Effect of material tramadol on some blood components of laboratory rats

: The current study deal with the effect of tramadol injections on body weight and some blood components in 40 male laboratory rats, divided into four groups. The first group, G1 represents the control group. The second group G2 and the third group G3 were injected with a concentration of 20 and 40 mg/100 g of body weight, respectively, and the fourth group G4 was injected at a concentration of 80 mg / 100 g of body weight for ten days respectively. The current study results showed a significant decrease in the weights of rats injected with tramadol compared to the control group. Also, there was a significant decrease in the numbers of RBC, PLT, MID#, and GRAN#, respectively. The concentrations of each of the following constants: HGB, HCT, MCV, MCH, and MCHC showed a significant decrease in their values; except for MCV, the decrease was not significant, and the results of this study also recorded a significant increase in the white blood cells count as well as the number of lymphocytes, and there was an increase in both RDW-CV & RDW-SD.


Introduction
Tramadol is an opioid synthetic opioid analgesic and is chemically trans-2 (dimethyl aminomethyl)-1-(m-methoxyphenyl)-cyclohexanol hydrochloride. It is a centrally acting analgesic, which is used orally or parenterally for including cancer and non-cancer pain also the pain of various other organs, as well as gynecologic and obstetric; it decreases moderate to moderately severe acute or chronic pain 1-3 the choice of tramadol is affected by the degree of the pain present. It is used parenterally and orally. the analgesic efficacy is attributed to its partial affinity for the mu-opiate receptor and inhibition of norepinephrine and serotonin reuptake; they lower perception and reaction to pain as well as increase pain tolerance [3][4][5][6][7] .
Tramadol has analgesic activity, which lies between codeine and morphine. It represents about 10-20 % of the standard morphine 8 . It reaches its highest level in the blood after oral administration in about 2 hours; the reason for this is its high oral bioavailability, which is about seventy to eighty percent. Opioids used for analgesia present undesirable effects such as respiratory depression, cardiovascular depression, and sedation. Additionally, to associated with several adverse effects, among which those on the gastrointestinal (GI) tract is most troublesome in terms of frequency and severity Like constipation 5,6,9,10 and other Clinical symptoms include abdominal pain, vomiting,gastro-oesophageal reflux, nausea, anorexia, delayed digestion, bloating, straining during intestinal movements, hard stool, and incomplete evacuation 9 . Most of their effects on gastrointestinal motility and secretion result from suppressing neural activity. Inhibition of gastric emptying, increased sphincter tone, changes in motor patterns, and blockage of peristalsis 11 .
Tramadol is converted in the liver into O-and N-demethylated five different metabolites by the cytochrome p450 5,12 , and by-products are excreted through the kidney. The central role of the liver and kidney in detoxification and drug metabolism increases the risks of toxic injury., Further, it may cause hepatotoxicity 5 furthermore, the drugs excreted from kidneys could cause cellular damage leading to kidney dysfunction 5,12,13 . They pointed out some studies that the term period and high doses of tramadol cause hepatic and renal damage and impotence and increase lipid peroxidation in the rats 14 .

Animals
Forty male rats (Rattus norvegicus) weighed between (120-170) grams and were used in this study. The rats were left in the animal's house for a week before the study, in ideal conditions, natural light 1 dark cycle at 25 + 2 C temperature, and given free access to a balanced diet and water, all over the experimental period.

Experimental design
On the eighth day of the breeding, the Animals were randomly divided into four groups ( 10 rats in each group). The group one (G1 ), the average weight of rats was 153.78 g, served as control and injection doses of saline solution for 10 days. In group two ( G2 ), the average weight of rats was 140 g, and in group three (G3 ), the average weight of rats was 142.5 g, and in group four (G4 ) average weight of rats was 142.9 g. These rats were injected with tramadol HCl at concentrations of 20 mg, 40 mg, and 80 mg/100 g body weight/day for 10 days, respectively 14,15 .
We have calculated doses like El-Gaafarawi (2006) with our modification by adding a concentration of 20mg / 100g of body weight and the period of experiment time was 10 days instead of a month.
The second weight for animals was on the 11th day.

Blood collecting
Blood samples were collected from the eye rats on the 11th day by using an EDTA tube for CBC 14,16 .

