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					A Journal of the Bangladesh Pharmacological Society (BDPS)                           Bangladesh J Pharmacol 2008; 3: 49-54
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ISSN: 1991-007X (Printed); 1991-0088 (Online); DOI: 10.3329/bjp.v3i2.835

Protein binding interaction of warfarin and acetaminophen in
presence of arsenic and of the biological system
Md. Ashraful Alam, Riaz Uddin and Shamsul Haque

Department of Pharmacy, Pharmacology and Pharmaceutical Analysis Laboratory, Stamford University
Bangladesh, Dhaka, Bangladesh.

Article Info                          Abstract
Received: 8 February 2008             Equilibrium dialysis was used to study in vitro binding of warfarin at
Accepted: 1 May 2008
                                      physiological pH and temperature in bovine serum albumin (BSA)
Available online: 9 May 2008
                                      along with the increasing concentration of acetaminophen and there
                                      interaction with the protein in presence of arsenic. The free fractions
Keywords:                             were determined by UV spectrophotometric technique. The binding of
 Acetaminophen                        warfarin to BSA depended on both drug and arsenic concentrations.
 Arsenic                              Free warfarin concentration increased due to addition of acetaminophen
 Bovine serum albumin
                                      which reduced the binding of warfarin to BSA. Free warfarin
                                      concentration also increased accordingly by increasing the concentra-
Number of Tables: 3
                                      tion of acetaminophen when only the BSA was present. When the
Number of Refs:   35                  binding site was blocked by sufficient amount of arsenic the increment
                                      of free concentration of warfarin decreased to a lower extent. This
                                      suggests that in the presence of arsenic the warfarin being slowly
Correspondence: MAA
                                      displaced from its high affinity binding site with increasing acetamino-                  phen concentration.

Introduction                                                  and poorly understood issues within clinical
The binding of drugs to plasma and tissue protein             medicine (Grymonpre et al., 1988). The impact of
is an important factor affecting their distribution           drug-drug interactions on patient safety should be
and rate of metabolism. Pharmacological effect is             elucidated, and the magnitude of the problem is
closely related to the free concentration of drug at          vast (Einarson et al., 2002). Detection and
its site of action. There are examples of many drug-          anticipation of these interactions is a daunting
drug interactions which have been reported to                 task, given the breadth of pharmacodynamic and
present displacement of the bound drug by a                   pharmacokinetic variables with which clinicians
second therapeutic agent. Acidic drugs commonly               must grapple. NSAIDs have high protein binding
bind to plasma albumin and concomitantly                      that may represent displacement of bound drugs
administered drugs may displace one another                   especially those bound to albumin (Amitava and
from their binding site. Basic drugs may bind to              Timothy, 1996). Mefenamic acid can displace
either albumin or α-acid glycoprotein. Despite the            warfarin from its binding sites (Zahra et al., 2006).
ubiquity and severity of drug-drug interactions,              Acetaminophen is the preferred analgesic over
this problem is one of the most poorly recognized             aspirin and a nonsteroidal antiinflammatory drug

                                                                                       Bangladesh Pharmacological Society
50                                 Bangladesh J Pharmacol 2008; 3: 49-54

