Final Lab Report

BIO2118: A quantitative report on how pH level can directly impact the absorption and excretion rate
of drugs.
A quantitative report on how pH level can directly impact the absorption and excretion rate of
drugs.
Word Count:
This experiment aimed to investigate the effect variable pH has on absorption and excretion
rates of drugs, and how a protein load/eating can affect the pH and excretion rates.
1|Page
of drugs.
Understanding the impact pH has on the absorption and excretion of a drug assists in
achieving a better bioavailability and therapeutic factor. Three experiments were conducted.
Ten subjects underwent each experiment. In the first experiment, all ten subjects conducted
the same tests with a sample of the ethyl acetate layer from each test taken for analysis. In
experiment two, five (n=5) subjects were randomly assigned to both the aspirin and aspirin
+NaHCO3 group, and urine samples were taken for measurement and analysis. In experiment
3 and five (n=5), subjects were randomly assigned to both the protein and water groups and
urine samples were taken for measurement and analysis.
The results from this experiment included:
1. That the pH highly influenced the level of absorption of a drug.
2. Increasing the alkalinisation of urine using NaHCO3 results in an increase in aspirin
excretion.
3. Protein consumption can cause an increase in pH level and urine output.
Our results show that in addition to pH influencing absorption and excretion, protein loading
can also affect the pH and excretion rates of a drug.
A drug is a chemical substance that, when administered to living organisms, produces a

