A Biomedical Application of Activated Carbon Adsorption An Experiment Using Acetaminophen and N-Acetylcysteine Thomas R. Rvboli. . . Derrick E. Burrell. J o s e ~ hM. Shults. and Andrew K. Kelley University of Tennessee at Chattanooga, Chattanooga, TN 37403
A commonlv nerformed nhvsical chemistrv exneriment utilizes the adiorption of acekcacid t o determihe tde surface area of a n activated carbon powder ( I ) . T h e spectrophotometric determination of the concentration of dye molecules in a solution t h a t has been exposed to a solid adsorbent is a n alternative method t h a t can be used to determine the surface areas of adsorbents (2). Described here is another type of experiment t h a t illustrates a n interesting hiornedicalapplication of adsorption from solution and demonstrates some of t h e factors t h a t influence the in vivo adsorption of drug molecules onto activated carhon. T h e in vitro adsorption and coadsorption of acetaminophen and N-acetylcysteine will serve a s our model system. Drug Adsorption T h e placement of a high surface area carhon powder into tract by either oral or nasogastric tube the administration of a water powder slurry is a procedure t h a t is frequently used as a part of the emergency treatment of drug overdose victims (3).Because of the high binding affinity of many drugs for activated carhon powder, a portion of the drug molecules are adsorbed and along with the carhon nowder nass throueh the intestinal tract without enterina i h e blodd stream. it has also been found t h a t subsequent doses of nowder can increase the rate of drue diffusion into the gastrbintestina~tract and thus speed removal of the drug from t h e body (4). Although therapeutic dosages of acetaminophen (active ingredient of Tylenol) are quite safe, a n overdose can cause liver damage, renal failure, and even death. Several sulfhydry1 compounds diminish t h e toxic effects of acetaminophen bv nrovidine a nrotective effect for t h e metabolite of aceta h h o p h e n ;ha; causes depletion of hepatic glutathione (5). Oral formulations of N-acetvlcvsteine tiold a s hIucomvitl - . are effective in preventing liver damage, renal failure, and death if aiven within 8 hours of a n acetaminonhen overdose (6).In viiro studies have shown t h a t both acetaminophen (7) and N-acetylcysteine (8)are adsorbed by activated carhon. The adsorption and coadsorption of acetaminophen and N-acetylcysteine under gastric (pH 1.2) and intestinal (pH 7.0) conditions were examined. This experiment demonstrates how charcoal powder can be used for drug removal and indicates something of the factors: aromaticlaliphatic and ionizedlun-ionized t h a t influence drug adsorption i n general and acetaminophen and N-acetylcysteine adsorption in particular. ?
Experlment Special chemicals (Aldrich) needed include N-(4-hydroxypheny1)acetamide (acetaminophen), N-acetyl-L-cysteine, and Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (see figure). Simulated gastric fluid was a pH 1.2 solution consisting of 0.05 M KC1 and 0.085 M HC1. Simulated intestinal fluid was a pH 7.0 buffer consistine of 0.05 M KH9POaand 0.029 M NaOH. Dalv a i d Coanev ha"; sueeested that ~ .... . e.o s i nbe omitted from in vrtrostud~es,hut that ns n rulpof thumhonr can arsumr adsorption in rivo wrll he less than half #,fthat in vmu 191. Although pepsin is ~~
~~
~
