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Corresponding author: [email protected]; +2348033119599
*1Iliya HA and 2Hawksworth GM
1Institute of Medical Sciences, University of Aberdeen, AB25
2ZD, Aberdeen, UK (New address: Department of Pharmacology, Faculty
of
Pharmaceutical Sciences, University of Jos, Nigeria)2Institute
of Medical Sciences, University of Aberdeen, AB25 2ZD, Aberdeen,
UK
Abstract
Clearance of salicylic acid is extremely sensitive to changes in
urine pH. When the urine pH is increased, there
is an increase in renal clearance and this has been exploited in
the use of sodium bicarbonate in salicylate
poisoning. The pharmacokinetics of an orally administered dose
of aspirin with, and without sodium bicarbonate
was studied on the relationship between urinary pH and salicylic
acid excretion in two volunteers. High
Performance Liquid Chromatography (HPLC) method was utilized to
determine the levels of salicylic acid inplasma and in urine. The
results showed that the volunteer who was administered an oral dose
of aspirin with
sodium bicarbonate had a different pharmacokinetics data from
the volunteer administered an oral dose of
aspirin without sodium bicarbonate. The ingestion of bicarbonate
with aspirin increased renal clearance of
salicylic acid from 2.88 ml/min to 15.06 ml/min. The bicarbonate
raises the pH of the urine and this increases
the renal excretion of free salicylate resulting in lowering of
plasma salicylate levels. The control of urinary pH
in studies of pharmacokinetics is, thus, vital as urinary pH can
be important in determining drug toxicity moredirectly. Results
obtained indicated that HPLC can be used for separation of
salicylates and their metabolites
and the technique can be applied in human bioavailability
studies.
Key words: Aspirin; sodium bicarbonate; pharmacokinetics;
salicylic acid
Introduction
spirin, also known as acetyl salicylic acid
is a salicylate drug belonging to non steroidal anti
inflammatory drugs
(NSAIDs). It is used as an analgesic to relieve
pains and minor aches, as an antipyretic to reduce
fever, and as antiinflammatory medication (Ranget al., 2007). It
also has anti platelet effect and is
used for longterm, in low doses to prevent heartattacks, strokes
and blood clot formation in people
at high risk for developing blood clots (Lewis et
al., 1983). Aspirin is also used as a chemo
preventive agent in cancer because of its anti
proliferative and apoptosis inducing properties
(Amin et al., 2003). The major adverse effects of
aspirin are gastro intestinal tract and renal toxicity
(Rang et al., 2007).
Aspirin acts by suppressing the production of
prostaglandins and thromboxanes by irreversibly
inactivating the cyclooxygenase (COX) enzyme.COX enzyme is
required for prostaglandin and
thromboxane synthesis (Tseeng and Arora, 2008).
Specifically, aspirin acts as an acetylating agent
where an acetyl group is covalently attached to a
serine residue in the active site of the COX enzyme
(Tseeng and Arora, 2008). There are two isoformsof COX: COX 1
and COX 2. Different tissues
express varying levels of COX 1 and COX 2.
COX1 is constitutive, present in nearly all cells
and COX 2 is inducible, undetectable in most
normal tissues but abundant in other cells at sites of
inflammation. Aspirin irreversibly inhibits COX
1 and modifies the activity of COX 2. Theinhibitions of
prostaglandins and thromboxane
synthesis results in the loss of thromboxane A2, a
potent platelet activator and this drastically reduce
platelet aggregation (Hankey and Eikelboom,
2006).Both aspirin and salicylic acid are moderately weak
acids and very little of it is ionized in the stomach
on oral administration; absorption from the
stomach will be greater at low pH because they areabsorbed
largely in their non ionized forms, such
as exists at the low pH in the stomach. Aspirin ispoorly soluble
in the acidic conditions of the
stomach and this can delay absorption of high
doses.
