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The effects of methylphenidate combined with exhaustive practice on the white cells of the male rat.


Medical doctors and athletes are aware of the close correlation between exercise and immune system; several years ago, exercise safety experiments have been included in the topics of Stress Immunology. (1)

On the other side, energy providing supplements and various performance enhancing unauthorized drugs, not widely used in the past, are increasingly used. As a result, novel diagnosing methods for doping have made their appearance. Nowadays, the exercise life has turned out to be a bitter reality. Athletes undertake this job to achieve championship titles and various bonuses earnt through winning. (2,3,4) Oechnological advances in medical diagnostics, competition among pharmaceutical companies and new discoveries in doping world, make up a dangerous and uncontrollable problem. (2,3,4,5)

Methylphenidate is classified in stimulant drugs. It is a pyridine derivative closely similar to Amphetamine in aspect of structure. It is induced by CNS (Central Nervous System) bearing a weaker effect than amphetamine and stronger than Caffeine, with the molecular formula C14H19NO2HCL. Its surface resembles to crystal powder in white with melting point of 224-226[degrees]C , soluble in the water, alcohol and chloroform. The lethal dose of this drug for adult humans is 200 mg per kg of weight or 376 mg/kg of weight of the body for rats. Ritalin (methylphenidate) is used by athletes due to its obstructive effect on the exhaustion, creating euphoria, increasing mental/psychic power of the individual and enhancing the performance in sports to athletes. Long term usage of methylphenidate may disrupt the cycles of growth in young individuals, accompanied by side effects as: headache, insomnia, nausea and dry mouth. Excessive usage of methylphenidate can raise the blood pressure, heartbeat, cause chest ache, heart and blood vessels' collapse, a convulsion, coma and finally result to death. (2,3,6)

There have been several studies on the effect of stimulant drugs over immune system. In et al (7) have investigated the regulation of Murine strange-eating function by Methamphetamine. In this study, the immunity regulating effect of Methamphetamine was tested on peritoneal macrophages of Murine after daily oral administration for 2 weeks (5 mg/kg weight of the body). The results of FACS analysis indicate that CD14 case was tangibly increased by Methamphetamine in the Lipopoly-saccharide (LPS)-stimulated macrophages. In addition to this, their results show the segregated immunity and the regulating effect on cell activities and macrophages sprinkle. (7)

An other study by Pacifici et al (8) examines the effects of repeated (3,4)-Methylenedioxymethamphetamine or Ecstasy (MDMA) doses on the immune response through cell interface in humans, when MDMA had been used into fractions separated by two short intervals.

Immunity response with cell interface was evaluated after consuming two repetitive doses of 100mg MDMA (stimulant drug), in 4 and 24 hrs distance in two random groups. (8) MDMA created a time-based reduction in cell ratio of CD4/CD8 lymphocytes leading to reduction in the number of contributing cells CD4 lymphocytes, reduction of lymphocytes' functional response to mutagenic stimulation and simultaneous increase in natural killer cells. These results showed that repeated usage of MDMA with two short intervals can in the long run seriously harm the integrity of immune system functioning. (8)

In another research by Connor et al (9), the acute usage of methylphenidate was examined. Although the duration of methylphenidate effect on the immunity function has mainly been investigated only in vitro experiments, it has been reported that the acute usage of methylphenidate in vivo (20 mg/kg intra- peritoneal injection) can produce a quick suppression (in 30 min) against lymphocyte reproduction and an intensive reduction in the total count of leukocytes in rats, all at least occurred in the first six hours following injection. The research of Pacifici et al (10) in the laboratory of Ecstasy (MDMA) presented that some immunity parameters including regulating T-cell functions can change the activity of cytotoxic T lymphocytes, macrophages function and the activity of natural killer cells. The usage ofMDMA in rats can supress lymphocyte reproduction response; reduce in lymphocytes circulation is followed with increase in plasma corticosteroids. This reduction happened in CD4 lymphocytes count and responding function while there was a raise in the percentage of natural killer cells. (10,11)

Research methodology

The current study was conducted on male rats, Vistar breed. In total, 110 male rats were used, with an average age of 3.3 months and an average weight of 282 [+ or -] 52 g (weight ranging 230-340). All tracked animals were in complete health, and were divided into pilot groups and 6 groups including 10 experimental divisions as below. The Control group (no exercise) animals were taken for blood sample after selection. The following groups were specified: exercise groups with no drug prescription, exercise group with 25% dose of [LD.sub.50], and pilot study groups comprised of 50 male rats in order to test dosing (LD) of methylphenidate. The rats exercised into a treadmill, and were divided into 10 groups, 5 animals per group. The blood sampling was held appropriately.

