Experimental determination of pharmacokinetic parameters of sulfadiazine sodium in normal and renal failure rabbits
The purpose of this experiment is to master the replication method of acute renal failure rabbit model. To master the surgical procedure of arterial cannulation and bladder cannulation in rabbits. Familiarize with the method of determining endogenous creatinine clearance. Familiarize yourself with the method of determining the blood concentration of sulfonamides. Understand the calculation of pharmacokinetic parameters and further understand their practical significance by comparing the differences in pharmacokinetic parameters between normal and renal failure rabbits.
Operation method
coupling method
Principle
Sulfonamides in the blood can react with certain reagents to produce colored substances, and the sulfonamide blood concentration can be quantified by colorimetry. The plasma half-life and other pharmacokinetic parameters are calculated according to the changing pattern of blood concentration at different times after administration. Reproduce the animal model of acute renal failure by injecting mercuric chloride, and compare the difference of endogenous creatinine clearance and pharmacokinetic parameters between normal and renal failure rabbits, so as to achieve the purpose of integrating the knowledge of physiology, pharmacology and pathophysiology organically. The reaction of iodopamine to generate colored substances is as follows: I. Diazotization: sulfanilamide reacts with nitrous acid to generate diazonium salts, because nitrous acid is easy to decompose, there is no ready-made preparation, so the experiment uses sodium nitrite, so that it reacts with hydrochloric acid to generate nitrous acid, and sulfanilamide must first be reacted with hydrochloric acid, so that the amino group on the nucleus of the phenyl nucleus (-NH2) is ionized to generate ammonium (-NH3+) compounds in order to have a diazotization reaction with nitrous acid. diazotization reaction with nitrous acid. Specific reactions are as follows: 1. NaNO2+HCl→HNO2+NaCl coupling reaction: diazobenzenesulfonamide hydrochloride and muscimol in alkaline solution undergo a coupling reaction to produce an orange-colored azo compound.
Materials and Instruments
Rabbit Move I. Replicating the renal failure model Caveat 1. The amount of reagents such as serum and standard solution should be accurate. 2. Boiling and cooling time should be accurate. Otherwise, the color reaction will fade. 3. the required reagents should be prepared within two weeks before use, after which the color of picric acid will deepen and the optical density value will increase. For more product details, please visit Aladdin Scientific website.
Sodium sulfadiazine Sodium trichloroacetic acid Sodium nitrite Sodium muscimol Sodium hydroxide Sodium hydroxide Sodium sulfadiazine Sodium heparin Mercury chloride Sodium sulphadiazine Sodium heparin Sodium chloride Sodium uratan Sodium chloride Sodium procaine Sodium heparin solution Creatinine standard solution Picric acid Sodium hydroxide Sodium carbonate Sodium hydrogen carbonate
Test tubes Suction tubes Samplers Syringes Blood cups Pipette balls Cotton balls Model 722 Spectrophotometer Centrifuges Constant temperature baths Centrifuge tubes Surgical instruments
One day before the experiment (18-20 h), two rabbits (in good condition) were weighed and one rabbit was injected subcutaneously with 1% HgCl2 solution at 1.2 ml/kg body weight to create an animal model of acute renal failure; the other rabbit was injected with an equal amount of saline solution at the same site to serve as a normal control.
Urine collection
1. Glucose injection: The above two rabbits were weighed and anesthetized by intraperitoneal injection of 5 ml/kg of urethane. The rabbits were fixed on the rabbit table, and 15% glucose solution was injected intravenously into the ear margin at 15 ml/kg body weight (finished within 5 min) to ensure sufficient urine output.
2. Bladder intubation: The abdomen is clipped and a mid-abdominal incision is made at l.5 cm above the pubic symphysis, about 4 cm long. The subcutaneous tissue is separated. The peritoneum is incised along the white line of the abdomen, the bladder is located, and a bladder cannula is inserted. The remaining urine is emptied first, and then the urine is collected for one hour and converted to urine output per minute.
Collecting blood samples
1. Carotid artery intubation: fix the rabbit on the rabbit table in the dorsal position, cut off the hair on the front of the neck, and incise the skin of the neck along the median line from the thyroid cartilage downward for about 5 centimeters. The subcutaneous tissue was bluntly peeled off with vascular forceps, the muscles were separated, and the common carotid artery located on the side of the trachea was freed and separated from the surrounding tissues and the accompanying nerves. To prevent coagulation in the arterial cannula, 1% heparin is injected into the ear vein at this point in the procedure, and the cephalic end of the common carotid artery is ligated and the cardiac end is clamped with an arterial clip. An imaginary knot is made in the middle of the two, and a heparin-filled tee tube is inserted into the carotid artery by making a small cut downward with small ophthalmic scissors proximal to the cephalic ligature. The ligature is tied and secured.
