Investigation of Aspirin Overdose Using Salicylate Assay

Aspirin, Acetylsalicylic acid, is a widely used over-the-counter OTC medicine that was officially approved by the FDA in 1939. It has anti-inflammatory, analgesic and antipyretic effects. It is administrated orally in different doses. Smaller doses and administrated for the prevention of heart diseases, especially in the elderly. It is important to know that aspirin should not be given to children under 16 years old.

1. Explain carefully what the results of the screening test indicate, ensuring that you refer to each of the five solutions tested. (hint: You may wish to insert a small table here in order to represent these qualitative findings). (5 marks)

Sample Colour Explanation

Positive Control Purple The sodium salicylate standard reacts with Trinders reagent and forms a purple colour. The other samples will be compared to this control.

Negative control Light yellow When salicylates are not present in the sample, no purple colour will form and we will observe light yellowish solution.

Sample A Cream yellowish

Aspirin was detected in stomach content.

Acetylsalicylic acid does not react with Trinders reagent unless hydrolysed. Therefore, no purple colour was formed in sample A whereas sample B shows a positive reaction indicating the presence of aspirin in the stomach content.

Sample B Purple Sample D Pink A small amount of salicylate was detected in the urine since a pink colour was formed.

2. Why did a precipitate form on addition of Trinders reagent to the serum sample but not to the urine sample? (150 words max; 4 marks)

Trinders reagent is an acidic solution that consists of Hydrochloric acid and mercuric chloride. Mercuric chloride precipitates the proteins in the serum sample by disrupting the disulphide bonds in the protein. This is a crucial step for eliminating the interfering serum proteins. Since the urine sample doesnt contain any proteins, no precipitate was formed after the addition of Tinders reagent (Shaw, 2001).

3. Your fully labelled graph of calibration data and estimates of the unknown salicylate concentration submitted separately, will be attached to the end of your paper copy. (6 marks)

4. Using Excel, tabulate the class results (from the module Blackboard site under Assessments) and calculate the mean and standard deviation. Insert your table at the end of this report (both paper and online submitted copies). Briefly comment on any difference between your results and the class mean result. In your answer, make sure that you show that you understand the meaning of standard deviation. (150 words max; 6 marks)

Based on the table at the end of the report, the standard deviation value is large (143 ug/ml) which indicates that the class results are spread out over a wide range of values and they are actually not close the mean. If we look closely at the results, we will see that there are two values that are too far from the mean, which are the results of students No. 17 (1 ug/ml) and No. 31 (942 ug/ml). Therefore, if we eliminate these two values and recalculate the mean and standard deviation, we will find out that the mean will be 283 ug/ml and the standard deviation will be smaller (61 ug/ml), meaning that all class results are relatively close to each other except the results of two students. In both cases, my result (194 ug/ml) is a bit far from the mean (295 ug/ml in the first case or 283 ug/ml in the second case). However, that does not mean that it is far from the true value.

5. Do your results indicate that the patient has taken an overdose of aspirin? Explain why your conclusion might be different if you were informed that the patient was suffering from rheumatoid arthritis.(200 words max: 6 marks)

Based on our results, the mean serum concentration of salicylate was calculated as 194 ug/ml which is considered to be higher than the therapeutic serum concentration. However, for patients taking aspirin for Arthritis, serum therapeutic concentration is 150-300 ug/ml. So, in this case the patient did not take an overdose of Aspirin. 6. List the side effects that might be experienced by a patient who has taken a suspected aspirin overdose. (20 words max; 3 marks)

Gastric discomfort and emesis, hematemesis, tachypnea, hypernea, dyspnea, tinnitus, deafness, lethargy, confusions and seizures might individually or together occur. (Chyka et al, 2007)

7. The pKa value of aspirin is 3.0. (i) Name the part of the GI tract where the environmental pH would be expected to favour rapid absorption. (ii) Is this where most absorption actually occurs? If not, identify an alternative site in the GI tract and briefly explain your answer. (150 words max; 1 + 5 marks)

(i) Generally, absorption of a molecule into the bloodstream occurs when the molecule is in its unionized form. Henderson-Hasselbalch equation for acids is as follows:

pH = pKa log ([HA] / [A- ])

Since aspirin is a weak acid, it is expected to favour rapid absorption in the stomach where the pH is acidic (approximately pH=1) and >99% of the aspirin is in its unionized form.

(ii) Aspirin is usually mainly absorbed in the upper intestines (duodenum) by passive diffusion. The big surface of the intestines and the high solubility of aspirin in the intestines allow better absorption (Schror, 2009).

8. Briefly explain the molecular mode of action for aspirin, when it is used as an anti-inflammatory and/or antipyretic drug. (200 words max; 5 marks)

Aspirin is deacetylated quickly in the body to produce salicylate, the active compound that works by inactivates cyclooxygenase by irreversible acetylation. The anti-inflammatory and antipyretic effects mainly occur due to inhibition prostaglandin synthesis at the thermoregulatory centres in the hypothalamus and at peripheral target sites. This mechanism is also useful in reducing the sensitivity of pain receptors to both mechanical and chemical stimuli. As an anti-inflammatory agent, aspirin inhibits cyclooxygenase activity which lowers the formation of prostaglandins. Therefore, it changes the aspects of inflammation where prostaglandins act as modulators.

Prostaglandin PGE2 synthesis is stimulated by an endogenrous fever-producing agent which causes fever. Aspirin inhibits PGE2 synthesis and release patients with fever, thus lowering the bodys temperature. (Finkel, Clarck & Cubeddu, 2009)

9. Provide one example each of the systematic (chemical), trivial, generic and trade names for aspirin. (4 words max; 2 marks)

Acetylsalicylic Acid, Aspirin, Aspirin, Acuprin.

References. List here (correctly cited using the Harvard format) (2 marks)

Chyka, P.A. et.al. (2007) Salicylate poisoning: An evidence-based consensus guideline for out-of-hospital management. Clinical Toxicology 45(2) 95131. Available from: http://www.tandfonline.com/doi/abs/10.1080/15563650600907140 [ Accessed 8 March 2016].

Finkel, R. Clarck, M.A. and Cubeddu, M.X. (2009) Pharmacology: Lippincotts illustrated reviews, 4th edition. Philadelphia, Lippincott Williams & Wilkins.

Shaw, L.M. (2001) The Clinical Toxicology Laboratory: Contemporary Practice of Poisoning Evaluation, Washington, AACC Press.

Schror, K. (2009) Acetylsalicylic Acid, Weinheim, John Wiley & Sons.

Unknown sample Absorbance at 540 nm Calculated Concentration (ug/ml)

0.190 27

0.759 362

Mean= 194

-177800760793500

Class Results

(18 Feb 2016)

Student No. Serum Concentration (ug/ml)

1 290

2 399

3 339

4 315

5 250

6 304

7 310

8 290

9 297

10 320

11 290

12 250

13 120

14 377.5

15 194.5

16 300

17 1.1525

18 329

19 262

20 330

21 292

22 270

23 285

24 227.5

25 112

26 325

27 290

28 285

29 292.5

30 268

31 942

Mean Concentration 295.4

Standard Deviation 143.1