One day before implementing any level of tests, the rats were delivered from the reproduction and preservation center of experimental animals in Jondi Shapoor Medical Sciences University and transferred to the Physiology Laboratory of the Medical School, in order to get accustomed with the laboratory environment. To start the tests, first of all, the rats' weight and unique experimentation number was recorded. Each sample received the dose of drug allocated to its weight, and was transferred into a cage separated from other animals. The drug administration is usually accompanied by behaviors including: rapid move toward the corners of the cage, standing on feet, physical activity that raises especially in high doses, appearing moves like hitting on the cage and biting the walls of the cage.

The exercise test was conducted with the use of a treadmill. The treadmill was regulated on 1 km/h for 5 min to get the animal accustomed with this movement method into the device and warming after locating the animal into the treadmill. The treadmill was adjusted and fixed on 2 km/h after the first 5 min, until the animal gets exhausted. In this situation, the animal is incapable to move, being located into the device, and falls down on the side or back. The animal is then transferred out of the device after recording the time needed for the exhaustion effect. The rat was delivered into a chamber (lacuna), used for anaesthesia; the blood samples were finally received with the rat lying on a surgical table. The rat was located on its back on the laboratory table for blood sampling. The chest was opened using surgical scissors and blood samples were received from the left ventricle by a 10 ml syringe.

Statistical Analysis

For the research hypothesis test, one way variance analysis (ANOVA) was used; to be aware of meaningfulness difference among research groups, Tukey's test was used. The total of needed statistical calculations were made using SPSS software. Alfa error coefficient was considered 0.05 in the current research.


Research findings

The average of leukocytes percentage in the research groups has been mentioned in Table 1 and Figure 1. The research groups comprised of the witness group, the exercise group, 25% [LD.sub.50] group, 50% [LD.sub.50] group, 75% [LD.sub.50] group and [LD.sub.50] group.

Table 2 includes the data needed to test the research hypothesis based on the effect of an exhaustive practice as well as methylphenidate usage over the number of rats' leukocytes and compare the average of leukocytes in witness, exercise, 25%[LD.sub.50], 50%[LD.sub.50], 75%[LD.sub.50] and LD groups. The F amount has been calculated in the level of a=0.05 using one way variance test (ANOVA). Based on the results of the table it can be stated that there is a meaningful statistical difference within the average number of leukocytes in witness, exercise, 25% [LD.sub.50], 50% [LD.sub.50], 75% [LD.sub.50] and LD groups. Tukey's pursuit test was also used to know in which groups there is difference (see Table 3).

In Table 3, the results show that:

1. There existed significant difference between witness group and exercise group so that the average number of leukocytes in the exercise group was higher than in the witness groups.

2. There was a significant difference between the witness group and the 25% [LD.sub.50] group, so that the average number of leukocytes was higher in the witness group.

3. There was a significant difference between the exercise group and the 25% [LD.sub.50] group so that the average number of leukocytes was higher in the exercise group.

4. There was a significant difference between the exercise group and the 50% [LD.sub.50] group so that the average of leukocytes was higher in exercise group.

5. There was a significant difference between the exercise group and the 75% [LD.sub.50] group so that the average number of leukocytes was higher in exercise group.

6. There was a significant difference among the exercise group & the [LD.sub.50] group so that the average number of leukocytes was higher in exercise group.

7. There was not significant difference among the witness group, the [LD.sub.50] group and the 50% [LD.sub.50] group.

8. There existed significant difference regarding various doses of drugs in the number of leukocytes among exercise groups.


According to the current study results, conducting one session of exhaustive exercise along with consuming 25%dose of [LD.sub.50] up to [LD.sub.50] methylphenidate has no effect on the percentage of rats leukocytes including a significant level of a=0.05 in statistical aspect. The results about the difference among witness and exercise groups agree with the findings of research studies on what kind of physical exercises can lead to increase of leukocytes. (12-20) In most cases, the possibility that mechanical parameters, as increase in heart output and injection into small vessels may change the balance between leukocytes and endothelium of blood vessels, leading to separation of leukocytes from the vessels' wall. In addition to this, immature leukocytes may be released during very severe practices through bone marrow. Some of these changes made happen due to alteration in stress hormones levels and inducing reactions like cytokine and acute level proteins production.