About 5080% of salicylate in the blood is bound
by protein which is concentration dependent and
the rest remains in active, ionized state. Saturation
of binding sites leads to more free salicylate and
increased toxicity. The volume of distribution is 0.1
0.2 l/kg and acidosis increases the volume of
distribution because of enhancement of tissue
penetration of salicylates (Levy and Tsuchiya,1972). About 80%
of aspirin is metabolized in the
liver through conjugation with glucuronic acid and
glycine and these pathways have only a limited
capacity (Levy and Tsuchiya, 1972). Salicylates are
excreted mainly by the kidneys as salicyluric acid,
free salicylic acid, salicylic phenol and acylglucuronides, and
gentisic acid. On ingestion of
small doses, all pathways proceed by first order
kinetics with elimination halflife of about 24.5
hours (Done, 1960). With large salicylates doses
the half life becomes much longer and kinetics
switch from first order to zero order because
metabolic pathways become saturated and renalexcretion becomes
increasingly important. Salicylic
acid is extremely sensitive to changes in urine pH.
A
INTERFERENCE OF SODIUM BICARBONATE ON THE
PHARMACOKINETICS OF AN ORALLY ADMINISTERED DOSE OF ASPIRIN
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When the urine pH is increased from 5 to 8, there is
a 10 to 20 fold increase in renal clearance and this
has been exploited in the use of sodium bicarbonate
in salicylate poisoning (Dargan et al., 2002).
In the study, HPLC method was utilized to
determine the levels of salicylic acid (SA) in
plasma and in urine of two set of volunteers, eachset initially
comprising of three volunteers to study
the pharmacokinetics of an orally administered
dose of aspirin with, and without sodium
bicarbonate. In the course of the study, only two
volunteers, one volunteer in each set stayed up to
the end of the study.
Materials
In the study, the following were used: aspirin
tablets, sodium bicarbonate tablets (all from J.T
Baker Chemical Company, Dagentiam, England),
High Performance Liquid Chromatography
(HPLC) system with a Hichrom C 18 reversephase column (250 x 4.6
mm) packed with 5 m
spherical particles with a solvent system
comprising acidified aqueous methanol and U.V.
spectrophotometer for monitoring of the columneffluent. All
other solutions/reagents were of
analytical grade.
The study was carried out at Institute of Medical
Sciences, University of Aberdeen. Six adult male
volunteers were recruited and divided into two sets
each set comprising of three volunteers. Writtenconsent from the
volunteers was obtained after
explaining study objectives to them but only two
stayed up to the end of the study. Institutionalethical approval
was obtained from the Research
and Ethical Committee of Institute of Medical
Sciences, University of Aberdeen.
MethodsPreparation of platelet poor plasma from blood
samples of volunteers
Volunteer 1 ingested 600 mg aspirin plus 10 gsodium bicarbonate
while volunteer 2 ingested 600
mg aspirin. 10 ml whole blood sample was taken
from each volunteer prior to aspirin and sodium
bicarbonate ingestion at time zero hour (t = 0 h)
and subsequently at t = 1, 2, 4, 6 and 7 h afteraspirin and
sodium bicarbonate ingestion. 9.0 ml of
whole blood from each volunteer taken at the
various time intervals was added to 2.0 ml
anticoagulant (3.8% w/v trisodium citrate) in a
plastic sterillin tube and gently mixed.
The two blood samples were first balanced before
centrifuging at 250 x g (800 rpm) for 10 minutes
and a plastic transfer pipette used to remove the
platelet rich plasma. The two samples were
centrifuged again at 1500 x g (2,500 rpm) for a
further 10 minutes. The platelet poor plasma was
removed with a plastic transfer pipette and placed
in a plastic tube, capped, labeled and stored at -200C.
HPLC determination of Salicylic Acid in plasma
HPLC conditions
Column: 250 x 4.6 mm Hichrom (5) RPB
Mobile phase: 50% (v/v) 30 mM sodium citrate,
pH 2.5 with HCl and 50% (v/v) methanol
Flow rate: 1 ml/min.
Detector wavelength: 247 nmInjection volume: 20 l
Preparation of standard curves for Acetylsalicylic
Acid and Salicylic Acid
Standard curves were prepared using water, ASA
stock solution 0.2 mg/ml, and SA stock solution 0.5mg/ml (Table
1).