Statistical Analysis
All data are presented as means ± SE. Differences between groups were analyzed by using the Duncan test, oneway ANOVA at the level of statistical significance P _ 0.01 by SPSS version 21 16,17 .

Effect of tramadol on body weight
The results showed that tramadol injections to animals showed a significant difference in weight when comparing the weights of animals in the three groups with the control group. But when comparing the weight of each group after tramadol injection with its weight before tramadol injection and separately, there were no significant differences in the weights of those groups, as in Tables (1 and 2). However, there is a percentage decrease in the weight values of the animals of these groups G2, G3, and G4. And in both comparison cases, when compared with the weight value of the control group or with the weight values for each group before the tramadol injection. As shown in Figures (1 and 2).
This part of the results agreed with what was found by The use of such materials in injecting animals leads to diseases. And of course, the result will be many dangerous complications for these injected animals, which are constipation, loss of appetite, abdominal pain, and excessive diarrhea. Such substances show an inhibitory effect on bowel movements and increase muscle tension, cramping, etc. Consequently, weight loss for these animals 6,7,9,10 .

Effect of tramadol on Hb concentration, PLT, and RBC numbers
The results are shown in Table (3) and Figure (3) that the injecting laboratory animals with such a chemical. It shows changes in the values and concentrations of some blood components, depending on the concentrations used for injection if the total number of red blood cells showed a decrease according to the concentrations used. 80 mg/100 g of animal weight had the highest effect decrease in the number of red blood cells amounting to 4.72 ± 0.23 compared to their numbers in the control group, while the effect decreased at the concentration of 20 mg/100 g of animal weight was 6.2 ± 0.21 compared with the control group 7.61 ± 0.18.
When calculating the percentage decrease in the number of red blood cells for groups G2, G3, and G4 compared to their numbers in the control group, it had found that it reached 38% in the blood of animals for the G4 group, and the percentage was 18.5% for the second group. While the hemoglobin concentration also showed a decrease of (13.36 ± 0.38, 10.2 ± 0.34, and 8.2 ± 0.12) during the different injection concentrations, which are (20, 40, 80) mg/100 g of body weight, respectively, compared with the hemog- The reason may be that the decrease in these blood components, due to the inhibitory effect of tramadol on the morphological differentiation of red blood cells, will affect the rest of the blood components, such as hemoglobin concentration and platelet count. Thus, the amount of energy produced by the metabolic processes in the body Shaimaa Obaid Mostafa, Fulla Kaydar Mohamad Salih Volume 7 / Issue 3 / 22 • http://www.revistabionatura.com Table 1. It showed the weight by grams of animals before and after injection by different concentrations of tramadol. For ten consecutive days, when compared with the control group. Table 2. Shows there was no significant difference in the three groups when comparing the weight of rats in grams before injection and after injection with tramadol for ten days.
will decrease 15,16,[18][19][20][21] . Or it may be due to the adverse effect of the mechanism of blood clotting in the body. In addition, it causes the use of various analgesics such as tramadol, which many studies in this field 8,15,18,19,21 have confirmed.

The effect of tramadol on the constants of RBC that represent by HCT, MCV, MCH, MCHC, RDW-CV& RDW-SD
The results showed that the concentrations of tramadol injection led to a significant decrease in most of these constants, except for MCV, where the different concentrations of tramadol did not register any significant differences compared with the control group. The 40 and 80 mg / 100 g body weight concentrations recorded significant differences for all the remaining parameters. When compared with the control group. The decrease for these constants is (38.    As for both RDWCV and RDWSD, the results of this research recorded a percentage increase for each of them, following the increase in the concentrations of tramadol used in injections compared to the control group. And the percentage value was an increase in RDW-CV to (17.81; 28.14; 35.14)% for groups injected with concentrations of 20, 40, and 80 mg / 100 g of body weight, respectively. As for the RDW-SD, the percentage value increase was (29.9%) at the concentration of 80 mg / 100 g of body weight. As for each of the concentrations of 40 and 20 mg / 100 g of body weight, the percentage was (23.6 and 12.9), respectively, as in Figure  The reason for the decrease in the volume of HCT may be due to the effect of this substance tramadol on the decrease in the activity or function of the sympathetic nerve during blood circulation in the spleen and thus will affect   the volume of HCT 15,19,22,23 . In addition to a decrease in hemoglobin concentration, this will lead to the inhibition of the biosynthesis of iron in the bone marrow, and this will subsequently affect MCH and MCHC 8,15,18,19 or because when the animal is injected with tramadol, HCT will decrease due to the transfer of bodily fluids from parts outside the blood vessels to the inside of the blood vessels, and many studies confirmed this in this field 8,15,21,22 Or it could be the reason raised anti-diuretic hormone and aldosterone secretion with resulting water and salt retention causes the expansion of the extracellular fluid leading to hemodilution and drop in HCT 6 .

