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Sanni et al. Effects of chronic administration of on the activity of alanine aminotransferase of rat kidney. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 May (Supplement 1); 30(30): S65-S71.

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ISSN NO- 2230 – 7885 CODEN JPBSCT NLM Title: J Pharm Biomed Sci.

Sanni et al. . Available at jpbms.info

Original article Effects of chronic administration of indomie noodles on the activity of alanine aminotransferase of rat kidney

1SANNI, Momoh, 1EJEMBI, Daniel, 2EMMANUEL, T. Friday, 1ABBAH, Okpachi C & *OGALA, Emmanuel

Affiliation:- consists six (6) rats (i.e both the males and the 1Department of biochemistry, Kogi State University, females).The rats in group I, for both sex, were Anyigba, . administered cooked indomie spiced with the 2Department of medical biochemistry, Kogi State seasoning, group II, were given raw indomie University, Anyigba, Nigeria. spiced with the seasoning, group III, were given both the cooked and raw indomie mixed Author’s contributions-All authors contributed together and spiced with the seasoning, group equally to this paper. IV, were given cooked indomie without the seasoning, group V, were given raw indomie *Correspondence to:- without the seasoning, group VI, were given both EJEMBI Daniel. the cooked and raw indomie, mixed together, Department of Biochemistry, Kogi State University, without the seasoning and, group VII, which Anyigba, Nigeria. served as the control, were given the normal fowl formulation feed. The administration was Abstract: carried out for a period of four (4) weeks, the The effects of chronic administration of Indomie result showed that, there was a decrease in instant noodles on the activities of alanine kidney activity of the enzyme. aminotransferase (ALT) of rat kidney was investigated. Fourty-eight (48) rats (both males and females) of average weight 210g were Key words: Indomie instant noodles; Alkaline divided into seven groups. Each group consists of phosphatase. three (3) rats each, except for the control, which

Article citation:- Sanni et al. Effects of chronic administration of indomie noodles on the activity of alanine aminotransferase of rat kidney. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 May(Supplement 1);30(30): S65-S71.Available at http://www.jpbms.info

INTRODUCTION ndomie, is the world largest instant people both at work, in school, and at home. Indofood produced by Indofood-the world largest is one the largest pre-packaged food companies I manufacturer of dried in founded in 1972 in Indonesia as Panganjaya (Indofood, 2010)13. It is sold throughout Indonesia, Intikusuma before changing to its current name in , Australia, Nigeria, the , and 1990 by Sudono by Salim under the Salims group among other numerous countries. Indomie noodle is (Witular, 2004)20. very nutritious, easy to make and can be eaten both as snacks, as well, a major meal. It is very versatile and this make it attracts the patronage of majority of

