INTRODUCTION:

Tramadol is a synthetic opioid and central nervous system analgesic that is used to treat moderate to severe pain all over the world. It has recently been reported as a drug of abuse, with intentional overdoses or intoxications¹.

The abuse of Tramadol, especially off-label, is well recognized as a public health issue around the world². Some off-label uses of Tramadol for euphoria and delayed ejaculation might be responsible for its abuse³, especially among adult male users. There have also been studies of Tramadol dependency in a number of countries, including China⁴, Egypt⁵, Germany⁶, India⁷, Italy⁸ and the United Arab Emirates⁹. Nigeria is not exempt from these countries, despite the government's strict measures to prevent off-label use and abuse.

According to publicly available community survey data in Nigeria, up to 43.8 percent of men in the community have erectile dysfunction (ED)¹⁰. As a result, self-medication with Tramadol for the treatment of ED is becoming more popular among middle-aged adults. Despite the fact that Tramadol is strictly a prescription-only medicine (POM), its availability and affordability make it vulnerable to abuse by young men, laborers, and artisan. Although Tramadol may be beneficial in premature ejaculation, as reported by¹¹ and Kurkar et al.,¹² it is not medically indicated for ED. As reported by Peprah et al., ¹³ the prevalence of Tramadol abuse in Nigeria is around 54.4%, and more than 91% of users obtain the medicine without prescriptions. Off-label usage of Tramadol for purposes other than pain relief has been connected to three major causes in Nigeria and Ghana: psychological, favorable effects for physical or manual labor, and economic factors based on availability and affordability¹³. This has also been reported by United Nations Office on Drugs and Crime(UNODC)¹⁴.

Abusers of Tramadol always dissolve it with Lacasera® soft drink, which conceals the bitter taste and enhances the euphoric effect.This has been a source of concern due to the fact thatLacasera® soft drink contains other chemicals used as additives. Given the growing number of young people in a population of two hundred million in Nigeria, these combinations may have a negative impact on their health and socioeconomic well-being¹⁵. According to Olalekan et al.,¹⁶ if nothing is done, by 2030, 7 out of every 10 young men on the street in Nigeria will become Tramadol addicts, particularly in major cities. The maximum therapeutic dose of Tramadol per day is 400mg in an adult administered in divided doses per day¹⁷^,¹⁸. Due to the combination's potent euphoric and severe craving effects, off-label Tramadol-Lacasera® users typically surpass the daily dosage. This necessitates the need for this animal study to investigate the effects of Lacasera®, the medium in which Tramadol is dissolved, using therapeutic and supra-therapeutic doses of this combination. The findings from this study is expected to produce similar effects on chronic abusers in humans.

MATERIALS AND METHODS:

Materials:

Instruments and Equipment:

Forceps, Bunsen burner, stop watch, hypodermic needles, hand gloves, test tubes, spatula, Standard biochemical kits (Randox, United Kingdom),analytical weighing balance (PA214, Ohaus, USA), electronic weighing balance (SPU 401, Ohaus, USA), Compound digital light microscope (Motic^TM-BA210, China) Centrifuge (Vanguard V 6000, Germany), Acurex Chemistry Analyzer (SR NO: 7047, England), Semi-auto analyzer, EMP 165, China), Humalyzer 2000 (China), Ion selective electrode (ISE) analyzer (ISE 4000, France), Selectra Junior (Semi- robotics, England), and Sysmex Automated Hematology Analyzer (Sysmex Kx-21N,United States).

Animals and Rodent Feed:

The study employed healthy young adult male Albino Wister rats weighing between 160 and 180g. The rats were inbred at the Animal House facility of Nnamdi Azikiwe University's Faculty of Pharmaceutical Sciences in Awka, Nigeria.The animals were fed with commercial standard rodent feed (Vital Feeds®, Nigeria Limited).

Tramadol and Lacasera® soft drink:

Tramadol was obtained from a licensed pharmacy in Port-Harcourt, Nigeria, for use in the study. Tramadol capsules B.P 50mg (B.G Tragesic ®; N-1339, Stallion Laboratory PVT. Limited, India) were approved by Nigeria's National Agency for Food and Drug Administration and Control (NAFDAC) with registration number A4-2740.The study used a crate of 50cl Lacasera® soft drink purchased in a grocery with NAFDAC certified number, 01-4278.

Methods:

Animal Housing and Feeding Conditions:

The study used Wistar rats for all of the animal testing, and all of the procedures were performed in accordance with the ARRIVE criteria¹⁹ and were conducted in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the EU Directive (2010/63/EU) for animal experiments, and the National Research Council's Guide for the Care and Use of Laboratory Animals²⁰.

