Effects of Phenobarbital and carbon tetrachloride on

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1 Odhiambo et al.. J. Appl. Biosci Effects of phenobarbital and carbon tetrachloride on liver enzymes Journal of Applied Biosciences 56: ISSN Effects of Phenobarbital and carbon tetrachloride on liver enzymes Odhiambo F., Chek J.B.L and Moro J.O. Department of Biochemistry, University of Nairobi, P.O Box , Nairobi, Kenya. Corresponding author: Odhiambo Felix Blair, address: Original submitted in on 18 th April Published online at on August 31 st ABSTRACT Introduction: Barbiturates and carbon tetrachloride (CCL4) are known to have effects on liver enzymes. Moreover, barbiturates which are used as sedative hypnotics, anticonvulsants generally cause induction of liver microsomal enzymes while CCL4 cause hepatoxity releasing various liver enzymes. Objective: To determine the effects of Phenobarbital and carbon tetrachloride on some selected liver enzymes. Methodology and results: Rats treated with Phenobarbital had higher levels of activity of transaminases, alkaline phosphatase and longer to gain writing reflex while CCl4 treated rats had reduced levels of the mentioned enzyme and shorter time to gain writing reflex Conclusion and application: Treatment with Phenobarbital needs continuous adjustment and regulation of dosage by the attending clinician. A history of the patient or a therapeutic drug monitoring to confirm toxicology. It was therefore concluded that barbiturates are inducers of liver enzymes while CCl4 is a liver toxicant INTRODUCTION Barbituric acid is a malonylurea obtained by condensation of urea and malonic acid. Barbituric acid is not a sedative hypnotic but its derivatives (V N Sharma, 1999). Barbituric acid was first synthesized on December 1864 by German researcher Adolf Von Bayer ( Barbiturates have been classified mainly according to the speed of onset of action and their duration of action as ultra short acting, short acting, intermediate acting, and long acting barbiturates. Barbiturates and carbon tetrachloride (CCL4) are known to have effects on liver enzymes (Sherlock et al 1974). Further barbiturates which are used as sedative hypnotics, anticonvulsants, generally cause induction of liver microsomal enzymes (Sherlock et al, 1974). The major enzymes induced include the enzyme cytochrome p450, which is the major enzyme involved in drug metabolism. Another enzyme that increases is the gamma glutamyl transferase (GGT) known to be induced by the anti- epileptics or anticonvulsants for example the Phenobarbitals (Srivastava et al, 2002). Hence with heavy intake of barbiturates, levels of cytochrome p450 and GGT rise and this will lead to an increase in the rate of drug metabolism. As a result, drugs administered after the body is exposed to barbiturates are expected to metabolize faster due to increase in levels of drugs metabolizing enzyme cytochrome p450. When liver is exposed to CCl4 it gets damaged. This may lead to increase in the levels of liver enzymes in the serum as a result of the damage to the liver cells. Hepatocytes, are therefore found the serum 4097

