Renal Excretion of Paracetamol | Experiment

Renal reasoning by elimination of Paracetamol auditionParacetamol, cognize as acetaminophen in the USA, is one of the most usually apply analgesic and antipyretic do do drugsss available over-the-counter. Its common name derives from the adequate chemical name para-acetyl-amino-phenol, with the chemical formula C8H9NO2and a molecular load of 151.17.Paracetamol does not have any signifi privyt anti-inflammatory action and so domiciliatenot be entirely described as a non-steroidal anti-inflammatory drug (NSAID), as was once thought. Its mechanism of action is still poorly unsounded merely some studies have suggested that it inhibits a variant of the cyclo-oxygenase enzyme COX-1, which has been designated COX-3 (Swierkosz et al., 2002). Paracetamol acts mainly in the central nervous system and endothelial cells, rather than in platelets and resistive cells. Boutaud and colleagues (2002) hypothesised that this may be explained by the high levels of peroxides set up in the last mentioned cell types, which inhibit the action of paracetamol. in that respect has been some debate on the subject, with other researchers proposing an inhibitory action against COX-2 (Graham Scott, 2005). Further research is essential to fully elucidate the mechanism of action at the molecular level. by-line oral administration and absorption from the gastrointestinal tract, paracetamol enters the derivation and is distri entirelyed throughout the body. It is metabolised by enzymes in the hepatocytes of the liver and the bulk is converted to in expeditious metabolites by trades union with sulphate or glucuronide. This is then filtered out of the blood by the kidneys and into the pissing, via active renal tubular secretion. A small portion of paracetamol remains unaltered and passes into the urine via glomerular filtration and passive absorption (Morris Levy, 1984). There is also a small proportion of the paracetamol that is metabolised by the cytochrome P450 system, wh ich leaves in the formation of cysteine or glutathione conjugates and mercapturic battery-acid conjugates. These products of oxidative metabolism are also excreted renally (Andrews et al. 1976).Paracetamol has a low remedy index, so the therapeutic acid is very close to the cyanogenic dose. toxicity can occur by-line a single large dose (10g) or with chronic lower doses (4-5g/d) and is usually seen as hepatotoxicity, which can result in death within several days (Wikipedia).Toxicity occurs when the enzymes obligated for catalysing sulphate and glucuronide conjugation become saturated, forcing metabolism to be increasingly dependant upon the cytochrome P450 system. This results in formation of a toxic metabolite, N-acetyl-p-benzo-quinone imine (NAPQI), which is normally mopped up by binding to the sulphydryl group of glutathione to form inactive conjugates and mercapturic acid. Toxicity occurs when the glutathione supply becomes faint and NAPQI binds indiscriminately to mo lecules within the cell, such as membranes, to cause cell upon and death, seen as acute hepatic necrosis.Major pathway for normal metabolismMinor pathway via cytochrome P450 system produces toxic metabolite (NAPQI), shown in red. Normally this is detoxified by binding to glutathione.Toxicity occurs when pathways 1 and 2 are overloaded and NAPQI binds to molecules of the cell, causation scathe.Modified from Rang et al. 1995.The aim of this try is to investigate the renal excretory product of paracetamol, by measuring the levels of paracetamol metabolites in human urine over 6 hours following an oral dose of 500mg. The full excretion pull up stakes be assessed apply the spectrophotometric method. From this data the elimination rate constant (KE) and the half-life (T1/2) will be calculated. Qualitative analysis of the various metabolites will be conducted utilise curb chemical identification techniques.METHODA standard stock ancestor of paracetamol was prepared at 1mg/cm3 a nd dilutions were made to give a range of known concentrations. 