Experimental determination of plasma half-life and apparent volume of distribution of drugs
In pharmacology experiments, there are many factors affecting the experimental results, some of which can be controlled, while some are difficult to control. In order to reduce the influence of multi-factors and difficult-to-control factors, a control group should be set up at the same time in the experiments, and comparisons between the control group and the experimental group should be used to eliminate the influences of various extraneous factors, so that the experimental results error can be minimized as much as possible, and to achieve the purpose of correctly evaluating the effect of the drug. Observe the changes of plasma drug concentration in different time after intravenous injection of phenol red. Master the method of blood concentration determination, plasma half-life (t1/2) and the calculation of apparent volume of distribution (Vd).
Operation method
phenol red indicator method
Principle
Phenol red (PSP) is an indicator that appears purplish-red in alkaline environments.PSP is not metabolized in the body after intravenous injection, and its elimination is mainly by secretion from the proximal tubules of the kidneys. PSP is not metabolized in the body after intravenous injection, and its elimination is mainly secreted by the proximal tubules of the kidney. Therefore, the optical density of PSP in plasma at different times of administration can be measured colorimetrically to quantitatively calculate the concentration of PSP in blood.
Materials and Instruments
Rabbit Move 1. Anesthesia and fixation: A healthy rabbit was taken, weighed, anesthetized with 3% pentobarbital sodium 1 ml /kg intravenously at the ear margin, and fixed in the supine position on the rabbit table. Caveat 1. when separating the common carotid artery, separate the nerves and do not ligate the nerves when ligating. 2. be careful not to remove blood when removing supernatant with a fluid extractor. 3. When centrifuging, balance the blood tubes and place them symmetrically. 4. do not touch the light side of the cuvette with your hands. if there is liquid, use only mirror paper. For more product details, please visit Aladdin Scientific website.
Phenol red solution Dilution solution Sodium hydroxide solution 75% alcohol Sodium heparin Saline
Spectrophotometer Centrifuge Rabbit table Rabbit fixation cord Surgical instruments Silk thread Injection Needles Anticoagulation tubes Centrifuge tubes Artery clips Mirror paper Filter paper Batch sampler
2. Surgery: Longitudinal incision of the skin in the middle of the neck, separation of the subneck tissues, finding the trachea, separating the common carotid artery on the side of the trachea, and threading two wires under the common carotid artery for use. Intravenous injection of sodium heparin 1 ml /kg from the ear margin, ligation of the distal end, the proximal end with an arterial clip, leaving a distance of 2 cm between the two ends, with ophthalmic scissors in the distal end of the proximal end of the proximal end of the direction of a 45-degree angle to cut a small opening, and then inserted in the direction of the heart of the plastic tube, advancing the ligation of 1 ~ 2 cm and fixed, the plastic tube with hemostatic forceps clamped, loosened arterial clips.
3. Take blank blood: take about 1 ml of blood from the arterial catheter, put it into a numbered dry anticoagulated test tube, shake gently and mix well.
4. Administration and blood sampling: 6 ml/kg of 0.6% phenol red solution was injected intravenously from the ear margin, and about 1 ml of blood was taken at 2, 5, 10, 20 and 30 minutes after administration and placed in a numbered dry anticoagulant test tube and shaken gently to mix.
5. Centrifugation and determination: Each quantitative blood sample was taken in a numbered centrifuge tube, centrifuged at 3000 rpm for 10 minutes, 0.1 ml of plasma was taken, added to a clean numbered tube, 2 ml of diluent was added, shaken well and left to stand for 5 minutes, and then the blood samples were used as blanks for zeroing before the administration of the drug, and then colorimetric determination was carried out at the wavelength of 560 nm of a spectrophotometer, and the value of its optical density was recorded. The optical densities at different times after administration were entered into a calculator to calculate the plasma concentration of phenol red at different times.
6. make a standard curve of phenol red solution 1. 2. 4. 8. 16 umol-L-1 (the teacher takes the lead in drawing)
Experimental results and analysis:
The standard curve was plotted with concentration as the horizontal coordinate and optical density as the vertical coordinate, and the regression equation.
Time-volume curves were plotted with the logarithmic value of plasma drug concentration as the vertical coordinate and time as the horizontal coordinate, and the time-volume relationship of the first-level elimination kinetics was linear.
The equation for this straight line is logCt=lgCO-k/2.303-t
Drug plasma half-life T1/2 = 0.693/k (in h or min)
Vd=A/C0 (A is the total amount of drug administered)
The plasma concentration of phenol red at different times was calculated using the FX-3600P calculator, using a linear regression arithmetic program.
Blood Sample Drug Concentration Record Sheet 