Ethical Approval:

The study was granted approval by the Animal Research Ethics Committee (AREC) ofNnamdi Azikiwe University, Awka with reference number: NAU/AREC/2021/00008A. All procedures that involved the use of animals in the study were also observed by a member of the AREC.

Experimental Design, Selection of Animals, Groupingand Dosing:

Animals(rats) used in the study were randomly selected and sample size was determined using equation provided by Charan and Kantharia²¹. A total of 48 male Wistar rats were used in the 28 days sub-acute toxicity study, with consideration of 16% attrition. The animals were divided into eight (8) groups of six rats per group (n=6). The animals' body weights were measured at the start of the study (initial weight), then after 7 days, 14 days, 21 days, and 28 days respectively. Group 1: this group received only deionized water based on their body weight in 5ml/kg. The number of animals used were six (n=6). This group served as control (1). Group 2: this group received Lacasera®soft drinkbased on their respective body weights 5ml/kg. The number of animals used were six (n=6). This group served as control (2). Groups 3, 4 and 5: this group received normal and supra-therapeutic graded doses of Tramadol (35mg/kgday^-1, 70.7mg/kgday^-1 and 106mg/kgday^-1) dissolved in deionized water (i.e.Tramadol + water) respectively. The number of animals used in each of this group were six (n=6). Groups 6, 7 and 8: this group also received graded doses of Tramadol (35mg/kg day^-1, 70.7mg/kgday^-1 and 106mg/kg day^-1) dissolved in Lacasera® soft drink (i.e.,Tramadol +Lacasera®), respectively. The number of animals used in each of this group were six (n=6).

Electrolytes test:

Procedure:

Electrolyte test were carried similar to Bishopet al.,²² procedure. Ion selective electrode (ISE) (analyzer ISE 4000) was used for the determination of serum electrolytes. The serum was separated from the supernatant using a centrifuge machine. Controls were determined to ensure accuracy and precision. The ISE was turned on, causing the machine to release the probe. The probe then automatically takes up the sample (serum) from the container, which has been properly positioned by the analyst. The probe moves inward, transferring the sample to the reaction tray, where the major electrochemical reaction occurs. Following the reactions, the electrolytes were displayed on the computer screen, and the results were printed using the print-out device. The results were generated one per sample as soon as the machine devices completed the reactions.

Renal function tests:

Serum Creatinine:

Creatinine was determined using were estimated using diacetyl monoxime methods similar to Michael et al²³.The test tubes were suitably labeled as blank, standard, controls, and test samples. In each clean tube, 500µl of distilled water and 2/3 H2SO4 were inserted. Following this, 500µl of distilled water was placed in a blank tube, 500µl of eliterol standard in a standard tube, 500µl of samples (serum) in a test tube, and 500µl of QC 1 in a control tube. This phase was completed by adding 5% Sodium Tungstate (a precipitant) to all of the tubes. The test tubes' contents were thoroughly mixed before being centrifuged at 12,000rpm for 10minutes.

The second phase comprised the passage of 1000µl of distilled water through the various tubes. Following that, 1000µl of the relevant supernatants were added to the appropriately labeled tubes. This second phase of the reaction was completed by adding 1000µl of picric acid solution and 0.75N sodium hydroxide to the relevant tubes in the order listed. The constituent tubes were carefully mixed and allowed to stand for 8-10 minutes before being analyzed at 560nm with the Accurex chemistry analyzer.

Serum Urea:

Urea was determined usingJaffes’ methodsin accordance with Michael et al.,²³. The test tubes were suitably labeled as blank, standard, controls, and test samples. Following that, 10µl of distilled water was placed in a blank tube, 10µl of eliterol standard was placed in a standard tube, 10µl of samples (serum) was placed in a test tube, and 10µl of QC 1 was placed in a control tube. This was followed by the addition of 1000µl of distilled water, urea color reagent, and urea acid reagent in the order listed across all test tubes. The contents of the test tubes were thoroughly mixed and boiled for 15 minutes at 100^oC. Afterwards, the tubes containing the contents were cooled with tap water. The Accurex Chemistry Analyzer was then used to read it at 560nm.

Liver function tests:

Aspartate aminotransferase (AST):

This was carried out with analytical kit. The ALT assay was performed using two ELITech Clinical Systems reagents (R1 and R2). In order to prepare the reagent, 4ml of R1 and 1ml of R2 were combined to produce 1:5 dilutions_.After preparing the reagents, 3ml samples of the standard (ELITROL 1 and 11), control, and test were transferred into small tubes and inserted into the Selectra proM's sample rotor. The AST working reagents (R1 and R2) were then inserted in the right positions in the Selectra pro analyzer's reagent rotor, and the tests were conducted for 20 minutes. The results were recorded and presented on the monitor's screen.