2 Odhiambo et al.. J. Appl. Biosci Effects of phenobarbital and carbon tetrachloride on liver enzymes (Sherlock et al, 1974). By noting the serum level of these enzymes; the level of liver damage can be assessed through the estimation of Serum oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) levels. Other enzymes such as lactate dehydrogenase found in the liver are expected to rise. Hence using good assay techniques the levels of these enzymes can be determined by calculating specific enzyme activity for each of them. Recent finding have documented a continued use of barbiturates as drugs of choice in anesthesia, treatment of epilepsy, and as anticonvulsants. There was need therefore to understand how barbiturates influence the body system. This is only possible through continuous studies ascertaining their long-term and short-term effects in human and animal systems. This study was therefore done to confirm the previous findings and also to find out if they have to be continuously used or other better alternatives can be applied. Exposure of the liver to various toxins in the current world has increased tremendously, the intake of preserved foods are contributing factors in to this debate of hepatotoxicity. CCl4 used a model to understand the basis of study of liver damage and hepatotoxicity. MATERIALS AND METHODOLOGY Phenobarbital. Thiopental sodium. Carbon tetrachloride (CCl 4). 2mM ketoglutaric acid. 1.0M hydrochloric acid. Citric acid. 200mM D-alanine. 200mM L-aspartate. 0.1 Phosphate buffer. Sodium hydroxide (0.4M and 0.02M). 2, 4-Dinitrophenyhydrazine. Glycine glycine. Experimental animals: Albino rats of one year in age with their weights as indicated in appendix VII. Procedure Administration of Phenobarbital to the rats: There were two rats in each group.the administration of the Phenobarbital and thiopental was given intraperitonearly. Group1: They were treated with Phenobarbital sodium 30mg/kg three weeks after every two days. Group2:These were treated with Phenobarbital sodium 15mg/kg for 3 weeks after every two days. Group 3: They treated with carbon tetrachloride (10% in vegetable oil) 24 hours previously before treatment with thiopental sodium. Group 4: They treated with CCl 4 (20%) in vegetable oil 24 hours previously before treatment with thiopental sodium. Group 5: This group was not treated with any drug for three weeks and served as control groups for the Phenobarbital treated rats. Group 6: Served as controls for CCl 4 treated rats Preparation of serum: After successful injection of the rats with Phenobarbital sodium and feeding with CCl 4solution, they were injected with thiopental sodium to study the physical effects. They were then sacrificed to secure blood from which serum was obtained. The serum was obtained by living the blood to clot for about 20 minutes by putting it in ice. After clotting the blood was then centrifuged for 20 minutes at 6000 rpm. The serum was then decanted using Pasteur pipettes to get pure non-haemolysed blood containing serum. The serum was then put into labeled eppendorf tubes according to their respective drug treatments. The serum samples were kept frozen at 20º before caring out enzyme assays. Enzyme assay procedures Enzyme assays principles: GGT catalyses the transfer of gamma glutamyl group from gamma glutamyl-p-nitroanilide to accept or glycylglycine according to the following reaction Gamma l-glutamyl-3-carboxy-4-nitroanilide+ Glycyl glycine GGT Gamma l-glutamyl-glycylglycine + 2-Nitro-5-aminobenzoic acid The rate of 2-nitro 5amino benzoic acid formation, when measured photometrically indicates and proportional to the catalytic concentration of the enzyme present in the sample. The assay procedure for both the AST and ALT were based on the colorimetric method whereby the reaction was allowed to take place by adding their substrates, alpha ketoglutarate and aspartic acid for AST and alpha ketoglutarate and alanine for ALT. The reactions were then stopped by dinitrophenyl hydrazine (DNPH)

3 Odhiambo et al.. J. Appl. Biosci Effects of phenobarbital and carbon tetrachloride on liver enzymes reagent. DNPH reacts with pyruvate to form a brown colored complex medium which is measured calorimetrically (DNPH can react with all oxoic acids). It gives a triose colour with pyruvate and oxaloacetate than with alpha ketoglutarate as evidenced by relative absorbance for pyruvate, OAA and 2-oxoglulurate being 1, 0.55 and 0.33 respectively. Because of the blink value, the reaction is carried out at the sub optimal concentration of oxoglutarate). Procedure of assay of gamma glutamyl-transferase: One ml of the working reagent was put into a cuvette then 0.1ml of the serum sample was added for both the controls and test. The contents were then mixed, waited for 1 minute and read the initial absorbance (A) of the sample at 405 nm. The stop watch was started and the absorbance was read at 1-minute interval for three minutes. Glutamate Pyruvate Transaminase (GPT) assays: A calibration graph of units/l against the optical density reading at 505 is required and this is used in determination of enzyme activity in sera the following set up was used to generate the standard curve was arranged as below: Table1: Glutamate pyruvate assay Layout Tube no. Distilled water Pyruvate standard (mls) GPT Substrate buffer Corresponding IU /L (mls) (ml) After arranging the tubes as above, the contents were then mixed well and incubated at 37 0 for 30 minutes. After which 1 ml of 2, 4- Dinitrophenylhyrazine reagent in 1M hydrochloric acid was added to each tube, mixed well and incubated for further 20 minutes. 10 ml of 0.4M sodium hydroxide solutions was added to each tube and allowed to stand for 10 minutes. The optical densities were read at 505 nm. Assay Alanine Transaminase (ALT): One ml of the buffered substrate was first put in the test tubes for the assay of both test serum and control serum. Of the test serum 0.2ml was added and incubated for 30minutes at 37ºC, 1ml of 2,4 DNPH was added to each of the tubes and 0.2 ml of the control serum was the added its respective tube. The contents were then mixed well and incubated further for 20 minutes then removed from the water bath and 10ml of 0.4M NaOH added to each tube. The tubes were left to stand for 10 minutes after which the optical densities were read at 505 nm against the Water blank. The AST activity in serums was assayed as follows: After arranging the tubes as above, the contents were then mixed well and incubated at 37 º for 30 minutes in water bath. After which 1 ml of 2, 4- Dinitrophenylhyrazine reagent in 1.M hydrochloric acid was added to each tube, mixed well and incubated for a further 20 minutes. Ten (10) mls of 0.4M sodium hydroxide solutions was added to each tube and allowed to stand for 10 minutes. The optical densities were read at 505 nm. Table 2: Aspartate aminotransferase assay layout. Tube no. Distilled water Pyruvate standard AST substrate Corresponding IU /L (Mls) (mls) buffer (ml) Buffer

