THE EFFECT OF SOME SYNTHETIC FOOD COLORANTS ON SELECTED BIOCHEMICAL INDICES OF MALE WISTAR RATS

Wopara, Iheanyichukwu,*1 Awarajih, Uwaezuoke C.2 and Ike, Amanda3 123 Department of Biochemistry, Faculty of Science, Madonna University Nigeria Corresponding Author: Wopara, Iheanyichukwu. Department of Biochemistry, Faculty of Science, Madonna University Nigeria

ABSTRACT

The aim is to evaluate the effect of tartrazine and erythrosine on serum glucose, cholesterol and triglycerides. A total of 25 adult male wistar rats were divided into five groups with 5 rats per group. Group 1 served as the control group and was given only water and feed. While group 2,3,4,5 were administered 5mg/kgb.wt, 10mg/kgb.wt, 20mg/kgb.wt, 40mg/kgb.wt of Tatrazine and Erythrosine respectively. At the end of the experimental period (21 days), blood samples were collected via ocular puncture and used to measure glucose, cholesterol and triglyceride concentration in the serum of the animals. The result revealed that there was a significant difference p<0.05 in the mean glucose concentration of Group 4 (5.81±0.40mmol/L) and Group 5 (5.28±0.25mmol/L), when compared with the control (3.27±0.11mmol/L). Similarly, Group 4 showed a higher mean cholesterol concentration (0.92±0.27mmol/L) when compared with the control (0.18±0.05mmol/L). However, the difference was not significant p<0.05. Furthermore, Group5 showed the lowest mean triglyceride concentration (2.01±0.15mmol/L) when compared with the control (2.95±0.04mmol/L). This result revealed that the colorants increased the glucose and cholesterol levels in the test group in a dose dependent manner. The increase glucose concentration could be linked to a condition known as pancreatic cancer caused by the hydrocarbons contained in the colorants. Keywords: Tartrazine , Erythrosine ,Triglyceride, Cholesterol and glucose

1.0 INTRODUCTION , or color additive, is any dye, pigment or substance that imparts colour when it is added to food or drink. They come in many forms consisting of liquids, powders, gels, and pastes. Food coloring is used both in commercial food production and in domestic cooking. Food colorants are also used in a variety of non-food applications including cosmetics, pharmaceuticals, home craft projects, and medical devices. [1]

Tartrazine is a synthetic lemon azo dye primarily used as a food coloring. [2]. It is also known as E102, C.I. 19140, FD&C Yellow 5, Acid Yellow 23, Food Yellow 4, and trisodium 1-(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-5-pyrazolone-3-carboxylate).

Comment [JFR Rev1]: IF not original image/picture of the authors, include citation, origin of the picture/image.

Fig 1.1: Structure of Tartrazine [3].

Products containing tartrazine commonly include processed commercial foods that have an artificial yellow or green color, or that consumers expect to be brown or creamy looking. It has been frequently used in the bright yellow coloring of imitation “lemon” filling in baked goods.

Types of pharmaceutical products that may contain tartrazine include , antacids, cold medications (including cough drops and throat lozenges), and prescription drugs.

Erythrosine, also known as Red No. 3, is an organoiodine compound, specifically a derivative of fluorone. It is cherry-pink synthetic, primarily used for food coloring [4]. It is the disodium salt of 2,4,5,7-tetraiodofluorescein. Its maximum absorbance is at 530 nm [5] in an aqueous solution, and it is subject to photo degradation. Comment [JFR Rev2]: IF not original image/picture of the authors, include citation, origin of the picture/image.

Fig1.2: Structure of Erythrosine [5] Erythrosine (E127) is commonly used in sweets such as some and popsicles, and even more widely used in -decorating gels.

These synthetic food colourant have been implicated with high concentration of serum glucose which could lead to diabetes [6].