for patients treated with warfarin because it lacks     Standard Curve Preparation: Standard curve was
the potential to induce gastrointestinal bleeding       prepared by using the various concentrations and
and has no antiplatelet activity (Chan, 1995).          their corresponding absorbance at pH 7.4. UV
Evidence      suggests,    however,      that  oral     spectrophotometric scanning of the drugs
anticoagulation     therapy   with     vitamin   K      acetaminophen and warfarin showed maximum
antagonists, such as warfarin, may be potentiated       absorbance of the UV light at 246 nm and 306 nm
by acetaminophen (Antlitz et al., 1968).                respectively. Acetaminophen has found linearity
                                                        at a concentration of 10- 80µM/ml with a
Moreover, epidemiological studies have revealed
                                                        confidence level of 0.9967 at pH 7.4 with linear
that chronic arsenic exposure in many countries
                                                        equation (Y= 0.0107 X). Similar standard curve has
caused the increased risk of mortality associated
                                                        also been prepared for warfarin having linear
with cardiovascular disease (Engel et al., 1994), but
                                                        equation Y= 0.1264 X + 0.0161 and calculated the
a plausible explanation for the development of
                                                        concentration of those drugs using corresponding
arsenic-induced cardiovascular disease has not
                                                        liner equation. Zero order absorption spectrum of
been previously examined. Cardiovascular effects
                                                        the various system containing warfarin (10µM),
in humans drinking arsenic-contaminated water
                                                        acetaminophen (10µM), acetaminophen: warfarin
include blackfoot disease (resulting from gangrene
                                                        (10:10µM), arsenic: warfarin (10:10 µM), arsenic:
caused by obstruction of peripheral blood vessels),
                                                        acetaminophen (10:10 µM) was plotted in the
atherosclerosis, cerebrovascular diseases, and
                                                        Figure 1 and Figure 2 using HACH 4000 UV/VIS
ischemic heart diseases (Tseng, 1977; Rahman et
                                                        Spectrophotometer (USA).
al., 1999). These diseases have been clinically
associated with abnormal platelet activity and
                                                        Effect of acetaminophen on warfarin binding to BSA in
thrombosis and, as a consequence, new drugs have
                                                        presence of arsenic: Three milliliter of previously
been developed to therapeutically control platelet
                                                        prepared 20 µM BSA solution was taken in each of
action and thrombosis (Hollopeter et al., 2001).
                                                        seven cleaned and dried test tubes. 12 µl of 1×10-2
In view of the above consideration we conducted         M arsenic trioxide solution was taken in each of six
an in vitro investigation on displacement               cleaned and dried test tubes. So that final ratio
interaction  of   warfarin    in   presence  of         between protein and arsenic trioxide was 1:2 (20
acetaminophen and arsenic. Protein binding              µM: 40 µM) in each of six test tubes. Then the site-
studies are carried out with several methods            II is sufficiently blocked by arsenic trioxide. The
among which equilibrium dialysis is a routine           seventh test tube containing only BSA solution was
method and was performed in our laboratory.             marked as blank. After that warfarin was added in
                                                        6 out of 7 test tubes so that protein, arsenic and
                                                        warfarin was 1:2:1 (20 µM: 40 µM: 20 M).
Materials and Methods                                   Acetaminophen was added with an increasing
                                                        concentration in to five out of six test tubes
Drug and reagents used in the experiment:
                                                        containing 1:2:1 mixture of protein- arsenic tri
Acetaminophen (General Pharmaceutical Ltd.,
                                                        oxide- warfarin to make the final ratio of protein,
Bangladesh). warfarin (Incepta Pharmaceuticals
                                                        arsenic, warfarin and acetaminophen 1:2:1:0,
Ltd., Bangladesh), disodium hydrogen phosphate
                                                        1:2:1:1, 1:2:1:2, 1:2:1:4, 1:2:1:6 and 1:2:1:8.
(Na2HPO4), potassium dihydrogen phosphate
                                                        Acetaminophen was not added to one test tube.
(KH2PO4), cellulose nitrate membrane (Medicell
                                                        The solution mixture were then properly mixed
International Ltd. Liverpool Road, London; mol.
                                                        and allowed to stand for 15 minutes for the
Wt. 1200 Daltons), Bovine Serum Albumin (BSA)
                                                        confirmation of maximum binding to BSA. After
(fatty acid free, fraction V, Mr. 66,500 from Sigma
                                                        that the solution was pipetted out and poured in to
Chemical Ltd.), arsenic trioxide (As2O3).
                                                        seven different semi permeable membrane tubes.
Instrument used: pH Meter (HANNA Microproce-            Two end of the membrane tube were clipped and
ssor pH Meter, Portugal), HACH-4000 UV/VIS              was ensured that there was no leakage. The tubes
Spectrophotometer (USA), Metabolic Shaking              containing drug mixture were immerged into
Incubator (Clifton Shaking Bath, Nical electro Ltd.,    seven 50 mL conical flasks containing phosphate
England.) Micro Syringe (well. Liang. Jin.              buffer solution of pH 7.4 and shaked continuously
Yang.q.I., China.)                                      for six hours uninterruptly to complete the
                                                           Bangladesh J Pharmacol 2008; 3: 49-54                                                                    51