Courtesy: www.dufil.com

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Ingredients: The food standards agency (FSA) in April 10, 2008, Noodles: , vegetable oil, iodized , called for voluntary removal of the colors (but not sodium polyphosphate, sodium carbonate, potassium sodium benzoate). In addition, it recommended that carbonate, guargum, tartrazine (CI 19140), the colors should be phased out in foods and drinks antioxidant (TBHQ) in the European Union (EU) over a specific period Seasoning powder: iodized salt, monosodium (FSA Board, 2008)11.Tartrazine has a noticeable glutamate (621), hydrolyzed vegetable , soy effect on the behavior of young mice; powder, pepper, garlic powder, flavor, Chili 1. Tartrazine inflamed the stomach lining (increased powder. the number of lymphocytes and eosinophils of Indomie noodles contain Tartrazine, a synthetic rats) when given in the diet for a prolonged lemon yellow azo dye primarily used as food time15. colouring agent (Food standards agency website, 2. Tartrazine was found to adversely affect and alter 2011)11. It has a batch category code of E102 C.I. biochemical markers in vital organs e.g. liver, 19140, FD&C Yellow 5, acid yellow 23, food yellow 4 kidney, small intestines etc of rat not only at and trisodium-1-(4-sulfonatophenyl)-4-(4- higher doses but also at low doses4. sulfonatophenylazo)-5-(pyrazolone-3-carboxylate).It is water soluble and has a maximum absorbance in Organ studied (The kidney): aqueous solution at 427±2nm14. Tartrazine is a The Kidney, a perfect example of homeostatic organ, commonly used colorant all over the world, mainly maintains the constancy of fluid in human internal for yellow, but can also be used with brilliant blue environment, much like a water purification plant (FD&C Blue; E133) or Green (E142) to produce that keeps a city’s water drinkable and disposes of its various green shades. waste10. Tartrazine was believed to cause most of the allergic In rat, the right kidney is slightly placed anterior to and intolerance reaction experienced amongst all the the left while in man; the right kidney is slightly azo dyes, particularly in asthmatic patients and those lower than the left18. Every day, the kidney filters with aspirin intolerance (UK food Guide, 2007). nearly 200 litres of fluid from the bloodstream, Symptoms of tartrazine sensitivity can occur from allowing toxins, metabolic wastes, and excess ions to either ingestion or cutaneous exposure to substances leave the body in the urine and returning needed containing tartrazine. Symptoms could either be mild substances to the blood10. to severe3. A variety of immunological responses have been attributed to tartrazine ingestion, External anatomy: including anxiety, migraine3, clinical depression, The bean-shaped kidney lies in a retroperitoneal blurred vision, itching, general weakness, feeling of position (i.e. between the dorsal body wall and the suffocation, purple skin patches and sleep parietal peritoneum). In the superior lumber region, disturbance17. People who are exposed to the dye they extend approximately from the level of the experience symptoms of tartrazine sensitivity even at twelfth thoracic vertebra to the third lumber extremely small dose, some for periods up to 72 vertebra. Thus, the kidney receives some protection hours after exposure6. In children, asthma attack and from the lower part of the cage. The right kidney is hives have been claimed, as well as supposed link to crowned by the liver and lies slightly lower than the thyroid tumors, chromosomal damage and hyper left kidney. reactivity7. Internal anatomy: Possible health effects of tartrazine: A frontal section of the kidney reveals distinct The British food standards agency on September 6th, regions, the cortex, medulla and the pelvis. The most 2007, revised, advice on certain artificial food superficial region, which is the renal cortex10, is light additives, including tartrazine (BBC news, 2007)5. in color and has a granular appearance. Deep down Professor Jim Stevenson of Southampton University, the cortex is the darker, reddish-brown renal the author of the report said, “This is an important medulla, which exhibits cone-shaped tissue masses, investigating area of research”. The result suggests called ‘Medularry or Renal pyramid’. The broad base that consumption of certain mixture of artificial food of each pyramid faces towards the cortex, and its color and sodium benzoate preservatives are apex or papilla (nipple) points internally. associated with increase in hyperactive behavior in The pyramids appear stripped because they are children. formed collecting tubules. The renal columns and