1 cm3 of the paracetamol solution was added to 1 cm3 blank urine and 4 cm3 4M HCl, and abstruse thoroughly. A blank duplicate was also prepared, using water quite of urine. After an hour in a boiling water john the tubes were cooled and water added, up to 10 cm3. 1 cm3 of this hydrolysed urine solution was added to 10 cm3of colour forming solution, mixed and allowed to stand for 40 minutes. The absorbance of each solution was measured, using the spectrophotometer, zeroing the instrument using the drug free urine sample in between solutions. This produced the readings for the calibration wind up. The collected timed urine samples were then process in the same way, adding 1 cm3 water instead of paracetamol solution.RESULTS AND DISCUSSION cognise concentrations of paracetamol underwent spectrophotometry to measure the absorbance at 620nm. These results were used to produce a calibration curve (figure 3). The timed uri ne samples were then analysed following the same protocol and the absorbance at 620nm was used, in conjunction with the calibration curve to ascertain the concentration of paracetamol in the urine. Unfortunately, half of the samples produced absorbances outside the range of the calibration curve. Because this curve is non-linear, extrapolation and dilution cannot be used to accurately deduce the concentration of paracetamol in the urine. For the purposes of this report the concentration for these samples has been declared as greater than 800ug/cm3. This is not very satisfactory and further experiments mustiness be done to extend the range of the calibration curve to the level best absorbancy of the timed samples. The values of KE and T1/2 have been calculated to demonstrate the procedure, only when are inaccurate and will need revising once accurate concentrations have been established form the calibration curve.Table 1 mensurate urine sampleMean absorbance 620nmConc. ug/cm3Vol . Urine (ml)Total drug (ug of paracetamol)Excretion rate mg/h0000001 hour0.25619224547040472 hours1.9188005040000403 hours1.769800383040030.44 hours1.0288005544000445 hours0.3492461353321033.26 hours0.2551921603072030.7Table 1 contains the absorbance results of the timed urine samples and the deduced concentration of paracetamol in the urine, as well as the hourly excretion rate. The total amount of paracetamol excreted over the 6 hour period was 225.3mg, which is 45% of the orally administered dose. Due to problems discussed above, this is an underestimate of the true percentage of dose excreted renally, which has been found to be 55-70% by other studies (Steventon et al., 1996).When log of the excretion rate (equivalent to total drug excreted per hour) is plotted against time, a linear plot should be achieved, from which KE can be estimated. This is shown in Figure 4, but is likely to need revising.The face of this straight line equates to KE /2.303, which gives a value for KE o f 0.094. Using the formula T1/2 =0.692/ KE , the value of T1/2 = 7.36 hours.This states that it takes the body 7.36 hours to excrete half of the drug administered. This is longstanding than the 1-4 hours usually quoted for paracetamol (Rang et al. 1995), and is not surprising presumptuousness the underestimation of the paracetamol urine concentration. With appropriate calibration, this would be expected to decrease to nearer the previously found results.There were no results for the qualitative studies for metabolite composition, but it would be expected that sulphate and glucuronide conjugates would force the majority of the sample, with a smaller quantity of unchanged paracetamol, cysteine/glutathione and mercapturic acid metabolites.These results only(prenominal) represent one individual on one day and replications of this experiment are crucial. Nutritional status, recent alcohol consumption, ethnic background, concurrent drug usage and illness must all be taken into forec ast as factors that may affect paracetamol metabolism and excretion (Riordan Williams, 2002, Patel Tang, 1992).Further analysis of paracetamol excretionHepatotoxicity and drug interactionsTable 2 shows how concurrent use of sodium thiopental, an anti-epileptic drug, can increase the severity of liver damage caused by paracetamol administration and its accompanying metabolism.Table 