dialysis. At the end of dialysis, samples were                                     only protein-warfarin-acetaminophen mixture
collected from each flask. The free concentrations                                 (1:1:1) and the rest of the experiment was done as
of warfarin were measured by a UV                                                  described above using phosphate buffer solution
spectrophotometer at a wave length of 306 nm                                       of pH 7.4. At the end of dialysis, samples were
(BP).                                                                              collected from each flask. The free concentrations
                                                                                   of warfarin were measured by a UV
Effect of acetaminophen on warfarin binding to BSA in
                                                                                   spectrophotometer at a wave length of 306 nm
absence of arsenic: To perform the experiment the
previously described procedure has been followed
successively in absence of arsenic. Acetaminophen                                                         Absorbance Arsenic:Paracetamol (10:10 microM /ml)
was added with an increasing concentration in to                                                          Absorbance Arsenic:warfarin (10:10 microM /ml)

five out of six test tubes containing 1:1 mixture of                                               0.35
protein- warfarin to make the final ratio of protein,                                               0.3
warfarin and acetaminophen 1:1:0, 1:1:1, 1:1:2,

1:1:4, 1:1:6 and 1:1:8. Acetaminophen was not                                                       0.2
added to one test tube. That is acetaminophen was                                                  0.15
not present into the first test tube which contained
only protein-warfarin mixture (1:1) and the rest of
the experiment was done as described above using
                                                                                                      190           240            290           340          390
phosphate buffer solution of pH 7.4. At the end of
                                                                                                                    Wavelength (nanometer)
dialysis, samples were collected from each flask.
The free concentrations of warfarin were measured                                  Figure 2: Corresponding zero order absorption spectrum of
by a UV spectrophotometer at a wave length of 306                                  arsenic: acetaminophen (10:10 µM/mL) and arsenic: warfarin
nm (BP).                                                                           (10:10 µM/mL)
                         Absorbance of Warfarin (10 microM /mL)
                         Absorbance of Paracetamol (10 microM /mL)
                                                                                   Results and Discussion
                         Absorbance of Warfarin:Paracetamol ( 10: 10 microM /mL)
                0.6                                                                Chronic ingestion of arsenic contaminated
                0.5                                                                drinking water is the major pathway posing

                0.4                                                                potential risk to human health (Bagla and Kaiser,
                0.3                                                                1996). Since the early 1990s, in Bangladesh alone,
                0.2                                                                arsenic exposure has caused more than 7000
                                                                                   deaths and uncounted thousands show symptoms
                                                                                   of long-term arsenic poisoning (Masibay, 2000).
                  220   240    260 280 300 320                       340     360
                                                                                   Significant portions of world populations are
                               Wavelength (nanometer)                              exposed to low to moderate levels of arsenic of
                                                                                   parts per billion to hundreds of parts per billion.
Figure 1: Corresponding zero order absorption spectrum of
                                                                                   As a consequence, the World Health Organization
warfarin (10 µM/mL); acetaminophen (10 µM/mL) and
                                                                                   and U.S. environmental health agencies, such as
warfarin: acetaminophen (10: 10 µM/mL)
                                                                                   the Environmental Protection Agency made
                                                                                   arsenic their highest priority (Goering et al., 1999).
Effect of arsenic on warfarin binding to BSA in presence                           Recently, the WHO established an acceptable level
of acetaminophen:: To performs the experiment the                                  of 10 ppb for arsenic in drinking water (Lok, 2001).
previously described procedure has been followed                                   The EPA also recently proposed to reduce its
successively. Arsenic was added with an                                            drinking water standard from 50 to 10 ppb;
increasing concentration in to five out of six test                                however, this newly proposed maximum
tubes containing 1:1:1 mixture of protein- warfarin-                               contaminant level is now being reevaluated for
acetaminophen to make the final ratio of protein,                                  scientific merit (Kaiser, 2001). Arsenic ingestion
warfarin, acetaminophen and arsenic 1:1:1:0,                                       may also aggravate the platelet aggregation and
1:1:1:1, 1:1:1:2, 1:1:1:4, 1:1:1:6 and 1:1:1:8 . Arsenic                           may cause induce vascular diseases (Lee et al.,
was not added to one test tube. That is arsenic was                                2002). The activation and resulting aggregation of
not present into the first test tube which contained                               platelets are believed to be important events in
52                                 Bangladesh J Pharmacol 2008; 3: 49-54