Diabetes mellitus (DM) is a glucose metabolism disease characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both [7]. Type 1 DM results from an absolute deficiency in insulin caused by the failure of secretion by the pancreas, while type 2 DM is characterized by resistance of insulin and relative deficiency in insulin, which may be present at the time diabetes diagnosis. DM poses a great threat to human health as well as a huge socioeconomic burden for governments. According to the updated data from the international diabetes federation (IDF), the estimated global prevalence of DM reached 8.8% in 2015 and 12% of global health expenditure was due to DM in that same year[8]. More so, recent research has also discovered the role of T cells in the development of insulin resistance[9] and various complications in T2 DM, including atherosclerosis[10], nephropathy[11] and neuropathy[12]

2.0 MATERIALS AND METHODS

2.1 EXPERIMENTAL ANIMALS A total of twenty male albino rats ……age……? weighing between 128-244 grams were used Formatted: English (United States) for this experiment. The animals were obtained from the animal house at Federal University of Formatted: English (United States) Technology, Owerri. The male albino rats were randomly divided into five groups with 4 rats per group according to their initial body weight in the animal house at Madonna University, Elele. Animals were acclimatized for 1 week under normal environmental condition. They were fed with rat chow and water throughout the study.

Ethical comittee Review? Approval? Protocols for sacrifice approved by whom? Formatted: English (United States)

How was the follow-up of regulations of animal care? Under which regulation? Ethical comittee?

Formatted: English (United States)

2.2 SAMPLE PREPARATION

The samples tatrazine (T0388-100G) and erythrosine (1159360025) were gotten from Sigma

(company, city, country) company United state of American 5mg/b.wt ,10mg/b.wt ,20mg/b.wt, Comment [JFR Rev3]: What? 40mg/b.wt of each sample were weighed, then it was dissolveds with 84ml of distilled water of Formatted: English (United States) Formatted: English (United States) 84ml in a ratio of 1:1, Which was administered to group 2, 3 ,4 and 5 respectively repeatedly. Formatted: English (United States) Formatted: English (United States) 2.3 COLLECTION OF BLOOD SAMPLE Formatted: English (United States) Comment [JFR Rev4]: Ratio of what? Not The blood of the male albino rats was collected and separated according to their groups. This clear. was done by ocular puncture using capillary tubes to the collect the blood samples. After Formatted: English (United States) collection…. was centrifuged? at 5000 rpm for 10 minutes (model? Supplier? Country?). The Comment [JFR Rev5]: When? How many times? blood was collected and transferred into plain sample containers. The serum was decanted into Formatted: English (United States) another sample container and kept in the refrigerator. The samples were used for cholesterol, Formatted: English (United States) triglyceride and serum glucose analysis. Formatted: English (United States) Formatted: English (United States) 2.4 ANALYSIS2.4 ANALYSIS Formatted: English (United States) Comment [JFR Rev6]: Plasma only? What The separated serum were used to carry out the following analysis sample? Formatted: English (United States)  Glucose  Cholesterol  Triglyceride

2.5 METHOD OF ASSAY AND ASSAY PROCEDURE Formatted: English (United States)

2.5.12.4.1. GLUCOSE ASSAY Comment [JFR Rev7]: Do not include the protocol as in a lab protocol. Please, rewrite the protocol in sentences. Method: Randox

Formatted: English (United States) Principle: [13] Formatted: English (United States) Formatted: English (United States) Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide formed reacts, under catalysis of peroxidase, with phenol and 4- aminophenazone to form a red – violet quinoneimine dye as indicator

GOD

Glucose + O2 + H2O------ gluronic acid + H2O2

2 H2O2 + 4- aminophenazone + phenol------ quinoneimine + 4H2O

Reagent Composition

CONTENT INITIAL CONCENTRATION OF SOLUTIONS

R1a. Buffer

Phosphate buffer 0.1mol/l, pH 7.0

Phenol 11mmol/l R2b. GOD-PAP REAGENT

4- aminophenazone 0.77 mmol/l

Glucose Oxidase ≥1.5kU/l

Peroxidase ≥1.5Ku/l

CAL STANDARD

Glucose See lot specific insert

Procedure

1. Label the test tube blank, standard, test. 2. Add 1000µl of reagent the test tube. 3. Add 10µl of sample and standard to the test and standard test tube. 4. Mix, incubate for 25mins at 15-25ºC or 10mins at 37ºC. 5. Read and record

Calculations

Glucose concentration (mmol/l) = Asample × Standard conc (mmol/l)

Astandard

(mg/dl) = Asample × Standard conc (mg/dl)

Astandard

2.4.2. DETERMINATION OF TOTAL CHOLESTEROL CONCENTRATION Formatted: English (United States) Comment [JFR Rev8]: Do not include the protocol as in a lab protocol. Please, rewrite the The concentration of cholesterol was determined according to the method of [14]. protocol in sentences.