Alanine Aminotransferase (ALT):

This was done using an analytical kit. The ALT assay was performed using two ELITech Clinical Systems reagents (R1 and R2). Reagent 1 contains Tris buffer, L-alanine; Lactate dehydrogenase (LDH), pH: 7.3 while Reagent 2 (substrate) contains 2-oxoglutarate, NADH and sodium azide. This was prepared bymixing 4ml of R₁ and 1ml of R₂to make 1:5 dilutions and poured into a tube. The standard (ELITROL 1 and 11), control, and test samples were transferred into small tubes and inserted into the Selectra proM's sample rotor. The ALT working reagents (R1 and R2) were inserted in the correct positions in the Selectra pro analyzer's reagent rotor, and the tests were run for 20 minutes. The results were recorded and presented on the monitor's screen.

Serum Bilirubin (total and conjugated):

Total serum bilirubin and conjugated bilirubin were measured in accordance with Michael et al.,²³. Two reagents (R1 and R2) from an ELITech Clinical Systems kit were used. Reagent 1 contains Sulfanilic acid, Hydrochloric acid and Cetrimide, while Reagent 2 contains Sodium nitrite. The reagent was prepared by mixing 4ml of R₁ and 1ml of R₂to make 1:5 dilutions and pour into a container.The standard (ELITROL 1 and 11) and test samples of 3ml each were transferred into tiny tubes and inserted in the sample rotor of the Selectra proM. The bilirubin working reagent (R1 and R2) was then inserted in the appropriate position in the Selectra proM analyzer's reagent rotor, and the tests were conducted for 20 minutes. The results were presented on the monitor's screen and recorded.

Total Protein (TP): Determination of serum total protein concentration was carried out similar toMichael et al.,.²³. The blank, standard, controls, and test samples test tubes were all labeled suitably. Then after, 0.2 ml ml of distilled water was added to the blank tube, 0.2 ml of Randox standard to the standard tube, 0.2 ml of samples (serum) to the test tube, and 0.2 ml of QC 1 to the control tube. This was followed by addition of 1 mL of biuret working reagent to each of the test tubes separately. The test tubes were combined and incubated at room temperature for 30 minutes before being read and recorded using an Accurex Chemistry Analyser at 540 nm. The apparatus performed all of the calculations automatically using the Beer-Lambert Principle, and the results were printed out.

Albumin (ALB): Serum albumin was estimated quantitatively using Bromocresol Green Method as modified by Randox Laboratories (United Kingdom) whose kit was used. The blank, standard, controls, and test samples test tubes were all labeled suitably. After that, 0.1 ml ml of distilled water was added to the blank tube, 0.1 ml of Randox standard to the standard tube, 0.1 ml of samples (serum) to the test tube, and 0.1 ml of QC1 to the control tube. This was followed by addition of 1 ml of BCG working reagent to each of the test tubes separately. The components were combined and incubated at room temperature for 10 minutes. The Accurex Chemistry Analyzer was then used to read the result at 540 nm.

STATISTICAL ANALYSIS:

The data was analyzed with the Statistical Package for Social Sciences (SPSS) software (SPSS Inc., Chicago, IL, USA; Version 27). The mean ± SD values obtained from administration of equivalent doses of treatments in both groups were compared using independent t-test and ANOVA graphically represented with histogram. The values obtained at p< 0.05 were deemed statistically significant.

RESULTS:

Electrolytes: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on serum sodium, potassium, chloride & bicarbonate.Figures 1a-d show graphical representation of effect of graded doses of tramadol dissolved in deionized water and Lacasera soft drink on serum electrolytes (Na+, Cl-, K+ and HCO3-).

Figure 1a: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on serum Na+.

Values are presented as mean±Standard Deviation, n =5. nsp>0.05: Not statistically significantly different from W (water group). *p<0.05: Statistically significantly different from W (water group). **: Significant at 0.01, ***: Significant at 0.005.

Key: W. Control= control group treated with deionized water. L. Control=control group treated with Lacasera soft drink

Figure 1b: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on serum K⁺

Figure 1c: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on serum Cl^_

Figure 1d: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on serum HCO3^_.

Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on Kidney function parameters:

The effects of graded doses of tramadol dissolved in deionized water and Lacasera soft drink on serum urea and creatinine are shown graphically in figures 2a and 2b, respectively. Tramadol-Lacasera treatment resulted in a dose-dependent statistically significant increase (p<0.05) in the mean serum urea concentrations compared to tramadol-deionized water treatment. Similarly, the Tramadol-Lacasera treated group shows a significant increase (p<0.05) in the mean serum creatinine values of both groups compared.

Figure 2a: Effect of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on serum Urea.

Figure 2b: Effect of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on serum Creatinine.

Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on liver function parameters:

Figures 3a-f shows graphical representation of effect of graded doses of tramadol dissolved in deionized water and Lacasera soft drink on liver function parameters. Figs.3a and 3b demonstrate that in both groups, serum means of AST and ALT increased significantly (p<0.05) in a dose-dependent manner in the tramadol-Lacasera treated group compared to the tramadol-deionized water treated group. In contrast, figures 3c and 3d demonstrate a dose-dependent decrease (p<0.05) in total protein and albumin in the tramadol-Lacasera group compared to the tramadol-deionized water group. Figures 3e and 3f demonstrate a dose-dependent significant increase (p<0.05) in total and conjugated bilirubin serum means in the tramadol-Lacasera treated group compared to the tramadol treated group.

Figure 3a: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on AST

Figure 3b: Effect of graded doses of tramadol dissolved in deionized water and Lacasera®soft drink on ALT

Figure 3c: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on total protein.

Figure 3d: Effect of graded doses of tramadol dissolved in deionized water and Lacasera®soft drink on Albumin

Figure 3 e: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on total bilirubin

Figure 3f: Effect of graded doses of tramadol dissolved in deionized water and Lacasera® soft drink on conjugated bilirubin

DISCUSSION:

Sodium plays a key role in normal nerve and muscle function²⁴ when the intake is normal.The elevated serum sodium in rats treated with Tramadol-Lacasera® combination could be as a result of either high sodium content of Lacasera® beverage or when the ability of the kidney to excrete sodium is impaired²⁵. Lacasera contains sodium compound additives such as sodium benzoate and citrates. Sodium benzoate is a common preservative found in soft drinks. When protonated, it produces benzoic acid²⁶, leaving sodium chloride behind. Excess or chronic use of sodium benzoate, according toHelal et al., ²⁷, could result in metabolic acidosis, convulsions, and hyperpnoea in humans. Furthermore, Lewis ²⁸ believes that such an increase in serum sodium may result in kidney dysfunction, dehydration, or adrenaline gland malfunction. In humans, this may predispose individuals addicted to this combination to hypertension or other hypernatremia-related symptoms, including electrolyte imbalance²⁹. On the other hand,there was dose-dependent decrease in Tramadol-Lacasera® treated group.

In fig.1b, our study shows effect of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on serum K⁺. It depicts a dose dependent statistically significant decrease (p<0.05) in serum K+ level obtained in Tramadol- Lacasera® treated group. Physiologically, potassium levels frequently fluctuate in relation to sodium levels. It is usually caused by an increase in potassium excretion or an intracellular shift, but it can also be caused by a decrease in potassium intake³⁰ . When sodium levels rise, potassium levels fall, and when sodium levels fall, potassium levels rise ³¹.This might be responsible for our results which shows dose dependent statistically significant decrease (p<0.05) in serum K+ level obtained in Tramadol- Lacasera® treated group fig. 1b.

The mean serum Cl- level comparison between the Tramadol-deionized water group and the Lacasera®-Tramadol group shows a statistically significant dose-dependent increase (p<0.05) in the Tramadol- Lacasera® group compared to the Tramadol-deionized water group in fig.1c. According to Burdett and Vercueil ³², such increase in Cl- concentration might have resulted from loss of bicarbonate in the blood.Given the low pH caused by the Tramadol-Lacasera® combination, metabolic acidosis could have resulted from increased Cl- and decreased HCO3- ion changes. According toWorld Health Organization(WHO)³³ andCaporuscio ³⁴, respiratory depression often develops when a person begins using Tramadol or increases their dosage.Even if metabolic acidosis was induced by increased acid production or decreased acid excretion, respiratory compensation is the physiological mechanism that assists in normalizing metabolic acidosis³⁵. However, compensation may never totally correct an academia³⁶, particularly when an acidic soft drink such as Lacasera® is consumed in excess or on a chronic basis.