2 Effect of Phenobarbital on paracetamol induced hepatotoxicity discussion Dose of Paracetamol (mg/kg) Severity of liver necrosisNone3751-2+Phenobarbital3752-4+_________This occurs due to metabolism of phenobarbital by enzymes of the P450 cytochrome system, which results in upregulation of their production. As explained in the introduction (see fig. 2), P450 enzymes also metabolise paracetamol, to form the toxic metabolite NAPQI. This is normally a minor pathway but as the amount of P450 enzymes available increases, the activity of this pathway also increases. This results in a larger than normal amount of NAPQI, which is mopped up and inactivated by glutathione. Glutathione supplies will eventually run out, which occurs sooner if the person is malnourished. When this happens the toxic metabolite binds to cell components, causation necrosis. To prevent this occurring, such as in cases of overdose, N-acetylcysteine can be given (Routledge et al., 1998), which is required for glutathione synthesis and helps to boost it. This allows a greater amount of the toxic metabolite to be mopped up and reduces cell damage.Paracetamol metabolism following hepatotoxicityTable 3Plasma paracetamolconcentrations (ug/cm3)PatientsPlasmaparacetamol4 hrs after12hrs afterHalf life (h)ingestioningestion_______________________________________________________________no liver damage (18)2.9 +/= 0.3163 +/=20 29.5 +/=6liver damage (23) 7.2+/= 0.7296 +/= 26 124 +/=22___Table 3 shows that, in a study, the ability of patients with liver damage to eliminate paracetamol from the blood is much decreased, compared to healthy people. This is seen by the prolonged half-life and the high levels of paracetamol in the plasma. The plasma level does come down by 12 hrs, which indicates that in that location is enough meshal liver reserve to metabolise some of the drug, but the level is still very high. To ascertain whether it is just conjugation that is affected, or whether all the pathways are affected equally it would be necessary to quantify the levels of different metabolites in the blood and urine. As conjugation is responsible for the majority of metabolism, damage to all systems will still show up as affecting conjugation the most.In theory reduced clearance of a substance is useful for monitoring the severity of liver damage, but in the case of paracetamol it would be unwise as it could potentiate the hepatotoxic personal effects and worsen the liver condition. It is also unnecessary as there are already a number of reliable blood tests for liver function and damage.REFERENCESAndrews, R. S ., Bond, C. C., Burnett, J., Saunders, A. Watson, K. 1976 Isolation and identification of paracetamol metabolites. J Int Med Res 4, 34-9.Boutaud, O., Aronoff, D. M., Richardson, J. H., Marnett, L. J. Oates, J. A. 2002 Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases. Proc Natl Acad Sci U S A 99, 7130-5.Graham, G. G. Scott, K. F. 2005 Mechanism of action of paracetamol. Am J Ther 12, 46-55.Morris, M. E. Levy, G. 1984 Renal clearance and serum protein binding of acetaminophen and its major conjugates in humans. J Pharm Sci 73, 1038-41.Patel, M., Tang, B. K. Kalow, W. 1992 Variability of acetaminophen metabolism in Caucasians and Orientals. Pharmacogenetics 2, 38-45.Rang, H. P., Dale, M.M., Ritter, J.M. 1995 Pharmacology Churchill Livingstone.Riordan, S. M. Williams, R. 2002 alcoholic drink exposure and paracetamol-induced hepatotoxicity. Addict Biol 7, 191-206.Routledge, P., Vale, J. A., Bateman, D. N., Johnston, G. D., J ones, A., Judd, A., Thomas, S., Volans, G., Prescott, L. F. Proudfoot, A. 1998 Paracetamol (acetaminophen) poisoning. No need to change current guidelines to possibility departments. Bmj 317, 1609-10.Steventon, G. B., Mitchell, S. C. Waring, R. H. 1996 Human metabolism of paracetamol (acetaminophen) at different dose levels. medicine Metabol Drug Interact 13, 111-7.Swierkosz, T. A., Jordan, L., McBride, M., McGough, K., Devlin, J. Botting, R. M. 2002 Actions of paracetamol on cyclooxygenases in tissue and cell homogenates of mouse and rabbit. Med Sci Monit 8, BR496-503.Wikipedia. http//en.wikipedia.org/wiki/Paracetamol.