4 Odhiambo et al.. J. Appl. Biosci Effects of phenobarbital and carbon tetrachloride on liver enzymes The Procedure for the assay of serum AST levels was as follows: One ml of the buffered substrate was first put in the test tubes for assay of both test serum and control serums. Of the test serum 0.2 ml was added into the test tube and all the tubes incubated at 37º C for 1 hour in water bath. A drop of aniline citrate was added in each tube. The samples were left in water bath for 5 minutes, still at 37º C. Of the control serums 0.2 ml was then added followed by addition of 1ml of 2, 4 DNPH additions to all tubes. The contents were mixed and incubated further for 15 minutes and then removed. 10ml of 0.4M sodium hydroxide was added to the reaction. The contents were mixed and allowed to stand for ten minutes. The optical density was at 505 nm against water blank. Preparation of the calibration graph for the alkaline phosphatase assay: The tubes were arranged as follows then filled with the reagent therein. Table 3: Assay of alkaline phosphatase Alkaline Phophatase Tube No 4 - Nitrophenol Solution 0.02M NaOH The contents were then mixed well and their absorbance read at 400nm against water blank. Assay activity of the alkaline phosphatase in serum: Two test tubes were prepared for each serum to be assayed i.e. both the test and control serum 1.0ml of the buffered substrate was put into each of the tubes. Test (here for both the control and the test serum) and the serum blank. This was then incubated for 37º C for 5 minutes and serum added only in the tube for test. Mixed well and incubate at 37ºC for 30 minutes including serum blank. After that 10ml 0.02M NaOH was added. Serum was then added in the serum blank tubes. The contents were then mixed well in each tube and the optical densities read at 400nm. RESULTS AND DATA ANALYSIS Table 4: Calculated values of Gamma Glutamyl-transferase activity after treatment with Phenobarbital and carbon tetrachloride. Test Group Rat OD Factor GGT activity (U/L) 1 a b a b a b a b a b a b

5 Figure 1: Calibration Graph for the determination of GOT activity. Figure 2: Calibration curve for determination ALT activity 4101

6 Figure 3: Calibration curve for alkaline phosphatase Table 5: Activity for Aspartate aminotransferase after treatment with Phenobarbital and carbon tetrachloride Test Group Rat OD AST activity (U/L) 1 a b a b a b a b a b a b Table 6: Activity for Alanine aminotransferase after treatment with Phenobarbital and carbon tetrachloride. Test Group Rat OD ALT activity (U/L) 1 a b a b a b a b a b b

7 Table 7: Results of the estimated activity of alkaline phosphatase after treatment with Phenobarbital and carbon tetrachloride Group Rat Test (OD) Serum blank (OD) OD difference Enzyme activity 1 a b a b a b a b a b a b

8 Table 8: The effects of thiopental sodium treatment on rats after treatment with Phenobarbital and carbon tetrachloride Rat Drug Dose per kg weight Time of administration Time of loss of righting reflex Time of regaining righting reflex 1a Thiopental sodium 30 mg 0.0 mins 4.0mins 20.00mins 35mins 1b Thiopental sodium 30 mg 0.0 mins 3mins 54 secs 17mis 20 secs 36mins Time of recovery (walking) 2a Thiopental sodium 30 mg 0.0 mins 5mins 23 secs 15mins10 secs 18mins 12secs 2b Thiopental sodium 30 mg 0.0 mins 4mins 12 secs 19mis 29 secs 23mins 3a Thiopental sodium 30 mg 0.0 mins 10secs 3hrs 48 mins 4hours 20 mins 3b Thiopental sodium 30 mg 0.0 mins 5secs Never regained Never recovered 4a Thiopental sodium 30 mg 0.0 mins 2mins 2hrs 13 mins 2 hours 30mins 4b Thiopental sodium 30 mg 0.0 mins 4 secs Never regained Never recovered 5a Thiopental sodium 30 mg 0.0 mins 10 secs 54secs 1 hr 25 secs 5b Thiopental sodium 30 mg 0.0 mins 9secs 1 min 5 secs 1 hr 10 mins 6a Thiopental sodium 30 mg 0.0 mins 25 secs 45secs 56mins 49 secs 6b Thiopental sodium 30 mg 0.0 mins 6secs 58 secs 42 mins Summary of the above results Table 9: Summary of the result on the effects of thiopental sodium treatment on rats after treatment with Phenobarbital and carbon tetrachloride. Group of rat according to treatment Time of loss of righting reflex Time of regaining righting reflex Time of recovery (walking) Phenobarbital 3 mins 54 secs-5 mins 23secs 10-20mins mins CCl4 4secs-2 mins 2.5hrs- never Control 6-25 secs 45 secs- 1 mins 5 secs 42mins- 1 hr 10 mins 4104

