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Laboratory work 3. Metabolism of nitrogen-containing compounds
Experiment 1. Qualitative determination of free hydrochloric acid in gastric juice using indicator congo red.
Normal gastric juice has a strongly acidic reaction because of the presence of hydrochloric acid. Gastric contents, extracted by the probe after a certain time after the test breakfast is used for analysis. It normally has a pH of 1,5-2,5, and the concentration of HC1 is 0,1-0,2%. The following types of acidity are distinguished in a clinical analysis of gastric juice:
1) "free hydrochloric acid", which is in the free state and dissociates into ions;
2) "bound hydrochloric acid" in the form of salt-like compounds with proteins and products of their digestion;
3) "total hydrochloric acid" which is a combination of free and bound hydrochloric acids;
4) "total acidity" - all acidic substances (bound and free hydrochloric acid, hydrogen phosphates, and in pathologies lactic acid and volatile fatty acids).
The total acidity of gastric juice after a test breakfast is 40-60 conventional units; free acidity is 20-40 units. In some dysfunctions of the stomach, the hydrochloric acid content of the gastric juice may increase or decrease to the point of complete absence (achylia).
In the acidic medium congo red has a blue color, in slightly acidic, neutral and alkaline media red color. The transition pH range of congo red is between pH 3.0 and 5.2. Such a pH value in gastric juice is a result of only free hydrochloric acid presence.
Pour 1 ml of hydrochloric acid in the test tube and add 2 drops of indicator congo red. The same experiment is done with a gastric juice samples (samples 1 and 2). Observe the change of indicator’s color, and infer the presence or absence of hydrochloric acid in the studied juices.
Experiment 2. Qualitative determination of free hydrochloric acid in gastric juice with a tropeolin solution.
The transition pH range of tropeolin indicator is between pH 1,3 and 3,0. When pH is above 3,0 the color of indicator is orange-yellow. Only in the presence of mineral acids (when pH is 1,3 and below), color is crimson-red.
Pour 1 ml of samples of gastric juice (1 and 2) and hydrochloric acid and 2 drops of tropeolin in test tubes. Compare the color of the indicator in all the samples and make sure that the crimson-red color appeared only in the presence of hydrochloric acid.
Experiment 3. Titration of acids of gastric contents.
Acids of gastric contents are titrated with 0,1 n. sodium hydroxide solution. Use a variety of indicators with different transition pH range: phenolphthalein - 8,2-10,0; congo-red - 3,0-5,2; alizarin red - 4,3-6,3 .
Pour 5 ml of gastric contents into a flask. Add 10 drops of phenolphthalein solution, 2 drops of congo red solution and titrate with 0,1 n. sodium hydroxide solution to the transition of the original blue to red. The amount of alkali, used for the titration, corresponds to the amount of free hydrochloric acid and identifies by indicator congo red. Not adding alkali into the burette until zero, continue titrating until transition of the color in the crimson. The total amount of alkali corresponds to the total acidity and is detected by phenolphthalein indicator.
Measure 5 ml of gastric contents in another flask, add 2 drops of alizarin red solution and titrate to the transition of the original yellow color to the purple one. The amount of alkali used for titration, corresponds to the sum of all the substances that give an acid reaction, except bound hydrochloric acid, and is detected by alizarin red indicator. Results are entered in table.
Results of the determination of gastric acidity
|
Volume of gastric juice, ml |
Volume of 0.1 n. sodium hydroxide solution consumed in titration, ml |
Number of conventional units, mol/l |
||||
|
to red |
to crimson |
to purple |
free hydro-chloric acid |
total acidity |
bound hydro-chloric acid |
|
Results of titration are expressed in ml of 0,1n. sodium hydroxide solution, used on neutralizing of free hydrochloric acid and other acidic compounds in 100 ml of gastric contents (conventional units). One conventional unit corresponds to the concentration of hydrochloric acid of 1 mmol/l.
Compare the received data with the norm. Make a conclusion about the gastric acid secretion (hyper-, norm-, hypo- or achlorhydria).
Experiment 4. Quantitative determination of creatinine in urine by Jaffe color reaction (the method of Popper).
Creatinine interacting with picric acid forms creatinine picrate which converts to its tautomeric orange-red form in an alkaline medium. The color intensity is proportional to the concentration of creatinine.
The control sample. Mix 3 ml of a saturated solution of picric acid and 0.2 ml of 10% sodium hydroxide solution in a volumetric flask of 100 ml. Leave it for 10 minutes at room temperature and then add distilled water until the total volume of the contents is 100 ml.
The standard sample. Mix 0,5 ml of the base standard solution of creatinine and 3 ml of a saturated picric acid solution in a volumetric flask of 100 ml. Shake the flask and add 0,2 ml of 10% sodium hydroxide solution. Leave it for 10 minutes at room temperature and then add distilled water until the total volume of the contents is 100 ml.
The experimental sample. Put 0,5 ml of the daily amount of urine and 3 ml of a saturated solution of picric acid into a volumetric flask of 100 ml. Shake the flask and add 0,2 ml of 10% sodium hydroxide solution. Leave it for 10 minutes at room temperature and then add distilled water until the total volume of the contents is 100 ml.
Measure the optical density with an electrophotocolorimeter in a cuvette of 1cm layer thickness at a 510-560 nm wavelength (green filter) against control.
Calculations are made according to the formula:
,
where x is the amount of creatinine in the daily urine, mg; Cst is the amount of creatinine in the standard sample, mg; Dexp is optical density of the experimental sample; Dst is optical density of the standard sample; Vday is daily amount of urine; V is volume of urine taken for analysis.
The obtained data are compared with normal level of creatinine in the urine a day: for men – 1-2 g/day (8,8-17,7 mmol /day), women – 0,8-1,8 g/day (7,1-15,9 mmol/day).
Test Questions
1. What factors determine the biological value of dietary proteins?
2. What conditions are needed for protein digestion in the stomach?
3. The presence of what substances in gastric juice determines the total acidity?
4. How to prevent the action of peptidases on the cells of the stomach and intestines?
5. What are functions of creatine and creatinine in the human body? Give the equations of the conversion of creatine into creatinine.