strongly adsorbed on charcoal, it does not fit into pores of less than 200 nm and it does not seem to inhibit adsorption of small drug molecules. Smaller pores account for the bulk of drug adsorption and make the major contribution to the total exposed surface area of a carbon powder. Six solutions were prepared and included a mixture of 0.02 M (3.02 mg/mL) acetaminophen and 0.02 M (3.26 mg/mL) N-acetylcysteine prepared at both pH 1.2 and 7.0,0.02 M acetaminophen at both pH 1.2 and 7.0, and 0.02 MN-aeetylcysteine at both pH 1.2 and 7.0. Initial solution concentrations were prepared within 0.0001 M. The carbon powder used as an adsorbent was Nuchar SA (Westvaco Chemical Division). Other activated carbon ~owders could also be used. ~ u c h ahra i~a ~ ~ ~ s u r f a c e a r eabout a o f~~-~ lfifi0m2/e. ~~~~.n This ~~powder wau degassed in vacuum n t 125 'C for two hour, prior tu use. Degassing lo remove water vapor gives a better measure uf the actual powder mass hut is not essential to illustrate the basic effect. Assays for acetaminophen and N-acetylcysteine were based on Beer's law calibration plots obtained using a Hitachi 100-80spectrophotometer for the concentration ranges 0.02-0.16 mM and 0.0060.036 mM, respectively. Extinction coefficients were found to be 9640 for acetaminophen and 13020 for the N-acetylcysteine assay usine Ellman's reaeent acid), (10). " 5.5'-dithiobis-(2-nitrobenzoic . , . In the acetaminophen assay an aliquot was diluted with buffer to a final pH of 7.0 and the absorbance determined at 243 nm. In theNacetylcysteine assay (11) an aliquot was diluted with pH 7.0 buffer, and then 1 mL of this dilution was combined with 1 mL of 0.4 mM Ellman's reagent. This 2 mL mixture was diluted to 10 mL with pH 8.0 buffer, allowed to stand for 10 min, and then the absorbance of the resultant yellowish green solution was determined at 412 nm. N-acetylcysteine causes a cleavage of the disulfide linkage hetween the two nitrobenzoie acid groups in Ellman's reagent. Cancurrently, theN-acetylcysteine is oxidized to a disulfide. However, it is the reduced Ellman's reagent, 2-nitro-5-thiolbenzoic acid, which gives rise to an absorbance at 412 nm. The amount of reduced Ellman's reagent is proportional to the concentration of N-aeetyl~).rreinein the crriginal solution. A 2,i.mL aliquot of each of the sax sample solutions was mixed withtl.5Our!ofcarbunand thenallowed rosrand for6Omin i n a X "C water bath. Shaking of the solutions can be used to maximize powder solution contact and shorten the time required to reach equilibrium. Peristalsis does cause the mixing of solids and fluids in the gastrointestinal tract. However, we found one hour in a standing solution to be adequate for the adsorption systems usedin this work. The mixtures were gravity filtered (Whatman 42 filter paper) and then analyzed, as described previously, to determine the adsorhate concentrations. Samples without carbon were also passed through the filter to assess and correct for the minor effects of the filtering nrocess. ~
~~~
~~
~
Rcetaminophen
N-Rcetyl-1-Cysteine
~~
Strunures of acetaminophen and Mcetyl-L-cysteine Volume 65
Number 11 November 1988
1009
Results and Discussion The table shows the percentages of drug in the starting solutions that were adsorbed for iach powder concentration under mixed and pure ronditions. The charcoal todrug ratio hv" weieht at 0.5 e of r a r h m is more than 6 to 1. This ratio is u in line with recommended therapeutic doses of carbon and the relative amounts of gastrointestinal acetaminophen and N-acetylcysteine that are apt to result from acetaminophen overdose and treatment. As shown in the table, acetaminophen was adsorbed murh more eft'erti\.elv under all conditions than N-acetvlcvsteine. The acetaminophen adsorption seem to he little gffected in going from the acidic conditions existing in the stomach to ihe alkaline conditions existing in the jntestines and was decreased only slightly by the presence of N-acetylcysteine. N-acetylcysteine is adsorbed less than the acetaminophen under all conditions. The amount of N-acetylcysteine adsorbed on the carhon was greater at pH 1.2 than 7.0 and is significantly decreased when acetaminophen is present. Since the acetaminophen and N-acetylcysteine molecules are similar in size with molecular weights of 151.2 and 163.2, respectivelv, we must turn to other ~hvsiochemicalproperties of the kdecules to explain the relative binding efficacy of the two molecules. I t has been