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Sanni et al. . Available at jpbms.info inward extensions of the cortical tissues separate the acid-base balance: to reabsorb bicarbonate from pyramids. Each medullary pyramids and its urine, and to excrete hydrogen ions into urine. surrounding capsule of the cortical tissue constitutes a lobe of a kidney lateral to the hilus within the renal Osmolarity regulation: sinus called the ‘Renal pelvis’. This flat, funnel- Any significant rise in plasma osmolarity is detected shaped tube is continuous, with the ureter leaving by the hypothalamus, which communicates directly the hilus10. Branching extension of the pelvis from with the posterior pituitary gland. An increase in two or three major calyces (singular) each of which is osmolarity causes the gland to secrete antidiuretic subdivided into 10, forming several mino-calyces, hormone (ADH), resulting in water reabsorption by cub-shaped areas that enclose papillae of the the kidney and an increase in urine concentration. pyramids. The two factors work together to return the plasma The calyces collect urine, which drains continuously osmolarity to its normal levels. from the papillae, and empty it into the renal pelvis urine, then flows through the renal pelvis into the Enzymes studied: ureter, which transports it to the bladder to be Alanine amino-transferase: stored. The walls of the calyces rhythmically and Alanine transaminase (ALT) test measure the propels urine along its course by peristalsis10. amount of the enzyme in the blood. It is found mainly in the liver, but also in small amount in the kidneys, Functions: heart, muscles, and pancreas. Alanine transferase The Kidney regulates simultaneously the volume and (ALT) was formally called serum glutamic pyruvic chemical makeup of the blood, maintaining the transferase (SGPT) or Alanine aminotransferase proper balance between water and and also (ALAT). It plays a role in protein metabolism; between acids and bases. The following are specific therefore, an abnormal high blood level of ALT functions carried out by the kidneys. They are; signifies damage in some organs of the body, mostly the liver (Liver inflammation), (McPherson et al., Metabolic activities: 2011)15. The Kidney plays an important role in gluconeogenesis, a role undertaken only during Distribution of alanine aminotrnasferase: prolonged fasting. At such times, the kidney can Alanine aminotransferase (ALAT) is mainly found in supply approximately one-fifth as much glucose to the liver, but in small amount in the kidneys, heart, the liver10. and muscles. It is less abundant compared to aspartate transaminase (AST) in all tissues and Secretion: mostly occur in the cytosol and mitochondria of the The Kidneys produce the enzyme ‘Renin’, which cells. helps to regulate and kidney function, also the hormone ‘Erythropoietin’, which stimulates Clinical importance of alanine aminotransferase: the production of red blood cells (RBC) in bone It is commonly measured clinically as part of a marrow. diagnostic evaluation of hepatocellular injury, to determine liver’s state. It is usually measured in Excretion: international units/litre (U/L)12,20 . They filter ; separate wastes from the While sources vary on specific normal range values useful chemicals, and also eliminates the wastes for patients, 10-40 U/L is the standard normal range while returning the rest to the bloodstream. for experimental studies20. Alanine transaminase shows a mark diurenal variation.For female, it ranges Acid-Base homeostasis: between 5-38 U/L, while for males; it ranges Two organ systems, the kidneys and lungs, maintain between 10-50 U/L. acid-base homeostasis, which is the maintenance of pH around a relatively stable value. The lungs Functions: It catalyses the transfer an amino group contribute to acid-base homeostasis by regulating from alanine to α-oxoglutarate, the products of this reversible transamination reaction is pyruvate and carbon dioxide (CO2) concentration. The kidneys have two very important roles in maintaining the glutamate.

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ISSN NO- 2230 – 7885 CODEN JPBSCT NLM Title: J Pharm Biomed Sci.

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ALAT Glutamate+ Pyruvate ⇌ α-Oxoglutarate + alanine

Glutamate Pyruvate α-Oxoglutarate Alanine

It also helps in maintaining a balance in the supply of amino acid building blocks for protein synthesis.

MATERIALS AND METHODS Enzyme substrate: Animal sacrifice: The enzyme has already been prepared and ready for All experimental rats were starved for 24hours prior use. to the day of sacrifice after the thirtieth day of Alanine aminotransferase enzyme (RANDOX KIT) feeding and then sacrificed. Part of the organ of consists of Phosphate buffer, L-alanine and 2, 4- interest was cut, removed and put in a 0.25M ice cold dinitrophenylhydrazine as substrate. sucrose solution.