Principle: Cholesterol esterase hydrolyses esterified cholesterol to free cholesterol. The free cholesterol is oxidized to form hydrogen peroxide which further reacts with phenol and 4- aminoantipyrine by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex as shown in the reactions below; Chol. esterase Cholesterol ester + H2O Cholesterol + Fatty acid

Chol.oxidase Cholesterol + ½ O2+H2O holestenone + H2O2

Peroxidase 2H2O2 + 4–aminoantipyrine + phenol Quinoneimine + H2O

Reagents: Biosystem cholesterol kit. Reagents A: (35 mMo/l) Sodium cholate, phenol 28 mmol/l, Cholesterol esterase > 0.2 U/ml, Cholesterol oxidase > 0.4 U/l, peroxidase > 0.8 U/ml; 0.5 mmol/l, 4-aminoantipyrine, pH 7.0

Procedure: Three test tubes were prepared for the reactions as follows; Reagents Blank Standard Sample Cholesterol -- 10 µl -- Sample -- - 10 µl Reagent A 1.0 ml 1.0 ml 1.0 ml The test tubes were mixed thoroughly and incubated for 10 minutes at room temperature.

The Absorbance of samples (Asamples) and standard (Astandard) were read against the reagent blank at 500 nm using a Turner(R) 390 spectrophotometer. Calculations: Cholesterol concentrations in the sample were calculated as follows:

2.5.32.4.3. DETERMINATION OF TRIACYLGLYCERIDES CONCENTRATION (TG) Comment [JFR Rev9]: Do not include the protocol as in a lab protocol. Please, rewrite the Principle: Lipoprotein lipase hydrolyses triacylglycerides to glycerol and free fatty acids. The protocol in sentences. glycerol formed reacts with ATP in the presence of glycerol kinase forming glycerol-3- Formatted: English (United States) phosphate which is oxidized to form hydrogen peroxide. The hydrogen peroxide further reacts Formatted: English (United States) Formatted: English (United States) with phenolic compound (chlorophenol) and 4- aminoantipyrine by the catalytic action of a peroxidase to form a red coloured quinoneimine dye complex. Lipase Triglycerides + H2O Glycerol + fatty acid Glycerol + ATP Glycerol kinase Glycerol-3-P + ADP Glycerol-3-P-oxidase Glycerol-3-P + O2 Dihydroxyacetone phosphate + H2O2 peroxidase 2H2O2 + 4-aminoantipyrine + 4- chlorophenol Quinoneimine + 4H2O Reagents: TG standard (glycerol 200 mg/dl) Reagent A (45 mmol/L glycerol kinase > 4 Umol/l; peroxidase> 0.8 Umol/l, 4-aminoantipyrene, 0.75 mmol/L, ATP µmol/ml, pH 7.0

Procedure: Reagents Blank Standard Sample TG standard -- 10 µl -- Sample -- -- 10 µl Reagent A 1.0 ml 1.0 ml 1.0 ml The test tubes were mixed thoroughly and incubated for 15 minutes at room temperature.

The absorbance of samples (Asamples) and standard (Astandard) were read against the reagent blank at 500 nm in a Turner(R) 390 spectrophotometer.