both hemostasis and the pathogenesis of various         et al., 1982). An increased concentration of
vascular diseases due to thrombus formation             unbound warfarin can be expected to increase
(Marcus and Safier, 1993). Platelet aggregation is      inhibition of vitamin K-dependent factors, as it is
initiated by physiological agonists, such as throm-     unbound warfarin that binds to the vitamin K
bin, and hemodynamic factors, such as shear stress      epoxide reductase enzyme in the liver (Thijssen et
(Clemetson, 1995). Under normal physiological           al., 1987).
conditions, hemostatic balance by platelets is
strictly regulated between pro-aggregation (active-     Our findings may be important when considering
tion of platelets) and anti-aggregation (inhibition     prior work that supports the potential severity of
of platelet activation) activity.                       warfarin interactions. Drug interactions are the
                                                        most common factor associated with a critically
However, co-prescribing interacting medications         high INR (Panneerselvam et al., 1998) and an
can result in serious patient consequences, particu-    increased risk of bleeding (Van der Meer et al.,
larly in patients who receive warfarin, because of      1993). Acetaminophen has been reported by Hylek
warfarin’s narrow therapeutic index (Greenblatt         et al to be an unrecognized hazard for warfarin
and von Moltke, 2005). Combined therapeutic use         takers—as little as 1300 mg/d for 7 days increased
of warfarin and acetaminophen is very common,           by 10-fold the odds of an INR greater than 6.
and a clinically significant drug interaction is a      However, it is important to note that the clinical
debated matter. Insufficient data have been             significance of the acetaminophen-warfarin inter-
acquired on this interaction in controlled experi-      action is not without controversy. The mechanism
ments using INR determinations to monitor the           of this interaction has only recently been
level of anticoagulation. Many drugs routinely co-      elucidated (Thijssen et al., 2004) and likely results
administered with warfarin interact by inhibition       from the independent inhibitory effect of an
or induction of hepatic cytochrome P450 (CYP)           acetaminophen metabolite on enzymes of the
oxidative enzymes (Buckley and Dawson, 1992).           vitamin K cycle. Although several case reports and
During concurrent administration of warfarin and        controlled studies have reported that acetamino-
acetaminophen, site to site displacement take place     phen potentiates the anticoagulant effect of
and acetaminophen displaced warfarin from its           warfarin, (Hylek et al., 1998) others have not found
binding sites more slowly (i.e. induces small free      a clinically relevant interaction. (Fattinger et al.,
concentration). But in the presence of probe arsenic    2002). NSAIDs such as Mefenamic acid (Holmes,
increment of acetaminophen to BSA, free                 1966)     etodolac (Ermer et al., 1994; ibuprofen
concentration of warfarin was less prominent. This      (Penner and Abbrecht, 1975) and Tenidap
displacement may be due to reduce the binding           (Apseloff et al., 1995) may also displace coumarin
site of warfarin and increasing the free drug           anticoagulants from protein binding sites.
concentration, whereas in the presence of arsenic,
                                                        Acetaminophen was associated with an increased
warfarin may form complex with arsenic or arsenic
                                                        hypoprothrombinemic effect of warfarin. This
may increases the binding affinity to its sites or
                                                        interaction is proposed to be due to inhibition of its
arsenic may form complex to BSA. As observed in
                                                        metabolism and interference with formation of
Figure 3, during concurrent administration,
                                                        clotting factors. Gingival bleeding and hematuria
acetaminophen displaced warfarin from its high
                                                        were observed in case reports when paracetamol is
affinity binding site-I. Thus free concentration of
                                                        given with warfarin (Hylek et al., 1998). However,
warfarin increased from 3.19 ± 0.64% and 0.83 ±
                                                        due to lack of a safer alternative, paracetamol is
0.09% to 86.82 ± 0.53 and 75.82 ± 4.91% in absence
                                                        still the analgesic and antipyretic of choice in
and presence of site-II probe (arsenic) respectively.
                                                        patients receiving warfarin therapy, as long as
It has been proposed that competition for CYP1A2        excessive amounts and prolonged administration
between acetaminophen and R-warfarin causes a           are avoided (Shek et al., 1999). A combination of
clinically significant drug interaction (Lehmann et     paracetamol and codeine has enhanced warfarin
al., 2000). Warfarin is highly protein-bound, and       activity (Bartle and Blakely, 1991).
despite its low tendency to bind to plasma
proteins, acetaminophen has the potential to            Thus it can be suggested that acetaminophen
increase the unbound fraction of warfarin (Forrest      displaced warfarin from its binding site-I and at
                                                              Bangladesh J Pharmacol 2008; 3: 49-54                                            53