Extraction procedure for standard and sample
The plasma samples obtained from the two
volunteers at different time intervals were vortex
mixed and 500 l of each plasma sample was
pipette into a test tube. To both the standards and
the samples, 50 l of 0.1 mg/ml phenacetinsolution and 60 l of
1.0 M HCl were added and
vortex mixed. 5.0 ml of diethyl ether was added,
capped and mixed for 15 minutes on rotary mixer
then centrifuged for 5 minutes at 3000 rpm. Theether layer
(upper) was transferred to fresh test
tubes using Pasteur pipette, evaporated using a
nitrogen vortex evaporator and reconstituted in 150
l of mobile phase containing 5% 1.0 M HCl.
Peak areas were used to determine the
SA/Phenacetin area ratios and a standard curve ofSA
concentration versus area ratio plotted. From
the graph, the concentrations of SA in the plasma at
various time intervals were calculated.Retention Times
Acetyl salicylic acid: 5.6 minutes
Phenacetin: 6.9 minutes
Salicylic acid: 6.5 minutes
Determination of salicylate levels in urine samples
of volunteers
Urine samples from the two volunteers were also
obtained at various time intervals (0, 0 1, 12, 2
4, 46, 67, 724 hours) and vortex mixed. 1
ml of urine or standard sodium salicylate solution
(0, 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, and 1 mg/ml)
was pipette into test tubes and 5 ml ferric nitrate
solution (40 mg/ml in 0.1 M HCl) was added,capped and vortex
mixed. The absorbance at 525
nm was read.
A standard curve of sodium salicylate
concentration against absorbance was plotted and
the concentration of salicylic acid in each urine
sample at various time intervals calculated.
Results
Calculation
Area ratio = peak area of SA/peak area of
phenacetin
Area ratios for standards at different concentrations
were calculated and tabulated (Table 2). It can be
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Corresponding author: [email protected]; +2348033119599
Table 5: Urine data of set 1 volunteer
Time (Hours) Vol. (ml) Absorbance SA
concentration
in urine(mg/ml)
Amount
excreted in
urine (mg)
Excretion
rate (mg/h)
Cumulative
amount
excreted(mg)
0 80 0.000 0.00 0.00 0.00 0.00
0 - 1 580 0.216 0.10 58.00 58.00 58.001 - 2 270 0.266 0.13 35.10
35.10 93.10
2 - 4 120 0.405 0.21 25.20 12.60 118.30
4 - 6 170 0.541 0.28 47.60 23.80 165.90
6 - 7 70 0.575 0.30 21.00 21.00 186.907 - 24 1080 0.332 0.17
183.60 10.80 370.50
DiscussionThe results obtained showed differences in the
mean pharmacokinetics data of the two set of
volunteers, particularly the renal clearance of set 1
volunteer was greater than that of set 2 volunteersuggesting
interference of sodium bicarbonate with
the pharmacokinetics of an orally administereddose of aspirin.
The ingestion of bicarbonate with
aspirin might have resulted in increase urinary pH
and this might have affected salicylate renal
reabsorption. The blood salicylate levels might
have been reduced by bicarbonate probably owing
to increased excretion of salicylates in the urine.
The bicarbonate raises the pH of the urine and this
increases the renal excretion of free salicylate
resulting in lowering of plasma salicylate levels(Dargan et al.,
2002).