The effect of tramadol on the WBC ;LYM%;MID% ;GRAN% ;LYM# ;MID# &GRAN#
The indication of the results shown in Table (5) and Figures (6 and 7) that injections with different concentrations of 20, 40, 80, and mg/100 gm of body weight have an effect on the immune system, of the injected animals through the increase and decrease in the total numbers of white blood cells and they are the other types. The increase in the number of white blood cells was (10.13 ± 0.43; 12.95 ± 0.47; 15.3 ± 0.9) respectively for the above concentrations compared with its number in the control group (9.84 ± 0.27). While the increase in the numbers of lymphocytes and LYM % reached (14.03 ± 0.73; 89.95 ± 1.31), respectively, at the concentration of 80 mg / 100 g of animal weight compared to the control group (7.1 ± 0.53; 72.16 ± 0.91). but the numbers of MID# and GRAN# were showed a significant decrease in the three groups injected with different concentrations of tramadol compared with the control group, which reached (0.99 ± 0.04; 1.75 ± 0.19) for each one parameter, respectively. where their numbers were at a concentration of 80 mg/ 100 g of animal weight were (0.57 ± 0.03; 1.0 ± 0.08) for MID# and GRAN#, respectively, and the numbers of MID# reached (0.65 ± 0.04; 0.64 ± 0.03) at concentrations of 20,40 mg/100 g of animal weight, respectively.
The numbers of GRAN# cells were (1.47 ± 0.12; 1.45 ± 0.123) for concentrations of 20 and 40 mg/100 g of animal weight, respectively. MID% and GRAN% also significantly decreased in all groups injected with tramadol. When they compared to the control group. Which reached (10.06 ± 0.49 ; 17.78 ± 0.77) for each of MID% and GRAN%, respectively. While the values of each of them at the concentration of 20 mg/100 g of animal weight were (6.5 ± 0.28; 14.4 ± 0.47). The value of GRAN% decreased to (6.4±0.22; 11.2±0.37) at concentrations of 80 and 40 mg/100g of animal weight, respectively. As for the MID% also decreased to ( The difference in the number of diverse white blood cells may be due to the activation of the defense mechanism of the immune system in rats, and thus will affect the white blood cells and induce them to form a positive response by monocyte and lymphocyte in addition to the increase in the WBC, which was the result of lymphocytes, This is one of the main features of variance in white blood cell counts The difference in the number of diverse white blood cells may be due to the activation of the defense mechanism of the immune system in rats, and thus will affect the white blood cells and induce them to form a positive response by monocyte and lymphocyte in addition to the increase in the number of white blood cells, which was the result of lymphocytes, This is one of the main features of variance in white blood cell counts 2,8,15,16,21 . Or it may be due to the effect of this substance tramadol on the differentiation of white blood cells as a positive response to survival due to the immune response of the cells of animals treated with tramadol 2,4,8,18,19,22-24 .

Conclusions
This study showed that daily use of tramadol for a relatively short period in young rats leads to significant changes in all blood components, especially hemoglobin, red blood cell count, and differentiation. As well as its disorders in the immune system. And the number of blood platelets affects the process of blood clotting. In addition to negatively affecting body weight, which may cause future growth disorders.

Acknowledgments
The authors wish to express their gratitude to the University of Mosul, College of Science, Biology department, for their provided facilities, which helped improve this work's quality. Table 5. Shows the values of WBC, LYM%, MID%, GRAN%, LYM#, MID#, and GRAN# for the three groups injected with tramadol for ten days.