Moreover, Thomas ³⁷states that metabolic acidosis develops mostly as a result of HCO3^- loss from the kidneys or as a result of a renal acidification deficit or pathological conditions. The decreased mean serum bicarbonate concentration in the Lacasera®-Tramadol treated group could be a result of increased plasma chloride supplied by the acidic Lacasera® soft drink and renal bicarbonate loss, as illustrated in the equation:

H ++ Cl⁻+ Na⁺ + HCO3 ⁻ → Na⁺ + ↑Cl⁻+ H₂CO₃(CO₂)³⁸. The effect of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on serum HCO3^_ were shown in fig.1d.The serum level of HCO3- in the Tramadol-Lacasera® treated group decreased dose-dependently and significantly (p<0.05) compared to the Tramadol-deionized water treated group. This confirms the loss of HCO3- in the renal bicarbonate equation as written by Nagami³⁸.

Our study recorded dose-dependent significant (p<0.05) increases in serum urea and creatinine analyzed from rats treated with Tramadol-Lacasera® combination than Tramadol-deionized water group as shown in figs.2(a and b). This also implies that the Lacasera®-Tramadol combination has caused kidney dysfunction, resulting in bicarbonate depletion³⁹as recorded in the results of electrolytes analyzed. This suggests that Tramadol-Lacasera® combination causes renal impairment or malfunction, which is predicted to worsen with continued use. This study, therefore agrees with the studies done byOwoade et al.,⁴⁰and Aldiwan ⁴¹.

Our study shows the effect of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on liver function parameters in figs.3a-f. Liver function tests assess how well the liver performs its normal functions of protein synthesis and bilirubin elimination. The mean values of AST recorded in both groups increased dose-dependently as the dose of Tramadol increases in fig.3a.Our findings are consistent with those Owoade et al., ⁴² who recorded a dose-dependent increase in AST in a previous study using only Tramadol. The Tramadol-Lacasera® treated group recorded a statistically significant(p<0.05) increase when compared Tramadol-only treated group. This demonstrates injurious impact of Tramadol-Lacasera® on liver cells. This finding suggests that persistent use of this combination (Tramadol + Lacasera®) could eventually disrupt the liver's normal function, even at low doses. The ALT enzyme is mostly found in kidney cells, with a higher quantity in liver cells⁴³.Moreover, the Tramadol-Lacasera® treated group shows dose-dependent significant ALT increase(p<0.05) compared to the Tramadol-deionized water treated group in fig.3b.Any sort of damage to the liver cells will result in an increase in serum ALT levels⁴⁴.This also suggests that, based on our findings, a normal therapeutic dose of Tramadol may not cause significant liver injury when administered over a 28-day period, as opposed to the increased toxicity of Tramadol-Lacasera® combination at therapeutic dose.

The liver produces total protein, and in the case of liver injury, production of these proteins is diminished or ceases entirely⁴⁵. Total protein (TP), albumin (ALB), total bilirubin (TB), and conjugated bilirubin (CB) all show evident abnormalities of the liver enzymes, as seen by decreased TP and ALB but increased TB and CB in our study.In humans and animals, total protein and bilirubin may play a role in liver function⁴⁵ While TP and ALB indicate hepatic synthesis function⁴⁶,⁴⁷, bilirubin (total and conjugated) rises in cholestatic and hepatotoxic liver dysfunction or disease⁴⁸.

Figures 3c and 3d shows the effects of graded doses of Tramadol dissolved in deionized water and Lacasera® soft drink on total protein and albumin. The results demonstrate a dose-dependent decrease (p<0.05) in total protein and albumin in the Tramadol-Lacasera® group compared to the Tramadol-deionized water group.In contrast, the Tramadol-Lacasera® treated group shows a dose-dependent significant increase (p<0.05) in total and conjugated bilirubin serum means as compared to the Tramadol treated group in figures 3e and 3f.In our study, the decreases and increases in these proteins were dose-dependent in both treatment groups, however the Tramadol-Lacasera® group was statistically significantly (p<0.05) different from the Tramadol-deionized water group.By comparing the results of liver function parameters in Tramadol-related studies, we established that our results were comparable to work done byAbbas et al., ⁴⁹. Their study focused on the acute effects of taking supra-therapeutic Tramadol doses for 28 days. Our study, on the other hand, focused on the effects of using Tramadol and Lacasera® off-label for 28 days at both minimum recommended therapeutic and supratherapeutic doses. At minimal therapeutic dose of Tramadol, our study showed enhanced toxicity of Lacasera® in liver function parameter. Tramadol-Lacasera® soft drink combination shows significant abnormality in liver function enzymes at supra-therapeutic doses, indicating the toxic effects of Lacasera® on liver.