9 DISCUSSION Barbiturates are known to be enzyme inducers while CCL 4 is a hepatotoxicant able to cause liver damage(.levels of GGT in the serum can therefore be used as an indication of either enzyme induction or liver damage, the later being more common(. The GGT levels also helps to detect liver damage and bile injury while in some cases it is used in people suspected of having liver diseases. It is also used to help explain the cause of other changes in the liver or if alcohol abuse is suspected Lab test online.org ( ). For example both ALP and GGT are elevated in diseases of bile ducts and in some liver diseases but only ALP will be elevated in bone diseases.if the GGT levels are normal in a person with high ALP, the cause most likely is bone disease. GGT can be used to screen for chronic alcohol abuse where it will be elevated in about 75% of chronic diseases (Lab test online.org ( ). The normal levels range form 4-18U/Lin males and in females 6-28U/L when the assay is done at room temperature (25ºC), )(Chrono lab AG Switzerland, 2008) From the results obtained, the rats that were not treated (controls) had lower levels of the GGT activity as compared to the ones treated with barbiturates. Although the GGT levels in rats treated with Phenobarbital were within the normal reference range, their activity was higher than the controls and this could be as a result of the induction made by the barbiturate (Phenobarbital). Rat 2 (b) treated with 15mg/kg of the drug had increased levels than others treated with the same drug including the one treated with higher dosage (30mg/kg) of the same drug. This could suggest other factors that could induce the levels of GGT that could be bone disease, bile duct injury or billiary obstruction. But this are just suggestive as this could be combined with other liver function tests involving other enzyme for example higher levels of GGT and lower levels of ALP rules out bone disease. In hepatocellular damage higher levels of GGT have always been observed (Lallit et al). In this case, rats treated with CCL 4 had increased levels, some even beyond the normal documented levels of 4-18 U/L in males (Chorono lab AG Switzerland, 2008), meaning that CCL 4 was responsible for the increased levels of GGT for both treated with 20% and 10 % CCL 4 in vegetable oil. From the results obtained it was evident that Phenobarbital is a good inducer of liver enzymes, in rat 1a with an estimated level of 17U/L, which is slightly above the normal levels. Rat 1b also had increased levels of ALT of 17.5 U/L. From this it, suggests that there was no hepatobilliary injury since the levels of GGT was falling in a normal range among these rats. Bone disease could also not be suggestive compared to the results of ALP levels among these rats, which were at normal levels. Rats in group two also had increased elevated levels too, though rat 2a was within the normal levels but on the higher side. Rat 2b had elevated levels above the normal range. This also confirmed the levels in rats of group one. Rats in group 3 and 4 had increased levels of both the transaminases above their normal ranges. This could not be due to induction but injury to the hepatocytes but when compared to the levels of GGT it could confirm that this rise in levels of transaminase was not due to induction but due to liver damage. ALT levels of rats of groups 5 and 4 fell within the normal range with rat 5b having the minimal value of the enzyme level. ALT is primary liver enzyme and its induction correlates to the very effect of these drugs. Serum level of AST, which is one of the enzymes most used in diagnosis of liver function test, is also suggestive of the pathologies of the liver. The rats in group 1 treated with 30mg/kg had induced levels of AST with level of rat 1a going up to 45 U/L much above the maximum normal level maximum of 20U/L. Rats in group in two also had increased levels of the enzyme with 2a being up to 40 U/L. Even though 2b had 18U/L, which falls within the normal range but on the higher side. Rats of group 3 had excessive levels with 3a going up to 55U/L and 3b 29.5U/L which when compared to the reference range was high. This was due to liver damage caused by the CCL 4. Group 4 animals especially had elevated levels and explanation was just as the group 3 rats. Rat 4a had normal levels. May be there was no damage to it or the experiment didn t give a cognitive figure but it is believed it also underwent liver damage as Levels of GGT indicates. On the other hand, the effect of Phenobarbital and CCl 4 on rat liver enzymes can be analyzed by physical examination of rats by treating them with a short acting barbiturate which can give immediate anesthetic effect (Brodie et al, 1958). In this case thiopental sodium was used. Rats on treatment with enzyme inducing agent such as barbiturates (Phenobarbital in this case) take shorter time to recover while those intoxicated with very active hepatotoxicants take too long to recover or might end up in dying Brodie et al, 1958). In this particular study group 1 and 2 rats treated with Phenobarbital previously, took longer time to lose their righting reflex, between 3 mins 54 secs to-5 mins 23 secs. While those on treatment with CCL 4 took a shorter time (4 sec