biological effect and is used to treat, prevent, or diagnose a disease or promote wellbeing.
2|Page
of drugs.
The ability of a drug to elicit a pharmacological and therapeutic effect is related to the
influence of the various physicochemical properties on the drug (Cheng and Wong, 2020).
The bioavailability of a drug product is known as the rate and extent of its absorption. Urine
pH is a significant factor in the absorption of a drug; previous studies have found that a drugs
solubility and its absorption are strongly dependent on the pH (Stevens et al., 2019).
Understanding how pH and changes in pH can affect the absorption and excretion of a drug is
crucial in determining adequate dosage, dosing regimen, and condition under which a drug
should be taken (with or without food). A better understanding of the drug absorption process
and affecting factors plays an essential role in achieving better bioavailability and better
therapeutic effect.
Most drugs are either weak bases or weak acids; in alkaline urine, acidic drugs become more
ionised, whereas basic drugs become more ionised in acidic urine. Ionised substances are
water-soluble and dissolve more readily for excretion in the body fluid (Garza, Park and
Kocz, 2021). The urine pH level can also be manipulated to control the excretion of a drug
from the body in some clinical situations (Abramson, 2010). In cases such as a drug
overdose, pH manipulation is important when the body amount has reached a toxic level and
must be excreted from the body rapidly.
The physiology of the GI tract can also influence drug absorption, and it is, therefore,
imperative to ensure that any situations deterring the digestive system from homeostasis are
explored (Cheng and Wong, 2020). Food intake is known to alter the gastrointestinal
environment through a condition known as the alkaline tide. This condition, generally
encountered after a meal, causes an increase in pH through the influx of bicarbonate ions that
diffuse into the bloodstream during the production of hydrochloric acid (Smith and Morton,
2010). This alteration to pH can affect the safety and efficacy of a drug. Therefore, it is
crucial to be aware of the state in which different drugs should be taken. Some medications
are recommended to be taken with food due to the changes that occur after eating, increasing
the amount of medicine that's absorbed by the body. In other cases, these changes can reduce
the effectiveness of a medicine (Enwerem, 2017).
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of drugs.
Three experiments were conducted to investigate the effect variable pH has on drug excretion
and absorption as well as how a protein load/eating can affect the pH and excretion rates. It is
hypothesised that pH level has an impact on drug absorption and excretion, specifically that
basic drugs will become more non-ionised and absorb better in an alkaline pH and acidic
drugs will become more non-ionised and absorb better in an acidic pH. It is also hypothesised
that a protein load will cause an increase in urine pH and as a result an increase in excretion
rates.
Three experiments were conducted as follows:
Please refer to the lab manual for the full procedure. Ellem S, Windus L, Brown L. BIO2118
Systems Physiology and Pharmacology Residential School. University of Southern
Queensland 2020; Semester 1.
Part A: Effect of pH of drug absorption
For the first experiment, ten (n=10) male and female volunteers with a mean age of 23.6 ±
1.29227 from the USQ BIO2118 class participated in this study. Participants undertook
testing in all three drugs being tested.
A simple model of the GI tract was created using test tubes containing aqueous solutions of
different pH (1.5, 3.5, 5.5 and 8pH). Drugs being investigated were aspirin, 3 – aminophenol
and paracetamol. A sample of these drugs was added to aqueous solutions before being
mixed with ethyl acetate. A sample from each test-tubes ethyl acetate layer was then added to
a silica sheet and evaporated. Drug absorption was measured by viewing the silica sheet
containing the sample from the ethyl acetate layer of each sample under ultraviolet light. The
concentration of drug present was then ranked on a scale from 0 (none) to 4 (very high).
All data was analysed by a single factor ANOVA test. When required, a two-sample t-test
assuming unequal variances post hoc analysis was performed. The level of statistical
significance was set at P<0.05. Data is expressed as mean ± SE.
A one-way ANOVA was conducted for each drug result to compare the presence of the drug
in the ethyl acetate layer in 1.5, 3.5, 5.5 and 8pH conditions. There was clear significance
(p<0.05) between these four groups in each drug. Post hoc comparisons using a two-sample t-
test assuming unequal variances indicated that the presence of aspirin in the ethyl acetate
layer 1.5pH was significantly different (p<0.05) than the presence at 3.5, 5.5 and 8pH (Figure
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of drugs.
1). Post hoc comparisons using a two-sample t-test assuming unequal variances indicated that
the presence of 3-aminophenol in the ethyl acetate layer at 8pH was significantly different
(p<0.05) than the presence at 1.5, 3.5 and 5.5pH (Figure 2). Post hoc comparisons using a
two-sample t-test assuming unequal variances indicated that the presence of paracetamol in
the ethyl acetate layer at 1.5 was significantly different (p<0.05) than the presence at 5.5pH
(Figure 3). No p-value was calculated for 3.5pH and 8 pH as there was no standard error of
the mean as results had no variance.
4.5
4
Aspirin drug concentration
3.5 **
3
2.5
2
**
1.5
1
**
0.5
0
1.5 3.5 5.5 8
pH of aqueous solution
Figure 1: Aspirin drug concentration in ethyl acetate layer across four aqueous solutions of
varying pH levels (1.5, 3.5, 5.5 and 8pH). Values are mean ± SEM. **Significantly different
from 1.5pH values (p<0.05).
4
3-aminophenol drug concentration
3.5
**
3
2.5
2 **
1.5 **
1
0.5
0
1.5 3.5 5.5 8
Figure 2: 3-aminophenol drug concentration in ethyl acetate layer across four aqueous
solutions of varying pH levels (1.5, 3.5, 5.5 and 8pH). Values are mean ± SEM.
**Significantly different from 8pH value (p<0.05).
5|Page
of drugs.
4.5
4
Paracetemol drug concentration
3.5
**
3
2.5
1.5
0.5
0
1.5 3.5 5.5 8
Figure 3: Paracetamol drug concentration in ethyl acetate layer across four aqueous solutions
of varying pH levels (1.5, 3.5, 5.5 and 8pH). Values are mean ± SEM. **Significantly
different from 1.5pH values (p<0.05).
Part B: Urinary Salicylate excretion
