1. DIAGNÓSTICO SITUACIONAL DEL CANTÓN MACARÁ, PROVINCIA DE LOJA
1.3. Ámbito ecológico territorial
1.3.3. Ecosistemas
Blood samples were collected by cardiac puncture using sterile syringe and needle. The collected blood was put into plain sample bottles (with anti-coagulant) and allowed to clot. The clotted blood meant for serum biochemistry were separated from clear serum by centrifugation at 2000 rpm for 15 minutes and properly labeled. All serum biochemistry determinations were carried out following standard procedure using Randox test kits (Randox United Kingdom). Blood and serum samples were packaged in ice and transported to the Department of Veterinary Medicine laboratory, Faculty of Veterinary Medicine, University of Nigeria, Nsukka.
3.5.1 Alanine amino transferase (ALT)
ALT assay was carried out using the ALT kit from Randox Laboratories, United Kingdom.
The kit is designed for the quantitative in vitro determination of ALT in serum. The kit contains two reagents R1 and R2, R1 contains buffer made up of phosphate buffer 100mmol/L,pH 7.4, L-Alanine 200mmol/l and 𝛼 −oxoglutarate 2.0mml/l. R2 contains 2,4- dinitrophenylhydrazine 2.0mmol/l.
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76 The principle is based on the following biochemical reaction;
𝛼 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 + 𝐿 − 𝑎𝑙𝑎𝑛𝑖𝑛𝑒𝑎𝑝𝑡 𝐿 − 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒
Alanine amino transferase is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4 -dinitrophenylhydrazine ( Oseni et al., 2018 ).Serum sample (0.7ml) was mixed with 0.5ml of R1 solution and incubated for 30minutes at 37oC, 0.5ml of R2 solution was added, the mixture was allowed to stand for 20 minutes at 20 to 25oC (room temperature). Sodium hydroxide (5ml) was added and mixed. The absorbance of the serum sample was read against the reagent blank, after 5 minutes at 540nm using a UV spectrophotometer. The reagent blank contains 0.5ml R1
solution, 0.1ml distilled water, 0.5ml R2 solution and 5ml sodium hydroxide. The ALT activity in the serum was obtained from the matching table and expressed in international unit per litre (iu/l).
3.5.2 Aspartate amino transferase (AST)
AST assay was carried out using the AST kit from Randox Laboratories, United Kingdom. The kit being designed for the quantitative in vitro determination of AST in serum contains two reagents R1 and R2.R1 contains buffer made up of phosphate buffer 100mmol/l, pH 7.4, L-Aspartate 100mmol/l.R2 contains 2,4- dinitrophenylhydrazine 2.0mmol/l.
The principle is based on the following reaction;
𝛼 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 + 𝐿 − 𝑎𝑠𝑝𝑎𝑟𝑡𝑎𝑡𝑒𝐺𝑂𝑇 𝐿 − 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒
Aspartate amino transferase (AST) is measured by monitoring the concentration of oxaloacetate by hydrazone formed with 2,4- dinitrophenylhydrazine (Oseni et al., 2018). Serum sample (0.1ml) was mixed with 0.5ml of R1 solution and incubated for 30minutes at 37oC. R2(0.5ml) was added, the
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77 mixture was allowed to stand for 20mins at 20-250C (room temperature). Sodium hydroxide (5.0ml) was added and the absorbance of the serum sample was read against the reagent blank after 5mins at 540nm using UV spectrophotometer. The blank reagent contains 0.5ml R1, 0.1ml distilled water, 0.5ml R2 and 5.0ml sodium hydroxide. The activity of the AST in the serum was obtained from the matching table and expressed in international unit per litre (iu/l).
3.5.3 Alkaline phosphatase (ALP)
ALP assay was carried out using the ALP kit from Randox Laboratories, United Kingdom.The ALP kit was designed for the quantitative in vitro determination of alkaline phosphatase (ALP) in serum and plasma. The kit employs a colorimetric method which is an optimized standard method according to Deutsche Geseilschaftfur Klinische chemie (DGKC) (1972) which is a kinetic method.The kit contains 2 reagents R1,a buffer which contains diethanolamine buffer 1mmol/L, pH 9.8 and magnesium chloride 0.5mmol/l and R2 which is p-nitrophenylphosphate 10 mmol/l. The principle is based on the following biochemical reaction;
𝑃 − 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑦𝑙𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐻20𝐴𝐿𝑃 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝑃 − 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑙
The alkaline phosphatase present in the serum sample catalyses the hydrolysis P- Nitrophenylphosphate (PNPP) during which P- Nitrophenol and Phosphate are released.Mg 2+ ions enhance the activity.The increasein absorbance at 405nm correlates with the activity of serum alkaline phosphatase according to assay kit manufacturer’s instructions. Serum sample (0.02ml) was mixed with 1.00ml of R1 at 30oC. The initial absorbance of the mixture was read at 405nm, after which other absorbances were read after 1, 2 and 3 minutes using a UV spectrophotometer to
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78 determine the change of optical density (∆A). The ALP activity was calculated using the formula below,to express the value of ALP in iu/L.