                                            %Release of warfarin after addition of acetaminophen in increasing presence of arsenic
                                            %Release of warfarin when arsenic was increasing in presence of acetaminophen
                                            % Release of warfarin when acetaminophen is in incresing conc. and no arsenic present

                    % Release of Warfarin
                                                  0                2                4                6                8               10
                                                                         Concentration (microgram/mL)
Figure 3: % Release of warfarin from protein binding site in presence of acetaminophen and arsenic

the same time a sufficient portion of the free drug                                     Bagla P, Kaiser J. India’s spreading health crisis draws
might have bound to site-II or increase the free                                          global arsenic experts. Science 1996; 274: 174–75.
drug concentration in plasma. The release pattern                                
of warfarin was some what lower in presence of                                          Buckley NA, Dawson AH. Drug interactions with
arsenic alone in increasing concentration in                                              warfarin. Med J Australia 1992; 157: 479-83.
presence of acetaminophen and the % release of                                          Chan TY. Adverse interactions between warfarin and
drug was 7.81 ± 2.13% to 0.81 ± 0.22% after                                               nonsteroidal antiinflammatory drugs: Mechanisms,
increasing the arsenic concentration from its initial                                     clinical  significance,    and   avoidance.  Ann
value of 10 µM to 80 µM. This results is quite an                                         Pharmacother. 1995; 29: 1274-83.
interesting findings considering our previous work                                      Clemetson KJ. Platelet activation: Signal transduction via
(Uddin et al., 2004)                                                                      membrane receptors. Thromb. Haemostasis 1995; 74:
Considering the above result and discussion it
would demand extensive research for the safety of                                       Einarson TR, Metge CJ, Iskedjian M, Mukherjee J. An
those patients who are taking warfarin along with                                         examination of the effect of cytochrome P450 drug
acetaminophen in a locality where arsenic free                                            interactions of hydroxymethylglutaryl- coenzyme A
                                                                                          reductase inhibitors on health care utilization: a
drinking water can not be avoided due to the lack
                                                                                          Canadian population-based study. Clin Thera. 2002;
of better alternatives.                                                                   24: 2126–36.
                                                                                        Engel RR, Hopenhayn-Rich C, Receveur O, Smith AH.
References                                                                                Vascular effects of chronic arsenic exposure: A review.
Amitava D, Timothy GT. In vitro displacement of                                           Epidemiol Rev. 1994; 16: 184–209.
 phenytoin from protein binding by non-steroidal                                        Ermer JC, Hicks DR, Wheeler SC. et al. Concomitant
 antiinflamatory drugs tolmetin, ibuprofen and                                            etodolac affects neither the unbound clearance nor the
 naproxen in normal and uremic sera. T.D.M. 1996; 18,                                     pharmacologic effect of warfarin. Clin Pharmacol
 97-99.                                                                                   Ther. 1994; 55: 305–16.
Antlitz AM, Mead JA, Tolentino MA. Potentiation of                                      Forrest JA, Clements JA, Prescott LF. Clinical
 oral anticoagulant therapy by acetaminophen. Curr                                        pharmacokinetics of paracetamol. Clin Pharmacokin.
 Ther Res. 1968; 10: 501-06.                                                              1982; 7: 93-107.
Apseloff G, Wilner KD, Gerber N. Effect of tenidap                                      Gadisseur AP, van der Meer FJ, Rosendaal FR. Sustained
 sodium on the pharmacodynamics and plasma                                               intake of paracetamol (acetaminophen) during oral
 protein binding of warfarin in healthy volunteers. Br J                                 anticoagulant therapy with coumarins does not cause
 Clin Pharmacol. 1995; 39: 29S–33S.                                                      clinically important INR changes: A randomized
54                                       Bangladesh J Pharmacol 2008; 3: 49-54