Test animals: Preparation of tissue homogenate: Fourty eight (48) albino rats of average weight, 210g The tissue was weighed and homogenized using an were obtained from Faculty of Agriculture farm, Kogi automated homogenizing machine in a 0.25M State University, Anyigba, Nigeria. sucrose solution; this was kept in a clean dry sample bottle and kept in the freezer before been used for Methods: enzyme activity determination2. Animal groupings: Forty eight (48) rats (Rattus novergicus) were Enzyme activity determination: randomly grouped into seven (7) groups. Groups 1-6 Determination of alanine aminotransferase being the test groups consists of 6 rats each, each (Reitman and frankel, 1957)17 group was further sub-grouped by sex into 3 males The colorimetric method of Reitman and Frankel was and 3 females respectively. Group 7 is the control employed of 1957 was employed. group and was fed with normal rat feed; the control group had twelve rats of which six were males and Principle: six females. The experimental design is as follows; α- oxoglutarate + L-alanine → L- glutamate + pyruvate  Group 1 was fed with indomie instant noodles cooked and spiced with the seasoning. 17  Group 2 was fed with raw indomie instant Procedure: (Reitman and frankel, 1957) noodle spiced with the seasoning. The reagent composition consist of 100mmol/L, of  Group 3 was fed with a mixture of both phosphate buffer and 200mmol/L, of L-alanine, cooked and raw indomie instant noodle 2.0mmol/L of α-oxoglutarate, and 2.0mmol/L of 2, 4- spiced with seasoning. dinitrophenylhydrazine, which is the substrate. 0.1ml  Group 4 was fed with indomie instant noodles of sample was pipette into a clean test tube, 0.5ml of cooked without seasoning. the solution R1 was also added into both the reagent  Group 5 was fed with raw indomie instant blank and the sample, 0.1ml of distilled water was noodle without seasoning. added into the blank, mixed and incubated for  Group 6 was fed with a mixture of both 30mins at 37 °C. 0.5ml of solution R2 was added to cooked and raw indomie instant noodle the blank and sample mixed and was allowed to without seasoning. stand for 20mins at 25°C. Then, 5.0ml of sodium Water was provided ad libitum. The rats were hydroxides was added into the blank and the sample examined within a thirty (30) days scheme in order mixed, the absorbance of the sample was read to ascertain levels of chronic toxicity. against the reagent blank after 5 minutes.

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RESULTS on rats kidney, in group 2 showed the greatest Alanine aminotransferase activity average decrease in the enzyme’s activity). The results obtained were carefully and adequately Generally, rats fed with indomie containing spice examined. From the chat presented below, the showed greater decrease in ALT (Alanine results indicates the effects of the test sample aminotransferase) activity than those fed without the (indomie) on the activity of the marker enzyme, spice. Also a greater decrease in the enzyme’s activity alanine aminotransferase (ALT), whose cellular and among males than females in group 1, group 3, group extracellular levels serves as indicators of 4 and group 5 except in group 2, where there was an pathological state. From the data obtained in the average decrease, and in group 6, with higher study of the effects of chronic administration of decrease in the enzyme activity in females than in indomie on the activity of alanine aminotransferase males was observed.

Figure 1. Graph of Alanine aminotransferase activity From figure 1 above, the blue charts represents the rats in group 4, indicating the possible role of the values for ALT activity in male rats while the green seasoning spice causing more toxic effect. charts represent the values of ALT activity in female Likewise, for female rats in group 1, the enzyme rats. activity in the kidney was also lower than that of the When indomie was administered into the rats, the female rats in group 4, indicating also a possible role following result was observed. of the seasoning spice causing toxic effect. For males, taking control rats as the reference group; Comparing and contrasting the results of group 2 and for group1, the activity of ALT was observed to group 5 with reference to the control result, it was decrease by 9.98%, in group2, it decreased by 22.0%, observed that there were greater activity of the in group3, it decreased by 18.97%, in group4, it enzyme in the kidney of rats in group 5 over the rats decreased by 46.20%, in group5, it decreased by in group 2 irrespective of their sex, this also shows 23.01% and in group6, it decreased by 70.00%. the possible role of the seasoning spice in causing a For females, taking control as the reference group; in more toxic effect and also reducing the activity of the group1, the activity of ALT was observed to decrease enzyme in the kidney over a time. by 36.10%, in group2, it decreased by 22.00%, in Comparing and contrasting the results of group 3 and group3, it decreased by 36.02%, in group4, it group 6 with reference to the control result, the decreased by 50.00%, in group 5, it decreased by result showed a greater activity of the enzyme in the 30.02%, in group6, it decreased by 34.15%. kidney of male rats in group 6 over the male rats in Comparing and contrasting the results of group 1 and group 3, indicating a possible role of the seasoning 4, in reference to the control results, it was spice. discovered that the enzyme activity in the kidney of However, in female rats of group 3, the activity of the male rats in group 1 was lower than that of the male enzyme in the kidney was greater than that of group

