Enzima extraction
- Tessuto
According to the tissue mass (G): the volume of Reagent I (ml) È 1:5~10 to extract. It is recommended to add 1 mL of Reagent I to 0.1 g of tissue, and fully homogenize on ice bath. Centrifuge at 10000g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
- Batteri o cellule
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 (placed on ice, ultrasonic power 300W, working time 3s, intervallo 7s, tempo totale 3 min). Centrifugare a, 10000g for 10 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.
- Culture medium or other liquid: Rileva direttamente.
II. Rilevamento
- Preriscaldare lo spettrofotometro per 30 minuti, regolare la lunghezza d'onda su 595 nm, azzerare con acqua distillata.
- Preheat reagent III at 30℃ for more than 20 minuti.
- 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) | Provetta (T) | Tubo standard (S) | Tubo vuoto (B) | |
| Campione (μL) | 100 | 100 | – | – |
| Soluzione standard (μL) | – | – | 100 | – |
| Reagente I (μL) | 450 | 400 | 400 | 500 |
| Reagente II (μL) | – | 50 | 50 | 50 |
| Mix and react for 10 min at 30℃ | – | – | ||
| Reagente III (μL) | 400 | 400 | 400 | 400 |
| Reagente IV (μL) | 50 | 50 | 50 | 50 |
| Mix thoroughly and detect the absorbance at 595 nm, record as AC, A, 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. Calcolo:
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. Calcolo
- Tissue protein concentration
Definizione di unità: 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
Definizione di unità: 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 (Peso U/g) = x×Vs÷(W×Vs÷Ve)÷T =0.0333x÷ W
- Liquido
Definizione di unità: 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
Definizione di unità: 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
Contro: Volume del campione (ml), 0.1 ml; Ve: Estrarre il volume della soluzione, 1 ml;
Cpr: Supernatant sample protein concentration (mg/ml); T: Tempo di reazione (min), 10 minuti;
W: Peso del campione, G;
Quantità di celle: 10 thousand as unit.
Nota:
- 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
- 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
- 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
Esempio sperimentale:
- 1g rat liver was taken for sample processing, and the supernatant is diluted 24 volte, then the operation is carried out according to the operation steps. Measured and calculated by 96 bene piatto: ΔA = AT-AB = 0.713-0.001=0.712, and the standard curve: y = 0.6381x – 0.0005, calculate x =1.1166
HL activity (Massa U/g) = x×VS ÷ (W×VS ÷ VST)÷ T ×48 = 53.597 Massa U/g.
- After the turkey serum was diluted 6 volte, the operation was carried out according to the operation steps. Measured and calculated by 96 bene piatto: ΔA = AT-AB =0.572-0.003=0.569, and the standard curve: y = 0.6381x – 0.0005, calculate x =892
HL activity (Massa U/g) = x×VS ÷ (W×VS ÷ VST)÷ T ×12 = 1.071 Massa U/g
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