     double-blind clinical trial. J Thromb Haemost. 2003; 1:   Marcus AJ, Safier LB. Thromboregulation: Multicellular
     714-17.                   modulation of platelet reactivity in hemostasis and
     7836.2003.00135.x                                          thrombosis. FASEB J. 1993; 7, 516–22.
Goering PL, Aposhian HV, Mass MJ, Cebrian M, Beck              Masibay K. Drinking without harm. Sci. Am. 2000; 283:
 BD, Waalkes MP. The enigma of arsenic                          22.
 carcinogenesis: Role of metabolism. Toxicol Sci. 1999;        Panneerselvam S, Baglin C, Lefort W, Baglin T. Analysis
 49, 5–14.               of risk factors for overanticoagulation in patients
Greenblatt DJ, von Moltke LL. Interaction of warfarin            receiving long-term warfarin. Br J Haematol. 1998;
  with drugs, natural substances, and foods. J Clin              103: 422-24.
  Pharmacol. 2005; 45:127-32.                                    2141.1998.00988.x                   Rahman M, Tondel M, Ahmad SA, Chowdhury I A,
Grymonpre RE, Mitenko PA, Sitar DS, Aoki FY,                     Faruquee MH, Axelson O. Hypertension and arsenic
  Montgomery PR: Drug-associated hospital admissions             exposure in Bangladesh. Hypertension 1999; 33, 74–78.
  in older medical patients. J Am Geriatr Soc. 1988; 36:       Schulman S, Henriksson K. Interaction of ibuprofen and
  1092–98.                                                       warfarin on primary haemostasis. Br J Rheumatol.
Hertz-Picciotto I, Arrighi HM, Hu SW. Does arsenic               1989; 28: 46–49.
 exposure increase the risk for circulatory disease? Am 
 J Epidemiol. 2000; 151, 174–81.                               Shek KLA, Chan LN, Nutescu E. (1999) Warfarin
Hollopeter G, Jantzen HM, Vincent D, Li G, England L,            acetaminophen      drug     interaction    revisited.
 Ramakrishnan V, Yang RB, Nurden P, Nurden A,                    Pharmacotherapy 1999; 19: 1153–58.
 Julius D, Conley PB. (2001). Identification of the    
 platelet ADP receptor targeted by antithrombotic              Thijssen HH, Soute BA, Vervoort LM, Claessens JG.
 drugs. Nature 2001; 409, 202–07.                                Paracetamol (acetaminophen) warfarin interaction:                              NAPQI, the toxic metabolite of paracetamol, is an
Holmes EL. Experimental observations on flufenamic,              inhibitor of enzymes in the vitamin K cycle. Thromb
 mefenamic, and meclofenamic acids. IV. Toleration by            Haemost. 2004; 92: 797- 802.
 normal human subjects. Ann Phys Med. 1966; 9                  Thijssen HHW, Baars LGM. Hepatic uptake and storage
 (Suppl), 36–49.                                                 of warfarin: the relationship with the target enzyme
Hylek EM, Heiman H, Skates SJ, Sheehan MA, Singer                vitamin K 2, 3-epoxide reductase. J Pharmacol Exp
 DE. Acetaminophen and other risk factors for                    Ther. 1987; 243: 1082-88.
 excessive warfarin anticoagulation. JAMA 1998; 279:           Tseng WP. Effects and dose–response relationships of
 657-62.                skin cancer and blackfoot disease with arsenic.
Kaiser J. Toxicology: Science only one part of arsenic           Environ Health Perspect. 1977; 19, 109–19.
  standards. Science 2001; 291: 2533–33.                            Uddin SJ, Shilpi JA, Murshid GMM, Rahman AA, Sarder
Lee MY, Bae ON, Chung SM, Kang KT, Lee JY, Chung                MM, Alam MA. Determination of the binding sites of
  JH. Enhancement of platelet aggregation and                   arsenic on bovine serum albumin using warfarin (site-
  thrombus formation by arsenic in drinking water: A            I specific probe) and diazepam (site-II specific probe).
  contributing factor to cardiovascular disease. Toxicol        J Biol Sci. 2004; 4: 609-12.
  Appl Pharmacol. 2002; 179: 83–88.                            van der Meer FJ, Rosendaal FR, Vandenbroucke JP, Briet                       E. Bleeding complications in oral anticoagulant
Lehmann DF. Enzymatic shunting: resolving the                    therapy: an analysis of risk factors. Arch Intern Med.
  acetaminophen-warfarin controversy. Pharmaco-                  1993; 153: 1557-62.
  therapy 2000; 20: 1465-68.                                          Zahra R, Ahmad R, Asghar MSA, Ali A, Soghra K. A
Lok C. Plans to reduce acceptable arsenic limit put on           study of the interaction between ropranolol and
  hold. Nature 2001; 410: 503–03.                                NSAIDS in protein binding by gel filtration method.
  doi:10.1038/35069212                                           Indian J Clin Biochem. 2006; 21: 121-25.