For the second experiment, ten (n=10) male and female volunteers with a mean age of 21.2 ±
1.314872 from the USQ BIO2118 class participated in this experiment. The subjects were
randomly assigned to either the Aspirin only group (n=5) or the Aspirin + NaHCO3 group
(n=5). Volunteers were not allowed to participate if they were allergic to sodium bicarbonate
or aspirin or had any breathing problems (asthma). Students with underlying health issues
like cardiac failure, hypertension, and kidney diseases could not participate as the ingestion
of sodium bicarbonate may exacerbate their symptoms. All subjects were requested to limit
their caffeine and ethanol intake in the 24 hours leading up to the experiment. The procedure
and potential risks of the experiment were explained in detail verbally and in writing to the
participants; participants written consent was obtained.
All participants ingested a single dose of aspirin (600mg) with a glass of water. Participants
then provided a urine sample where the pH was used as the baseline sample. An hour later,
participants in the aspirin + NaHCO3 ingested a single dose of NaHCO3 (0.2g/kg),
participants then measured total urine output and pH, and a sample was provided for analysis.
An hour later, another sample was provided for analysis; this action was then repeated every
30 minutes for the next 2 hours.
Participant’s urine output, pH and salicylate excretions were measured at 0, 60, 120, 150,
180, 210 and 240 minutes. pH was measured using Clinistix and pH strips and a Trinder’s
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of drugs.
reagent test, and a standard curve was used to estimate salicylic acid concentration in urine
samples.
Comparison between test groups for the dependent variable (pH and salicylate secretion)
were made by a two-sample t-test assuming unequal variances. The level of statistical
significance was set at P<0.05. Data is expressed as mean ± SEM.
A two-sample t-test with unequal variances was conducted to compare urine pH and
salicylate secretion in aspirin and aspirin + NaHCO3 test groups. There was a significant
difference (p<0.05) in cumulative salicylate excretion between the test groups at three of the
time points recorded (Figure 4). There was also a significant difference (p<0.05) in pH levels
between the test groups at three of the time points recorded (Figure 5).
250.00
**
Cumalative Salicylate secretion(mg)
200.00
**
150.00
**
100.00
50.00
0.00
0 50 100 150 200 250 300
Time in minutes
Asprin Asprin +NaHCO3
Figure 4: Changes in cumulative salicylate secretion in urine across 120 minutes after taking
either a single dose of aspirin (n=5) or a single dose of aspirin combined with a single dose of
sodium bicarbonate (n=5). Values are mean ± SEM. **Significantly different from aspirin
values (p<0.05).
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of drugs.
9.00
8.00 ** ** ** **
7.00
6.00
5.00
Urine pH
4.00
3.00
2.00
1.00
0.00
0 50 100 150 200 250 300
Time in minutes
Asprin Asprin=NaHCO3
Figure 5: Changes in urine pH across 120 minutes after taking either a single dose of aspirin
(n=5) or a single dose of aspirin combined with a single dose of sodium bicarbonate (n=5).
Values are mean ± SEM. **Significantly different from aspirin values (p<0.05).
Part C: Measuring the ‘alkaline tide.’
For the third experiment, ten (n=10) male and female volunteers with a mean age of 26.3 ±
3.235395 from the USQ BIO2118 class participated in this experiment. Participants were
assigned to the water test group (n=5) of the protein test group (n=5). Participants in the
protein test group were required to consume a protein shake that contained whey protein, as
whey protein contains lactose, students with lactose intolerance were not able to be in the
protein test group.
All participants recorded their initial urine pH level and urine output to be used as a baseline
measurement. Participants then ingested either 500ml of water or 500ml of protein shake;
participants in the protein group should consume 1.5g of protein per kg of bodyweight.
Participants then measured total urine output and pH every twenty minutes for two hours.
Participant’s urine output and pH level were measured after water or protein shake
consumption at 0, 60, 120, 150, 180, 210 and 240 minutes. pH was measured using Clinistix
and pH strips.
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of drugs.
All data was analysed by a two-sample t-test assuming unequal variances. The level of
statistical significance was set at P<0.05. Data is expressed as mean ± SEM.
A two-sample t-test with unequal variances was conducted to compare urine pH and output
between the water and protein test groups. There was a significant difference (p<0.05) in pH
between the test groups at two of the time points recorded (Figure 6). There was also a
significant difference (p<0.05) in urine output between the test groups at five of the time
points recorded (Figure 7).
6
** **
5
Urine pH level
0
0 20 40 60 80 100 120
Time in minutes
Water Protein
Figure 6: Changes in pH of urine across 120 minutes after consuming either a 500ml whey
protein shake (n=5) or 500ml of water (n=5). Values are mean ± SEM. **Significantly
different from protein values (p<0.05).
9|Page
of drugs.
200
180
160
140
Urine Output (ml)
120
100
80
60 ** ** **
40 ** **
20
0
0 20 40 60 80 100 120
Time (minutes)
Water Protein
Figure 7: Changes in urine output across 120 minutes after consuming a drink of either
500ml of water (n=5) or 500ml of a whey protein shake (n=5). Values are mean ± SEM.
**Significantly different from protein values (p<0.05).
Firstly, we looked at the effect of pH on drug absorption using a model of the GI tract. A
model of the GI tract was used as the pH of its contents vary from 1.2 to 3 in the stomach to
about 8 in the intestine. The drugs tested in this experiment were paracetamol, 3-aminophenol
and aspirin. It was expected that the ionisable drugs (3-aminophenol and aspirin) solubility
would be influenced by pH. As aspirin is a weak acid, it was anticipated that it would have a
higher concentration in the ethyl acetate layer at a pH<3, which was verified with the results.
The aspirins absorption rate was significantly higher at 1.5pH when compared to 3.5, 5.5 and
8 pH (Figure 1). It has been observed that weak acidic drugs like aspirin are more easily
absorbed by the stomach due to the more acidic pH (Stevens et al., 2019). With 3-
aminophenol being a weak base, it was expected to have a higher concentration in the ethyl