CONCLUSION:

The sub-acute toxicity study on electrolytes, liver and kidney effects of graded doses of Tramadol dissolved in Lacasera® soft drink was carried out to investigate and extrapolate the potential health effects of Tramadol-Lacasera® combination on biochemical parameters of humans using Wistar rats. Findings of our study indicate that the Tramadol-Lacasera® combination could cause both liver and kidney failure on chronic use. It could also cause electrolyte imbalance, resulting in hypernatremia, which may predispose chronic users to hypertension in humans even at the recommended dose. The toxicity of this combination increases dose-dependently and highly toxic at supra-therapeutic doses. However, neither irregular Tramadol nor Lacasera® users will experience toxicity at minimal therapeutic doses.

ACKNOWLEDGMENT:

The authors would like to express their appreciation to the Pharmacology Department, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Nigeria, for granting permission to use their animal facility and laboratory for the purpose of conducting this research.

FUNDING SOURCES:

No government, private, or nonprofit organization provided any specific financial support for this study.

COMPETING INTEREST:

There are no competing interests to declare in this study.

REFERENCES:

1. Nakhaee S, Hoyte Christopher, Dart RC Askari M Roland Lamarine J, et al A review on tramadol toxicity: mechanism of action, clinical presentation, and treatment. Forensic Toxicol. 2021; 39: 293–310. doi.org/10.1007/s11419-020-00569-0

2. Rostam-Abadi Y Gholami J Amin-Esmaeili M Safarcherati, Mojtabai R Ghadirzadeh MR, et al Tramadol use and public health consequences in Iran: a systematic review and meta-analysis. 2020; Blackwell Publishing Ltd. https://pubmed.ncbi.nlm.nih.gov/32196801/

3. Peprah P Agyemang-Duah W Appiah-Brempong E Akwasi AG Morgan AK “with tramadol, i ride like a Jaguar”: A qualitative study of motivations for non-medical purpose tramadol use among commercial vehicle operators in Kumasi, Ghana. Subst Abus Treat Prev Policy. 2020; 15(1): 1–15. doi.org/10.1186/s13011-020-00292-4

4. Zhang H Liu Z. The investigation of tramadol dependence with no history of substance abuse: A cross-sectional survey of spontaneously reported cases in Guangzhou City, China. Biomed Res. Int. 2013; https://pubmed.ncbi.nlm.nih.gov/24151592/

5. Mohamed N El Hamrawy L Shalaby A El Bahy M Abd Allah M An epidemiological study of tramadol HCl dependence in an outpatient addiction clinic at Heliopolis Psychiatric Hospital. Menoufia Med J. 2015; 28(2): 591. http://www.mmj.eg.net/text.asp?2015/28/2/591/163924

6. Soyka M Backmund M Hasemann S Tramadol use and dependence in chronic noncancer pain patients. Pharmacopsychiatry. 2014; 37(4): 191–2. https://pubmed.ncbi.nlm.nih.gov/15467978/

7. Sarkar S Nebhinani N Singh SM Mattoo SK, Basu D. Tramadol dependence: A case series from India. Indian J Psychol Med. 2012; 34(3): 283–5. https://pubmed.ncbi.nlm.nih.gov/23440178/

8. Pollice R Casacchia M Bianchini V Mazza M Conti CM Roncone R Severe tramadol addiction in a 61 year-old woman without a history of substance abuse. Int J Immunopathol Pharmacol. 2018; 21(2): 475–6. https://pubmed.ncbi.nlm.nih.gov/18547496/

9. Alblooshi H Hulse GK El Kashef A Al Hashmi H Shawky M Al Ghaferi H et al The pattern of substance use disorder in the United Arab Emirates in 2015: Results of a National Rehabilitation Centre cohort study. Subst Abus Treat Prev Policy. 2016; 11(1). https://pubmed.ncbi.nlm.nih.gov/27177422/

10. Olugbenga-Bello Adeoye O Adeomi A Olajide A Prevalence of erectile dysfunction (ED) and its risk factors among adult men in a Nigerian community.Niger Postgrad Med J. 2013; 20(2). https://www.npmj.org/article.asp?issn=1117-

11. Manraj D Kenia A. Maldonado Maani VC Tramadol. StatPearls Publishing LLC. 2020; 1–6. https://www.ncbi.nlm.nih.gov/books/NBK537060/

12. Kurkar A Elderwy AA Abulsorour S Awad SM Safwat AS Altaher A A randomized, double-blind, placebo-controlled, crossover trial of «on-demand» tramadol for treatment of premature ejaculation. Urol Ann. 2015; 7(2): 205 https://www.urologyannals.com/article.