Calculations: The cholesterol concentration of the sample was calculated as follows:

2.4.4. STATISTICAL ANALYSIS ??

3.0 RESULTS

3.1 Effect of tartrazine…….Do not use uppercase fonts. Formatted: English (United States)

FFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON THE Formatted: English (United States) CONCENTRATION OF TRIGLYCERIDE

As shown in the Ffigure 3.1, below Group5 showed the lowest mean average triglyceride Comment [JFR Rev10]: ? concentration (2.01±0.15 mmol/L) when compared with the control (2.95±0.04 mmol/L).**** Formatted: English (United States) Formatted: English (United States) Do not repeat data in text and in figures. Formatted: English (United States) Formatted: English (United States)

Comment [JFR Rev11]: Decimal points are marked with “point” not commas! 3.5 Statistical analysis? P value? 3

2.5

2

1.5 (Mmol/L) 1 0.5

TRIGLYCERIDE CONCENTRATION 0 control 5mg of E+T 10mg of E+T 20mg of E+T 40mg of E+T TREATMENT

Figure : 3.1 EFFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON

THE CONCENTRATION OF TRIGLYCERIDE Comment [JFR Rev12]: Do not use uppercase fonts!

3.2 EFFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON THE CONCENTRATION OF CHOLESTEROL

Figure 3.2 revealed that Group 4 showed a higher mean cholesterol concentration (0.92±0.27 mmol/L) when compared with the control (0.18±0.05 mmol/L). As seen in the other groups they were slightly higher than the control. However, the difference was not significant p<0.05.

1.4 Comment [JFR Rev13]: Decimal points are marked with “point” not commas!

1.2 Statistical analysis? P value?

1

0.8

0.6 (Mmol/L) 0.4

0.2 CHOLESTEROL CONCENTRATION 0 control 5mg of E+T 10mg of E+T 20mg of E+T 40mg of E+T TREATMENT

Figure 3.2: EFFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON Comment [JFR Rev14]: Do not use uppercase fonts! THE CONCENTRATION OF CHOLESTEROL

3.3 EFFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON THE CONCENTRATION OF SREUM GLUCOSE

This figure 3.3 below showed that there was a significant difference p<0.05 in the mean glucose concentration of Group 4 (5.81±0.40 mmol/L) and Group 5 (5.28±0.25 mmol/L), when compared with the control (3.27±0.11 mmol/L).

7

6

5

4

3

2

1

GLUCOSE GLUCOSE CONCENTRATION MMOL/L 0 Gcontrol E+T 5mg E+T 10mg E+T 20mg E+T 40mg TREATMENT

Figure 3.3:

Do not use uppercase fonts! Formatted: English (United States)

EFFECT OF TARTRAZINE AND ERYTHROSINE ADMINISTRATION ON THE CONCENTRATION OF SREUM GLUCOSE

4.1 DISCUSSION

Tatrazine and erythrosine is one of the most commonly used synthetic food colourant all over the world. As it gives good colouration to food as it makes it enticing [2]. These food colourants have being implicated with higher concentration of serum glucose in the body which may be an indicative of the presence of diabetes which means that glucose is not regulated in the body which could be as a result of insulin not being present or present of insulin but cannot bind to its receptor site [15].

This study have shown that rat administered with different concentration of tatrazine and erythrosine showed a significant difference p<0.05 in the mean glucose concentration of Group 4 (5.81±0.40 mmol/L) and Group 5 (5.28±0.25 mmol/L), when compared with the control (3.27±0.11 mmol/L), there was no significant difference in group 2 and 3 when compared to that of the control. Tartrazine and erythrosine significantly increased the concentration of serum glucose demostrating hyperglycemic effect.This result is in correspondance with [3] and [6] whom reported that high dosage of tatrazine elevate the serum glucose concentration level in rat after administration .

From the results, the activities of cholesterol concentration shows Group 4 had the highest mean cholesterol concentration (0.92±0.27 mmol/L) when compared with the control (0.18±0.05 mmol/L), although group 3 also showed a high concentration of cholesterol when compared with that of control. However, the difference was not significant p<0.05.

The activities of triglyceride concentration shows that group5 showed the lowest mean Comment [JFR Rev15]: These are results, NO discussion at all. triglyceride concentration (2.01±0.15 mmol/L) when compared with the control (2.95±0.04 Comment [JFR Rev16]: Workers of what? Technicians? Farmers? Other researchers? mmol/L).There was a slight difference between group 2,3,and 4 when compared to the control. Formatted: English (United States) This result revealed that the colorants increased the glucose and cholesterol levels in the test Formatted: English (United States) Comment [JFR Rev17]: Then…. Where that group in a dose dependent manner. information is extracted from?