Table 7: Pharmacokinetics data of the two sets
of volunteersPharmacokinetic
parameter
Volunteer 1 Volunteer 2
Elimination
constant plasma
(Kel)
0.27 hr-1 0.17 hr-1
Elimination half
life (t1/2)
2.57 hrs 4.08 hrs
Area under the
curve (AUC)
0.41 mg.h/ml 0.66 mg.h/ml
Renal clearance 15.06 ml/min 2.88 ml/min
Volume of
distribution (vd)
5.40 L 5.40 L
Bioavailability(F)
0.9999 0.9999
Total clearance 24.15 ml/min 15.00 ml/min
Elimination
constant renal
(K)
0.51 hr-1
0.65 hr-1
Absorption rate
constant (Kabs)
0.199 hr-1
0.165 hr-1
Absorption half
life
3.50 hrs 4.2 hrs
Change in the pH of urine will change the rate of
urinary excretion. When a drug is in its unionized
form it will more readily diffuse from the urine to
the blood. In acidic urine, aspirin being an acidic
drug will diffuse back into the blood from the
urine. The control of urinary pH in studies of
pharmacokinetics is, thus, vital as urinary pH can
be important in determining drug toxicity more
directly. Bicarbonate alkalinizes the urine raisingthe urine pH
and in alkaline urine, aspirin being an
acidic drug will be in ionized form and will notdiffuse back
into the blood from the urine but will
be excreted.
Aspirin also undergoes first pass (pre systemic)
elimination where it is extracted so efficiently by
the gut wall and /or liver which lead to reduced
blood levels of the parent drug or even some of its
active metabolites when the drug is given orally.
The amount reaching the systemic circulation is
considerably less than the amount absorbed andthis reduces
bioavailability even when aspirin is
well absorbed from the gut. The first pass
elimination of aspirin results in a much larger dose
of the drug being needed when it is given orallythan when it is
given by other routes. Also, marked
individual variations can occur in the extent of firstpass
metabolism of aspirin and this can result in
unpredictability when aspirin is taken orally
(Kotani et al., 2010). This might also be another
reason behind the different pharmacokinetics data
obtained from the two set of volunteers.
The occurrence of first pass effect in the
metabolism and distribution of orally administered
aspirin in man is also important in respect of the
dosage form of the drug. Thus, enteric coated
aspirin is absorbed to an appreciable extent in the
upper intestinal tract (compared with plain tabletsof the drug
which are absorbed largely in the
stomach). Consequently, aspirin released from
enteric coated tablets may undergo appreciably
more hepato- intestinal metabolism to salicylate
than evident with plain aspirin tablets. This is quite
important therapeutically, example, in achieving an
optimal aspirin/salicylate ratio for prophylaxis of
platelet regulated thrombogenesis (Kotani et al.,
2010).
In the study, HPLC was used for the separation of
salicylic acid because of its simple application in
the analysis of salicylates and their metabolites
(Kees etal., 1996). The procedure utilizes a C 18reverse phase
column with a solvent system
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comprising acidified aqueous methanol and U.V.
spectrophotometer for monitoring of the column
effluent. Thus, HPLC separation of salicylic acid
can be applied in human bioavailability studies.
The study has shown that both aspirin and its active
metabolite salicylic acid being weak acids are
extremely sensitive to changes in urine pH, and sowhen the urine
pH is increased, renal clearance is
also increased, thus, it can be concluded that
sodium bicarbonate interferes with the
pharmacokinetics of an orally administered dose of
aspirin and this can be exploited in salicylate
poisoning. Also, the first pass elimination of aspirin
can lead to reduced blood levels of aspirin and
salicylic acid when given orally and this can be
important in respect of the dosage form of the drug.
Acknowledgement
The authors are grateful to Mr. Camaroo of theInstitute of
Medical Sciences, University of
Aberdeen, UK for technical assistance and Dr. N.
N. Wannang of the Department of Pharmacology,
University of Jos, Nigeria for critical reading of the
manuscript.
Table 6: Urine data of set 2 volunteer
Time (h) Vol. (ml) Absorbance SA concentration
in urine (mg/ml)
Amount excreted
in urine (mg)
Excretion
rate (mg/h)
Cumulative
amount
excreted (mg)
0 60 0.000 0.00 0.00 0.00 0.00
0 - 1 560 0.165 0.07 39.20 39.20 39.20
1 - 2 250 0.218 0.10 25.00 25.00 64.202 - 4 100 0.358 0.18 18.00
9.00 73.20
4 - 6 150 0.480 0.25 37.50 18.80 92.00
6 - 7 50 0.515 0.27 13.50 13.50 105.50
7 - 24 1060 0.288 0.14 48.40 8.70 114.20
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