13. Peprah P Agyemang-Duah W Appiah-Brempong EAkwasi AG Morgan AK “With tramadol, I ride like a Jaguar”: a qualitative study of motivations for non-medical purpose tramadol use among commercial vehicle operators in Kumasi, Ghana. Subst Abus Treat Prev Policy. 2020; 15(1): 1–15. https://substanceabusepolicy.biomedcentral.com/articles/10.1186/s13011-020-00292-4

14. United Nations Office on Drugs and Crime(UNODC) At The Crossroads of Licit and Illicit. United Nations Office on Drugsand Crime. 2021. 1–78. https://www.unodc.org/documents/nigeria/Tramadol_Trafficking_in_West_Africa.

15. Chikezie UE Ebuenyi ID Tramadol misuse in the Niger Delta; A review of cases presenting within a year. J. Subst Use. 2019; 24(5): 487–91. https://www.tandfonline.com/doi/full/10.1080/14659891.2019.1604842

16. Olalekan RM Funmilayo AA Okoyen E Oyinlola BO Public Health Impact of Substance Use on Adolescent: A Snapshot of Yenagoa in Bayelsa State. Nigeria. Am J Biomed Sci Res. 2019; (3): 2019–23. www.biomedgrid.com

17. British National Formulary 74th ed. London: Royal Pharmaceutical Society; 2018: 1–1565. www.bnf.org

18. Nakhaee S Hoyte C Dart RC Askari M Lamarine RJ Mehrpour O A review on tramadol toxicity: mechanism of action, clinical presentation, and treatment. Forensic Toxicol. 2021; 39(2): 293–310. https://link.springer.com/article/10.1007/s11419-020-00569-0

19. du Sert NP Ahluwalia A Alam S Avey MT Baker M Browne WJ et al Reporting animal research: Explanation and elaboration for the arrive guidelines 2.0. Vol. 18, PLoS Biology. 2020. 1–65.

20. National Research Council(NRC) Guide for the Care and Use of Laboratory Animals . 8th ed. Ballinger MB, Philippe J.R. Baneux, Stephen W. Barthold, Linda C. Cork, Hau J, Huerkamp MJ, et al., editors. National Academies Press; 2011. 1–246. http://www.nap.edu.

21. Charan J Kantharia N How to calculate sample size in animal studies? J Pharmacol Pharmacother. 2013; 4(4): 303–6.

22. Bishop ML Fody EP Schoeff LE Clinical chemistry : principles, techniques, and correlations /. 7thEdition ed. Wolters Kluwer Health/Hippincott Williams & Wilkins; 2013. 752–754.

23. Michael B Fody E Schoeff L Clinical Chemistry: Principles Techniques Correlations | Michael Bishop, Edward Fody, Larry Schoeff. 8th Editio. Lippincott Williams & Wilkins; 2017. 1–1860 p. https://ng1lib.org/book/3651959/8a66f7

24. Lewis JL Overview of Sodium’s Role in the Body - Hormonal and Metabolic Disorders - MSD Manual Consumer Version. MSD Manual. 2021. p. 1–5. https://www.msdmanuals.com/home/hormonal-and-metabolic-disorders/electrolyte-balance/overview-of-sodiums-role-in-the-body

25. Grillo A Salvi L Coruzzi P Salvi P Parati G Sodium Intake and Hypertension. Nutrients. 2019;11(9). http://pmc/articles/PMC6770596/

26. Solano DM The Effect of pH on Sodium Benzoate. California State University, Bakersfield. 2000. 1–4. http://www.csub.edu/chemistry/organic/manual

27. Helal EGE Abdelaziz MA El-Shenawe NSA Helal E Adverse Effects of Two Kinds of Food Additive Mixtures (Sodium benzoate + Monosodium glutamate, Monosodium glutamate + Chlorophyllin and Sodium benzoate + Chlorophyllin) on Some Physiological Parameters in Male Albino Rats. Egypt J Hosp Med. 2019; 75(4): 2736–44. https://ejhm.journals.ekb.eg/article_32065.html

28. Lewis JL Hypernatremia (High Level of Sodium in the Blood) . MSD Manual. 2020 https://www.msdmanuals.com/home/hormonal-and-metabolic-disorders/electrolyte-balance/hypernatremia-high-level-of-sodium-in-the-blood

29. Shrimanker I Bhattarai S Electrolytes - StatPearls - NCBI Bookshelf. 2021. 1–7. https://www.ncbi.nlm.nih.gov/books/NBK541123/

30. Kardalas E Paschou SA Anagnostis P Muscogiuri G Siasos G Vryonidou A Hypokalemia: A clinical update. Vol. 7, Endocrine Connections. BioScientifica Ltd.; 2018; R135–46. https://pmc/articles/PMC5881435/