observed that drug adsorption onto activated carhon is greater for aromaticthan aiiphatic compmmds and greater for un-ionized than imi7ed compounds (12, 19). With this basic information it is possible to offer an explanation for the relative adsorption of the two molecules. Based on the relative binding efficacies of aromatic and nonaromatic comuounds. we would expect. . . as observed. more acetaminophen to bk adsorbed than N-acetylcysteine: Since the DK. for acetaminophen is above 7.0. reported as 9.5 (13).the ~ e i d e r a o n - ~ a s s e i h a l cequation h shows that aretaminnohen is un-ionized at both eastric and intestinal pH, and thus we would predict no p~-effecton the acetaminophen adsorption. However, since the pK, for N-acetylcysteine is 3.3 (lo), N-acetylcysteine is un-ionized a t gastric pH and ionized a t intestinal pH. Thus we would predict, as ohserved, that N-acetylcysteine should be adsorbed more a t the gastric than the intestinal pH. I t should he noted that current theraoeutic orocedure utilizes N-acetylcysteine without activateh carbon powder for the treatment of acetaminoohen overdose. This practice has been based on the fact that^-acetylcysteine is adsorhed by carhon powder. However, it is interesting to note that the relative binding efficacies of acetaminophen and N-acetylcvsteine for activated charcoal are in the desired ratio for a poison and its antidote. In studies on acetaminophen-poisoned mice, charcoal was not found to reduce protection by N-acetylcysteine (14). In these studies on mice, charcoal and N-acetylcysteine were found to offer more ~rotectionthan N-acetvlcvsteine alone. In vivo human studiks have shown that the"p1asma levels of orallv ineested N-acetvlcvsteine are not sianificantlv reducei by-activated charcoal (15,16). In contrast, ~ o r d o net i al. (17) found a sienificant in vivoremoval of acetaminophen by charcoal if th; charcoal was given within 60 minutes of the ingestion of acetaminophen. In vitro studies have also ~
1010
~
Journal of Chemical Education
Percent of Acetaminophen and KAcetylcystelne Removed from Solution by Adsorptlon as a Function of pH and Presence (Mixed) or Absence (Pure) of a Competlng Adsorbate Adsorbate
PH
Acetaminophen KAcetylcysteine Acetaminophen
7.2 7.2
KAcetvlcvsteins
7.0 7.0
Percent Adsorbed Pure Mixed 97 77 97 54
91 66 96 40
suggested that the concomitant use of N-acetylcysteine and charcoal is an appropriate therapv for acetaminophen overdose (10). Other Experiments Other student activities based on the absorption of acetaminophen are possible. For more quantitative work, experiments can be conducted using a range of amounts of powder and the results plotted usine a Lanemuir adsorution isotherm. ~cetamiiophenadsorption f ~ t the s ~ a n ~ m umodel ir very well at pH 1.2 and 7.0 and with or without N-acetvlcvsteine. The average moles adsorbed under these various conditions have been reported as 2.0 mmollg for Nuchar SA powder (10). We have found the relative amounts of acetaminophen and acetic acid required for monolayer coverage will vary considerable from one powder to another. I t is anticipated that the acetaminophen molecule will maximize pi-electron and van der Waals interaction by adsorbing on the surface in a flat configuration (18). The N-acetylcysteine surface configuration seems to change depending on pH conditions and presence of acetaminophen which may hlock surface sites (10). Tylenol tablets can also he used as a convenient source of acetaminophen to illustrate the basic effect of drug removal by carhon powder. Acknowledgment We would like to thank Robert L. Smith of Southeast Missouri State University for his encouragement to present this adsorption work as an experiment for this Journal and Robert C. Mehane for helpful discussions of this work. Literature Cited I .Sh r m a l c r . 1) I' Garand. C \I.Stc8nkla J I . Ulokr I $4 E c p r r n m i . .n I ' n i ' r o ('nea