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6, indicating the other factors such as varied There was a fall in the activity of the enzyme in the metabolic patterns between sexes, which could be kidney. This could be due to the inhibition of the responsible for this difference. enzyme activity by some agents which are present in the indomie, one of which may be Tartrazine. DISCUSSION The continuous decrease in the enzyme activity could Defect to metabolic route could possibly be reasons make the kidney become permeable, thereby for causes of several diseases of which hypo activity, allowing the enzyme to escape into the extra-cellular hyperactivity and inhibition of metabolic enzymes fluid, preventing the kidney from maintaining are enormously involved. Increase or decrease in constancy of fluid in the internal environment. amount of these metabolic enzymes serves as biomarker for clinical diagnosis of some illness9. CONCLUSION This research work had shown that chronic Biochemical parameter like enzyme assay indicates consumption of indomie instant noodles could be that cellular damages could be by conventional dangerous to health. Much intake of it could lead to historical techniques1,2. diseases like kidney failure, Cancer, and high blood Only one (1) ‘marker’ enzyme was assayed for this pressure. The effect also could vary depending on the present study to determine the effects of indomie mode of preparation, sex of the animal, and also the instant noodles on the activity of Alanine physiological state of the animal. aminotransferase (ALT) in rat kidney. Alanine aminotransferase is a transaminase enzyme RECOMMENDATION (EC 2.6.1.2). It is mostly associated with the liver, but I therefore, suggest that further research be carried also found in the serum and kidney. out on indomie noodles for a more prolong time, with the same method used above under more favourable Tissue enzyme: conditions. Alanine aminotransferase I also suggest that, even though indomie should be The effect of Indomie instant noodles on the activities consumed, it should be done occasionally, especially of alanine aminotransferase was observed in the when the need for a fast food arises. This is because it kidney and the result is as follows; contains the necessary nutrients for healthy living in moderate proportion. Table1. Alanine aminotransferase activity results GROUP MALE AST(nm/min) I/U FEMALE AST(nm/min) I/U 1 10.40 34.85 2 22.85 22.85 3 18.02 35.70 4 44.72 48.17 5 25.50 30.66 6 73.10 34.85 CONTROL 53.04 40.04

Table 2. Analysis of variance (Anova) For ALT activity In male albino rats Descriptive male 95% Confidence Interval for Mean Std. N Mean Deviation Std. Error Lower Bound Upper Bound Minimum Maximum Group1 1 10.4000 . . . . 10.40 10.40 Group2 1 22.8500 . . . . 22.85 22.85 Group3 1 18.0200 . . . . 18.02 18.02 Group4 1 44.7200 . . . . 44.72 44.72 Group5 1 24.5000 . . . . 24.50 24.50 Group6 1 73.1000 . . . . 73.10 73.10 Control 1 53.0400 . . . . 53.04 53.04 Total 7 35.2329 22.44439 8.48318 14.4753 55.9905 10.40 73.10

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Table 3. Analysis of variance (Anova) For ALT activity In female albino rats Descriptive Female 95% Confidence Interval for Mean N Mean Std. Deviation Std. Error Lower Bound Upper Bound Minimum Maximum Group1 1 34.8500 . . . . 34.85 34.85 Group2 1 22.8500 . . . . 22.85 22.85 Group3 1 35.7000 . . . . 35.70 35.70 Group4 1 48.1700 . . . . 48.17 48.17 Group5 1 30.6600 . . . . 30.66 30.66 Group6 1 34.8500 . . . . 34.85 34.85 Control 1 40.0400 . . . . 40.04 40.04 Total 7 35.3029 7.80173 2.94878 28.0875 42.5182 22.85 48.17

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