Where are the citations? Some workers [6] observed significant increases in serum total lipids, cholesterol and triglycerol Where IS THE DISCUSSION ABOUT YOUR DATA AND levels in rats whose diets were supplemented with mixtures of four synthetic dyes. THE DATA OF THOSE “PREVIOUS WORKS”?????

Formatted: English (United States)

4.2 CONCULSIONCONCLUSION!

This experiment revealed a high possible effect of hyperglycemia, due to the increased level of Comment [JFR Rev18]: Not clearly defended/demonstrated serum glucose concentration. High cholesterol concentration in blood could lead to the accumulation of cholesterol on the walls of the arteries (artherosclerosis) and could reduce blood Formatted: English (United States) flow through the arteries. There was a slight difference in triglycerides level but the tendency of Comment [JFR Rev19]: Not a conclusion of this work. a high difference may be seen in a long term administration. Formatted: English (United States) Comment [JFR Rev20]: Just guessing, again, not proven in your work.

Formatted: English (United States)

REFERENCES

1. Jeannine .D. (2003). “The impact of perceptual interactions on perceived flavor”. Food Quality and Preference. 14(2); 137-146.

2. Elhkim M.O., Héraud F., & Bemrah N, (April 2007). "New considerations regarding the risk assessment on Tartrazine: An update toxicological assessment, intolerance reactions and maximum theoretical daily intake in France" Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim 9(3):123-453.

3. Amin K.A., Abdel H., & Abd E. A.H. (2010). Effect of food azo dyes tartrazines and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food chemistry Toxicology Journal. 48(10):2994-9.

4. Phyllis, A. L. (2005) Iodine and Iodine Compounds in Ullmann's Encyclopedia of Industrial Chemistry. Wiley-VCH, Weinheim. 9(3):123-453.

5. Gurr .E. (2012). Synthetic dyes in Biology, Medicine and Chemistry. Elsevier. 197,198.

6. Aboel-Zahab H, El-Khyat Z, Sidhom G,& Awadallah R,(1997). Physiological effects of some synthetic food colour additives on rats. Boll Chim Farm. 136:615-27.

7. Skyler JS, Bakris GL, Bonifacio E, Darsow T, Eckel RH, Groop L, et al. (2017) Differentiation of Diabetes by Pathophysiology, Natural History, and Prognosis. Diabetes 66: 241–255

8. Yates T, Khunti K (2016) Epidemiology: The diabetes mellitus tsunami: worse than the 'Spanish flu' pandemic? Nat Rev Endocrinol 12: 377–378.

9. Jagannathan-Bogdan M, McDonnell ME, Shin H, Rehman Q, Hasturk H, Apovian CM, et al. (2011) Elevated Proinflammatory Cytokine Production by a Skewed T Cell Compartment Requires Monocytes and Promotes Inflammation in Type 2 Diabetes. Journal Of Immunology 186: 1162–1172.

10. Zetterqvist AV, Berglund LM, Blanco F, Garcia-Vaz E, Wigren M, Duner P, et al. (2013) Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice. Plos One 8: 14. 11. Zhang N, Tai JD, Qu ZH, Zhang ZH, Zhao SC, He JX, et al. (2016) Increased CD4(+)CXCR5(+)T follicular helper cells in diabetic nephropathy. Autoimmunity 49: 405– 413.

12. Tang W, Lv Q, Chen XF, Zou JJ, Liu ZM, Shi YQ (2013) CD8(+) T Cell-Mediated Cytotoxicity toward Schwann Cells Promotes Diabetic Peripheral Neuropathy. Cellular Physiology And Biochemistry 32: 827–837.

13. J ulio .R.J., & Mottola .H.A. (1995). “Glucose oxidase as an analytical reagent ” Analytical Chemistry Journal.25(1); 1-42.

14. Allain C.C., Poon L.S., & Chan C.S.G.,(1974). Enzymatic cholesterol determination of total serum cholesterol.Clin.Chem. 20,1282.

15. Kitabchi A.E., Umpierrez G.E., Miles J. M., & Fisher J.N.,(2009).”Hyperglycemic crises in adult patients with diabetes. Diabetes care.32(7); 1335-43..

.