acetate layer in a more alkaline environment, verified with the results where the absorption
rate was significantly higher 8pH compared to 1.5 3.5 and 5.5 pH (Figure 2). Similar results
have been found in other studies, where it was observed that the absorption rate of 3-
aminophenol was higher in a more alkaline environment (Alharbi, 2018). The intestine more
easily absorbs weak basic drugs like 3-aminophenol due to the higher pH. It was expected
that paracetamol would have a reasonably high concentration in the ethyl acetate regardless
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of drugs.
of the pH as it is a neutral drug that requires a pH of well above eight before it can ionise.
Paracetamol achieves a high concentration in ethyl acetate regardless of the pH. The results
showed that paracetamol had a high concentration across all pH's with the 1.5 and 3.5pH
yielding the highest result and slowly starting to decrease at the 8pH (Table 3). Drugs like
paracetamol can be absorbed from both the stomach and the intestine.
A better understanding of the drug absorption process and affecting factors plays an
important role in achieving better bioavailability and a more safe and effective therapeutic
effect (Alagga and Gupta, 2021).
Secondly, we looked at the effect of NaHCO3 on the urinary excretion of aspirin. It was
expected that the Aspirin + NaHCO3 test group would have higher rates of salicylate
excretion and urine output. As aspirin can ionise, its solubility and excretion is largely
dependent on the pH level.
The aspirin + NaHCO3 had the highest amount of cumulative salicylate secretion in the
results, with there being a significant difference between the groups at 180, 210 and 240
minutes (Figure 4). Comparing the pH of the two groups also finds that the pH was higher in
the aspirin NAHCO3 group, with there being a significant difference at 150, 180,210 and 240
mins (Figure 5). As aspirin is a weak acid, it is more non ionised and lipid-soluble in an
acidic environment. In the group testing aspirin + NaHCO3, the dissociation of sodium
bicarbonate forms sodium and bicarbonate ions. This ion formation increases plasma
bicarbonate and buffers excess hydrogen ions, resulting in a raised pH (Carr et al., 2011). In a
more alkaline pH, acidic drugs like aspirin become more ionised due to the basic
environment seen in previous experiments and (Al-Khalaf et al., 2019), which showed a
decrease in aspirin concentration as the pH of the sample solution became more basic. This
pH change leads to aspirin becoming more water-soluble and more available for excretion
leading to higher amounts of salicylate acid in excretion, as seen in the results.
Whilst sodium bicarbonate’s ability to increase pH may reduce the effect of aspirin; it can be
helpful in the management of certain pharmacological overdoses (Mirrakhimov et al., 2017).
If a toxic amount of a drug has already been absorbed in the body, the only option to remove
the drug is to increase the rate of excretion, which can be done by manipulating the pH.
As the average age in this experiment was 21.2 ± 1.315, it would be beneficial in future
studies to incorporate participants of a higher age group to study the effects NaHCO3 has on
managing pH levels in the elderly. This would be beneficial as urine pH decreases with age
11 | P a g e
of drugs.
and becomes more acidic (Menezes et al., 2019); this can lead to acidic drugs become much
more lipophilic, leading to changes in the absorption and excretion rate.
Thirdly, we looked at the alkaline tide and investigated whether hydrochloric acid's (HCl)
secretion caused an increase in urine pH and if protein affected this. Histamine,
parasympathetic system (acetylcholine) and gastrin stimulate the parietal cell to trigger the
secretion of HCl into the stomach. Secretion of HCl after a meal is accompanied by an influx
of bicarbonate ions (HCO3) which are absorbed by the gastric venous blood resulting in a
temporary increase of pH(alkaline tide). The alkaline tide is then neutralised by the secretion
of H+ (Smith and Morton, 2010).
It was expected that the protein group would have a higher pH and urine output due to the
consumption of protein triggering the alkaline tide making the pH more alkaline. The protein
test group had the highest pH readings on average, with a significant difference between
groups at 40 and 60 minutes (Figure 6). Comparing the urine output of the groups also finds
that protein had the highest output, with there being a significant difference between the
groups at 40, 60, 80, 100 and 120 minutes (Figure 7).
This alteration to pH can affect the safety and efficacy of a drug therefore as the pH is altered
with food it is important to know whether a drug should be taken in a fed or starved state.
Some drugs are best taken on an empty stomach to not reduce the effectiveness of a drug
whilst others are best taken with food as the changes in the pH increase the effectiveness as
pH effects the absorption and bioavailability of drugs. As found in this study the rate of
absorption of the drug increased when taken with food (fed state) compared to without (fasted
state) (Abuhelwa et al., 2016).
To further this study, an investigation into the effects different types of drinks/food have on
the urine pH, and excretion rate could be conducted.
One general limitation for all these experiments is the small sample size (n=10). A larger
sample size needs to be tested to ensure the results and significant relationships are accurate
before they can be generalised to a whole population.
The purpose of these experiments was to see the effect pH had on drug absorption and
excretion and if a protein load would influence the pH and urine excretion rate. The data
obtained from these experiments demonstrates that drug absorption and excretion is highly
12 | P a g e
of drugs.
influenced by the pH and that a protein load is a factor that can influence the pH and
excretion rate.
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of drugs.
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BIO2118: A quantitative report on how pH level can directly impact the absorption and excretion rate

A drug is a chemical substance that, when administered to living organisms, produces a

Three experiments were conducted as follows:

Part A: Effect of pH of drug absorption

Asprin Asprin +NaHCO3

Part C: Measuring the ‘alkaline tide.’

Abramson, S., 2010. Extracorporeal Treatment of Poisonings. Chronic Kidney Disease,

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