3.4.1.3 Procedure


Mix well and read the optical density against purified water in a photometer at 505 nm, within 15 min.

Calculation:

AST GPT ð Þ activity IUð Þ =L

<sup>¼</sup> absorbance of test � absorbance of control � concentration of standard absorbance of standard � absorbance of blank

(3)

### 3.4.2 Estimation of SGOT (Span Diagionostics Ltd., Surat, India)

#### 3.4.2.1 Principle

Aspartate aminotranferase (AST) catalyses the transamination of L-aspartate and α-ketoglutarate to form L-glutamate and oxaloacetate. Oxaloacetate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured colorimetrically [14].

Mix well and read the optical density against purified water in a photometer at

Chemoprotective Effect of Edible Gastropod, Xancus pyrum and Its Usefulness…

<sup>¼</sup> absorbance of test � absorbance of control � concentration of standard absorbance of standard � absorbance of blank

Urea is converted quantitatively to ammonia and CO2 in the presence of urease. The ammonium ions react with the phenolic chromogen and hypocrite to give a green coloured complex. The intensity of the colour formed is measured at 578 nm and is directly proportional to the concentration of urea in test specimen [15].

Ammonia þ phenolic chromogen þ hypochlorite ! green coloured complex

i. Transfer the contents of one vial of Reagent 1 to the bottle of Reagent 3.

Pipette into clean and dry test tubes labelled as blank (B), standard (S) & test (T):

Addition sequence Blank Standard Test Working reagent 1000 μL 1000 μL 1000 μL Distilled water 10 μL — —

ii. Rinse 1 Urea vial properly with 3 Urea diluents and mix gently.

iii. Store these working reagents at 2–8°C when not in use.

iv. 2 Urea reagent and Urea standard are ready to use.

Urea þ H2O ! ammonia þ CO2 (7)

3.5 Effect of Xancus pyrum on the biochemical parameters after cisplatin

(6)

(8)

505 nm, within 15 min. Calculation:

administration

3.5.1.1 Principle

3.5.1.2 Reagents

3.5.1.4 Procedure

59

Reagent 1: Urea (Enzyme reagent). Reagent 2: Urea (Chromogen reagent). Reagent 3: Urea diluents (buffer). Reagent 4: Urea standard (50 mg/dL).

3.5.1.3 Working Reagent preparation

AST GOT ð Þ activity IUð Þ =L

DOI: http://dx.doi.org/10.5772/intechopen.88655

where concentration of standard = 114 IU/L.

3.5.1 Estimation of Urea (Span Diagnostics Ltd., Surat, India)

L � aspartate þ α � ketoglutarate ⇄ oxaloacetate þ L � glutamate (4)

Oxaloacetate þ 2, 4 � DNPH ⇄ corresponding hydrazone brown colour ð Þ (5)

#### 3.4.2.2 Reagents


Working reagent preparation:


## 3.4.2.3 Procedure


Chemoprotective Effect of Edible Gastropod, Xancus pyrum and Its Usefulness… DOI: http://dx.doi.org/10.5772/intechopen.88655

Mix well and read the optical density against purified water in a photometer at 505 nm, within 15 min.

Calculation:

3.4.2 Estimation of SGOT (Span Diagionostics Ltd., Surat, India)

Invertebrates - Ecophysiology and Management

Aspartate aminotranferase (AST) catalyses the transamination of L-aspartate and α-ketoglutarate to form L-glutamate and oxaloacetate. Oxaloacetate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured

L � aspartate þ α � ketoglutarate ⇄ oxaloacetate þ L � glutamate (4) Oxaloacetate þ 2, 4 � DNPH ⇄ corresponding hydrazone brown colour ð Þ (5)

2. 2,4-DNPH colour reagent 2,4-dinitrophenyl hydrazine

3. Sodium hydroxide, 4 N Sodium hydroxide 4. Working pyruvate standard, 6 mM (114 IU/L) Sodium pyruvate

• Solution I: Dilute 1 mL of Reagent 3 to 10 mL with purified water.

Pipette into tube marked Blank Standard Test Control

Reagent 1 0.25 0.25 0.25 0.25 Serum — — 0.05 — Standard — 0.05 — —

Reagent 2 0.25 0.25 0.25 0.25 Deionized water 0.05 — —— Serum — —— 0.05

Solution I 2.5 2.5 2.5 2.5

Mix well and allow to stand at room temperature (15–30°C) for 20 min

Phosphate buffer Preservative Stabilizer

Preservative Stabilizer

Preservative Stabilizer

Reagent no. Reagents Composition 1. Buffered aspartate-α-KG substrate, pH 7.4 L-aspartic acid α-KG

3.4.2.1 Principle

colorimetrically [14].

Working reagent preparation:

Mix well and incubate at 37°C for 30 min

• Reagent 1, 2 and 4 are ready to use.

3.4.2.2 Reagents

3.4.2.3 Procedure

Volume in mL

58

$$\begin{aligned} & \text{AST (GOT) activity (IU/L)}\\ &= \frac{\text{absolute of test} - \text{absolute of control} \times \text{concentration of standard}}{\text{absolute of standard} - \text{absolute of blank}} \end{aligned} \tag{6}$$

where concentration of standard = 114 IU/L.