10 mins) due to enzyme induction and liver toxicity (damage) respectively. Comparing the time of loss of righting reflex with the control rats (6 secs-25secs) gives an indication of barbiturate and CCL 4 effects. Rats treated with Phenobarbital took a shorter time (10 to 20 mins) to regain their righting reflex as compared with those treated CCL 4 which took longer time to regain their righting reflex ranging from 2 hours for rat 4b and 4 hrs 20 mins for rat a. Others treated with CCL 4 CONCLUSION AND RECOMMENDATIONS From the results obtained from this experimental analysis, it was important to conclude that barbiturates are inducers of liver serum enzymes, which move to the blood in excess. This was shown through the serum enzyme analysis and evident in the results. It was established that Phenobarbital, a barbiturate induces liver enzymes. It was also found that CCL 4 leads to increase in the levels of serum enzymes but this was due to liver damage it causes. There was evidence that treatment with Phenobarbital leads to a shorter action of thiopental sodium. This could suggest tolerance obtained by these animals after continuous administration. Patients therefore who have been on treatment with drugs (barbiturates) need to increase their dosage in order to get treated with other drugs to the due to the induction of microsomal enzymes increases. It was also confirmed that CCl4 is a liver toxicant with severe hepatic damage according to the enzyme levels of obtained and also after treatment with thiopental sodium which indicated long time of gaining the righting reflex even lack of recovery leading to death. Patients, who have been on treatment with Phenobarbital especially the epileptics, need a REFERENCES Brodie, B. B., Gillette, J.R., and Ladu, B.N., Enzymatic mechanisms of drugs and other foreign compounds. Ann. Rev. Biochem. 27, , 1958 Gendler S. Gamma GT. Kaplan et al. Clinical Chemistry. The C.V Mosby Co. St Louis. Toronto, Princeton; , Herbert Remmer, M.D and Karl Walter Bock, M.D The role of the Liver in Drug Metabolism, The liver and its diseases, Sherlock, http//. gamma glutamyl transferase. facesheet. 14. com.12/8/2008. http//. eet. 2. com.12/8/2008. never recovered and ended up dieing. The rats treated with Phenobarbital were able to metabolize the drug faster to corresponding induction of liver microsomal metabolizing enzyme system as compared to those under normal condition. This effect can make those who have been in continuous treatment with barbiturate to develop tolerance to drugs. continuous adjustment and regulation of the dosage by the attending clinician. Therapeutic drug monitoring is therefore needed before administration of other drugs especially those metabolized in the liver. Exposure of the liver to toxicants renders it either functionless to metabolize drugs or with reduced liver microsomal enzyme activity. Hence before giving drugs it is appropriate for clinician to ask the patient if at any time they were exposed to any of the liver toxicant or carry a clinical therapeutic drug monitoring to ascertain the toxicity levels in an individual. The estimation of serum enzyme levels originating from the liver was indicative examples that liver enzymes are affected with exposure of the subjects (both humans and animals) but it was thought that genetic analysis using polymerase chain reaction of the genes should be done for other liver microsomal enzymes to see if genes of these enzymes are activated on exposure to these drugs. This is quite had due inadequacy of liver microsomal enzyme primers especially the major enzyme involved in drug metabolism-the cytochrome p-450 or mixed function oxidase. James M.Perel and Lester C. Mark. The interaction of anesthetic Agents with Hepatic Microsomal enzymes. Francis F.Foldes. Enzymes in anesthesiology , Lab test online.org ( ) International Federation of Clinical Chemist, Lab test online.org ( ), International Federation of Clinical Chemist, Lalit, D. S., Organ function tests, liver function tests, Essentials of practical Biochemistry, , Lalit, Das subrata, Organ function tests, liver function tests, Essentials of practical Biochemistry,

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