Laboratory 8. Blood biochemistry

1. Buffer properties of blood serum.

Buffer systems maintain blood pH at a constant level close to 7.4. These include bicarbonate, phosphate, protein and hemoglobin buffer system. The pH of plasma 7.4 depends on the physiological concentrations of the bicarbonate and phosphate buffer systems components. The main extracellular buffer is bicarbonate buffer.

In one test tube pour 1 ml of serum, in the other - 1 ml of water. In both test tubes add 3-4 drops of the indicator bromophenol blue solution and then add dropwise 0,1 n. hydrochloric acid solution from a burette. Note how much acid has gone into both cases to achieve the same color change.

2. Quantitative determination of total protein in serum according to biuretic reaction.

Proteins of blood serum by the interaction with copper sulphate in alkaline solution form purple complexes with copper ions due to their peptide bonds. The intensity of the solution staining is directly dependent on the concentration of protein in it.

To 0.1 ml of serum add 5.0 ml of working solution of biuretic reagent. At the same time make control sample. To 0.1 ml of 0.9% sodium chloride solution add 5 ml of the biuretic reagent. After 30 minutes measure the optical density of the solution on photoelectrocolorimeter with a wavelength of 540-560 nm (green light filter) versus control.

To draw a calibration curve prepare working standard solutions from the standard 5% protein solution, according to the Table 8. Take 0,1 ml of working solution from each dilution and add 5 ml of the biuretic reagent. After 30-60 minutes the optical density is measured. According to the obtained data draw the calibration curve. Norm is 6.5-8.5% of protein. When the concentration of protein in the serum is more than 10% serum is diluted with saline, and the results are multiplied by the dilution factor.

Table 8

The composition of mixtures for the ployying of the calibration curve

Number	The standard protein solution, ml	0,9% sodium chloride solution, ml	The protein content in the sample, g	Protein concentration, %
1	0,4	0,6	0,02	2
2	0,6	0,4	0,03	3
3	0,8	0,2	0,04	4
4	1,0		0,05	5

3. Determination of calcium in serum by the method of de Waard.

In the centrifuge tube measure 2 ml of distilled water. Add 1 ml of serum. Then add 1 ml of saturated ammonium oxalate solution, and leave for 10 minutes, then centrifuge it for 5 minutes. Calcium oxalate forms a dense white precipitate at the bottom of the test tube. Supernatant is decanted. The precipitate is washed by adding 4 ml of 2% ammonium hydroxide solution in the test tube. It is again centrifuged for 5 minutes. Pour solution from the sediment and add into a test tube 2 ml of 1 n. sulphuric acid. Stir the sediment with a glass stick. Take another test tube and pour 2 ml of 1 n. sulphuric acid (control sample). Immerse test tubes for 1-2 minutes in boiling water bath. The hot solution is titrated from microburette with 0.01 n. of potassium permanganate solution until a pale pink color, disappearing within minutes. The amount of permanganate that went on the titration of control sample is subtracted from consumed amount of potassium permanganate in the test sample.

Example of calculation: 1 ml of 0.01 n. potassium permanganate solution is equivalent to 0.0499 mmol of calcium. For titration of serum 0.54 ml of potassium permanganate was spent; for the titration of control experiment – 0.05 ml of potassium permanganate, hence the amount of calcium in serum is:

(0.54-0.05)*0.04990*100 = 2.044 mmol/l.