𝑖𝑢
𝐿 = 2760 × ∆𝐴405𝑛𝑚/𝑚𝑖𝑛.
3.5.4 Determination of serum urea
Serum urea was determined using the urea Randox Kit from Randox Laboratories United Kingdom. The method is called Urease-Berthelot Colorimetric method for the quantitative invitro determination of urea in serum, plasma and urine.The kit contains four reagents R1a 1.0ml Urease R1b 37ml sodium nitroprusside ,R2 110ml phenol concentrate,R3 22ml hypochlorite concentrate, and R4 5.5ml standard calibrator. It is based on the principle of hydrolysis. Serum urea is hydrolysed to ammonia in the presence of urease. The ammonia is then measured photometrically by Berthelot’s reaction;
𝑢𝑟𝑒𝑎 + 𝐻20𝑢𝑟𝑒𝑎𝑠𝑒 2𝑁𝐻3+𝐶𝑂2
𝑁𝐻3+ ℎ𝑦𝑝𝑜𝑐ℎ𝑙𝑜𝑟𝑖𝑡𝑒 + 𝑝ℎ𝑒𝑛𝑜𝑙 → 𝑖𝑛𝑑𝑜𝑝ℎ𝑒𝑛𝑜𝑙 𝑏𝑙𝑢𝑒 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑
10µl of the serum sample was mixed with reagent 1 and incubated at 37oC for 10 minutes. Reagent 2(2.50ml) and reagent 3(2.50ml) were added immediately and incubated at 37oC for 15 minutes. The standard calibrator(10µl) was mixed with 100µl of reagent 1 and incubated at 37oC for 10 minutes.
The absorbance of sample (A sample) and standard (A standard) was read against the blank using a UV spectrophotometer at 540nm.Serum urea concentration was obtained by applying the formula below and the serum urea was measured in mmol/l or mg/dl.
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79 𝑠𝑒𝑟𝑢𝑚 𝑢𝑟𝑒𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝐴 𝑠𝑎𝑚𝑝𝑙𝑒
𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑× 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑜𝑛𝑐.
= mg/dl or mmol/L
3.5.5 Determination of lead concentration in Clarias gariepinusmuscle
Lead level analysis in the fish muscle was conducted using Agilent FS 240 Atomic Absorption Spectrophotometer (AAS) according to the method of American Public Health Association APHA (2012).Fish sample (5g) was weighed into a digestion flask and 20ml of acid mixture (650ml conc. HNO3, 80ml Perchloric acid and 20ml conc. H2SO4) was added to the flask.
The flask was heated until a clear digest was obtained. The digest was diluted with distilled water to the 100ml mark. 100ml of the diluted digested sample was transferred into a glass beaker of 250ml volume, to which 5ml of concentrated nitric acid was added and heated to boil till the volume reduced to about 20ml. Adding concentrated Nitric acid in increments of 5ml resulted to complete dissolutions of all residues. The mixture was cooled, transferred and made up to 100ml using metal free distilled water. The sample was aspirated into the oxidizing air-acetylene flame, to observe the sensitivity for absorption. Standard lead solutions in the optimum concentration range were prepared.
The quantity of lead in the digested fish sample was read from the AAS in µg/l (ppm) 3.6 Histopathological examinations
The gills, liver and stomach of the experimental fish were collected and fixed in 10% formal saline. They were dehydrated in graded alcohol, cleared in xylene and embedded in paraffin wax.
Cut sections (5µm thick) were stained with haematoxylin and eosin (H&E) stain for histological
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80 examination using light microscope (Bancroft and Stevens, 1977). Photomicrographs of histological sections were taken with a Moticam image plus 2.0 digital cameracameras.