But there is no conformity between those groups exercised and drug administration groups with the witness group in the mentioned studies, and as mentioned, such phenomena may result from methylphenidate usage. (10,21,22,23) Also, the results of this study differ to the study of Pacifici et al (8) which investigates the ecstasy (MDMA) use, with two short intervals between doses, and results that it endangers the capability of immune system. In addition, Son EW (5) et al with their study over methamphetamine present the effect of segregating immune regulation on the cell and secretory function of macrophages. Methamphetamine shows a meaningful effect on phagocytes as Interleukin- (1,6) on macrophages. In addition to this, in the research of Connor et al (9) in rats, they concluded that although duration of methylphenidate effect on immune system is limited to in vitro researches, the acute effects of methylphenidate in alive peritoneum injection (20 mg/kg) produces a fast suppression (within 30 min) against lymphocytes reproduction and an extensive reduction in the counting of leukocytes of rats which is at least more obvious in the first 6 hrs of injection. (23)

Also, Pichini et al, in their Biochemical Laboratory in Rome, have shown thatMDMA changes affect some immune parameters including regulator T cell function, cytotoxic T lymphocyte function, macrophages function and natural killer cell activity (NK) accompanying with corticosteroid plasma raise. (10, 17, 20) Several of the referenced studies have also reported a significant relationship among blood cortisol and the number of white cells during and after exercise. But some others did not succeed to detect such a relationship.


The general conclusion of the current study reflects that physical activities lead to leukocyte increase in the blood, but while using methylphenidate some reverse results are achieved; as using other compatible drugs i.e. CNS stimulant drugs have led to leukocytes reduction in the peripheral blood. Immune system regulated mechanisms can describe and explain the differences found between witness and exercise groups, as other researchers have already described, with the use of other stimulant drugs or/and exercise. (10, 23, 24, 25, 26, 27, 28)

Based on the results of the current study, the combined use of methylphenidate and exercising is expected to reduce the number of leucocytes. Thus, it is recommended to athletes to refrain from using stimulant drugs of central nervous system, and especially methylphenidate, in order to be protected from severe reduction in the number of leukocytes and the possibility of consequent vulnerability to infectious diseases.


[LD.sub.50]Abbreviation for Lethal Dose, 50%

Conflict of interest: None declared.


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Corresponding author: Abdolhamid Habibi,

Department of Sport Physiology,

Shahid Chamran University,

Golestan Boulevard,

Ahvaz, Iran

Table 1. Statistical description for the WBC measures.

Independent    Group      1 Number of mean   N       SD
variable                    [micro]/

               witness    0900/6             10      17988/1
               Exercise   7800/7             10      47633/1
               50LD 25%   8500/3             10      15109/1
White Cells    50LD 50%   5600/4             10      97434/0
               50DL 75%   1100/4             10      01811/1
               50LD       9700/4             10      63778/1
               Total      2267/5             60      81919/1

Table 2. The ANOVA results of the WBC measures.

Independent     Sum of                 Mean
White Cell      squares      fd      squares       F          P

Between group   173/109      5        835/21     697/13     000/0
Within group    084/86       54       549/1
Total           257/195

Table 3. The results of TUKEY test for the WBC measures.

                     Dependent Variable: y = % perception

Independent    Group      Group       Deferent       Standard    p
  variable      (I)         (J)         mean (J-I)     error

               witness    Exercise    6900/1- (*)    46565/0     045/0
               Witness    50LD 25%    262/2 (*)      56465/0     003/0
               Witness    50LD 50%    5300/1         56465/0     090/0
               witness    50LD 75%    9800/1 (*)     56465/0     011/0
               witness    50LD        1200/1         56465/0     365/0
               Exercise   50LD 25%    9300/3 (*)     56465/0     000/0
WBC            Exercise   50LD50%     2200/3 (*)     56465/0     000/0
               Exercise   50LD75%     6700/3 (*)     56465/0     000/0
               Exercise   50LD        8100/1 (*)     56465/0     000/0
               50LD 25%   50LD50%     7100/0         56465/0     806/0
               50LD 25%   50LD75%     2600/-         56465/0     997/0
               50LD 25%   50LD        1200/1         56465/0     365/0
               50LD50%    50LD75%     4500/0         56465/0     967/0
               50LD50%    50LD        4100/ -        56465/0     978/0
               50LD75%    50LD        8600/0-        56465/0     651/0

Figure 1. mean values of WBC in the studied groups.

                10 * 3 Microlitter

Control    6.09
Training   7.78
25% LD     3.85
50% LD     4.56
75% LD     4.11
LD         4.97

Note: Table made from bar graph.
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Author:Habibi, Abdolhamid
Publication:Archives: The International Journal of Medicine
Article Type:Report
Geographic Code:7IRAN
Date:Oct 1, 2009
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