31. Adam H Gregory T Martins JG Potassium (K) in Blood Test. University of Michigan Health. 2020; 1–2. https://www.uofmhealth.org/health-library/hw202677

32. Burdett E Vercueil A Hyperchloremic Acidosis. In: Alternatives to Blood Transfusion in Transfusion Medicine: Second Edition. Wiley-Blackwell; 2010; 194–202. https://www.ncbi.nlm.nih.gov/books/NBK482340/

33. WHO Tramadol. Pre-Review Report. 2017. http://www.who.int/medicines/access/controlled-substances/PreReview_Tramadol.pdf

34. Caporuscio J. What are the side effects of tramadol?. Medical News Today. 2019; 1–5. https://www.medicalnewstoday.com/articles/325278

35. Burger M, Schaller DJ. Physiology, Acidosis, Metabolic. Stat Pearls Publishing; 2018; http://www.ncbi.nlm.nih.gov/pubmed/29489167

36. Burger M Schaller DJ Metabolic Acidosis. StatPearls. 2021. https://www.ncbi.nlm.nih.gov/books/NBK482146/

37. Thomas CP. Metabolic Acidosis: Practice Essentials, Background, Etiology. Medscape. 2020; 1–5. https://emedicine.medscape.com/article/242975-overview#a6

38. Nagami GT Hyperchloremia-Why and how. 2016: www.revistanefrologia.comhttp://dx.

39. Sharma S, Hashmi MF, Aggarwal S. Hyperchloremic Acidosis. Altern to Blood Transfus Transfus Med Second Ed. 2021; 194–202. https://www.ncbi.nlm.nih.gov/books/NBK482340/

40. Owoade AO Adetutu A Sinbad Olorunnisola OHematological and Biochemical Changes in Blood, Liver and Kidney Tissues under the Effect of Tramadol Treatment. J Alcohol Drug Depend. 2019; 7(5): 1–7. https://www.longdom.org/open-access/hematological-and-biochemical-changes-in-blood-liver-and-kidney-tissues-under-the-effect-of-tramadol-treatment.pdf

41. Aldiwan MA Hassan AM Mohammed A Younis A The effect of Tramadol on some blood and biochemical parameters of male rats (Rattus norvegicus). Baghdad Sci. J. 2015; 12(3): 496–502. https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/view/2094

42. Owoade AO Adetutu A Sinbad Olorunnisola O Hematological and Biochemical Changes in Blood, Liver and Kidney Tissues under the Effect of Tramadol Treatment. J Alcohololism Drug Dependence. 2019; 7(5): 1–7. https://www.longdom.org/open-access/hematological-and-biochemical-changes-in-blood-liver-and-kidney-tissues-under-the-effect-of-tramadol-treatment.pdf

43. Nadim HH (PDF) Liver Function tests: Significance of ALT (Alanine aminotransferase) and AST (Aspartate aminotransferase) as markers of Liver injury. North South University. 2020; 2–4. https://www.researchgate.net/publication/342477925_Liver_Function_tests_Significance_

44. Gowda S Desai PB Hull V V Math AAK A review on laboratory liver function tests - PubMed. Pan Afr Med J . 2009; 3(17): 2–6. https://pubmed.ncbi.nlm.nih.gov/21532726/

45. Chukwudoruo CS Iheme CI Serum total protein concentration and liver enzymes activities in albino rats model administered with ethanolic leaf extract of Ficus capensis. 2021; 20(4): 164–8. https://academicjournals.org/journal/AJB/article-full-text-pdf/F29B1F266716

46. Teshome G Ambachew S Fasil A Abebe M Prevalence of Liver Function Test Abnormality and Associated Factors in Type 2 Diabetes Mellitus: A Comparative Cross-Sectional Study.The electronic Journal of the International Federation of Clinical Chemistry and Laboratory Medicine. 2019; 30(3): 303. https://pmc/articles/PMC6803769/

47. Harris EH Elevated Liver Function Tests in Type 2 Diabetes. Clin Diabetes. 2005; 23(3): 115–9. https://diabetesjournals.org/clinical/article/23/3/115/1694/

48. Coates P Liver function tests. Aust Fam Physician. 2011; 40(3): 113. www.rcpamanual.edu.au

49. Abbas RN Jouda J Alshammary AG Jumaa MS Toxicity of Liver and Kidney Induced by Different Concentrations of Tramadol in Young and Adult Mice. 2020. http://doi.org/10.36295/ASRO.2020.23219

Received on 07.12.2022 Modified on 20.10.2023

Research J. Pharm. and Tech 2024; 17(6):2761-2768.

DOI: 10.52711/0974-360X.2024.00433