2. Normal blood collection: About 0.5 ml of blood should be drained from the tee tube and shaken well for pharmacokinetic parameters; about 3 ml of blood should be taken for creatinine clearance.
3. Rapidly inject 1 ml/kg of 20% sodium sulfadiazine into a vein on one ear margin of the rabbit.
4. At the 1st, 3rd, 5th, 15th, 30th, 60th, 90th and 120th minutes after administration, collect about 0.5 ml of blood from a three-way tube connected to the carotid artery and shake it well (the residual blood in the arterial cannula should be drained off first).
Measurement of endogenous creatinine clearance
Blood and urine creatinine measurement (see Table 5-2 below) and calculation of endogenous creatinine clearance. Dilute the urine sample: After mixing the urine thoroughly, take out 1 ml of urine and dilute it 100 times with distilled water for the determination of creatinine content in urine.
Centrifuge the blood sample: put 3 ml of blood into the centrifuge, 3000 r/min, centrifuge for 20 minutes, take out the plasma for spare. Add the drug according to the table below: 
4. Mix well, put in 37 ℃ water bath for 20 min, and then put into the cold water basin and turn for 1 min to cool. At 520 nm wavelength each with its corresponding blank tube zero, colorimetric determination of the standard tube and the optical density of the tube.
5. Calculate the creatinine level in blood:
6. Notes
(1) The amount of serum, standard solution and other reagents should be accurate.
(2) Boiling and cooling time should be accurate. Otherwise the color reaction fades.
(3) The required reagents should be prepared within two weeks before use, after which the color of picric acid will deepen and the optical density value will increase. Affect the test results. At this time, the need to do reagent blank is more positive. The method is as follows (Table 5-3): 
The optical density of tube B was determined using B0 as the blank. The optical density of a freshly prepared test blank is around 0.01. Calculate according to the following formula: 
(4) The 0.23 in the formula is the protein interference coefficient for plasma protein content in the normal range; if the plasma protein content is too high or too low, the traditional protein-free filtrate assay is appropriate.
(5) Picric acid is explosive. When preparing it, a little distilled water should be added to the container first to prevent accidents.
V. Determination of pharmacokinetic parameters of sulfadiazine sodium
1. Deproteinization: accurately aspirate 0.2 ml of each blood sample, add 1.8 ml of distilled water (1.6 ml of standard tube), 4.0 ml of 5% trichloroacetic acid, shake well and then put the liquid into the centrifuge tube for centrifugation: 1000 r/min, 3 minutes.
2. Diazotization and coupling: Pipette 2.0 ml of supernatant after centrifugation at different times into the corresponding test tubes, and add the following reagents to each test tube in turn:
2N hydrochloric acid 0.5 ml.
0.5 ml of 0.5% sodium nitrite.
0.5% muscimol 1.0 ml, shake well.
3. Handling of standard tubes: Unlike other sample tubes, it is necessary to accurately add 30mg% sodium sulfadiazine 0.2 ml to ensure the accuracy of the calculation results.
4. Colorimetry: Place the liquid in each tube into a 0.5 cm colorimetric cup of the 722 spectrophotometer, and compare the color at a wavelength of 480 nm. Adjust the zero point with the sample tube before the administration of the drug, and then measure the optical density value of each sample tube. Recording: 
6. Calculations:
(1) If all other conditions (wavelength, colorimetric cup, absorption coefficient, dilution of solution, etc.) are the same, the concentration of the same solution is directly proportional to the optical density, so the following formula can be used to find the concentration of sulfonamide blood (mg%) in the sample tube. 
(2) Plotting: Convert the above calculated blood concentration at each time into logarithmic concentration and plot the time on grid paper to find the inflection point between the elimination phase and the distribution phase.
(3) Calculate the pharmacokinetic parameters of sodium sulfadiazine by the residual method.
(1) Use a calculator to carry out linear regression on each time of the elimination phase and the corresponding blood concentration after the inflection point, and find out the intercept a and slope b of the straight line; then find out B=10a, β=-2.303b (if it is an atrial chamber, β will be K); according to t1/2 =0.693/β,find out the half-life of the elimination phase, tl/2; and finally, write the equation of the elimination phase of the elapsed time, Ct=B-e -βt .
(ii) Calculate the blood concentration at each time from Ct=B-e -βt.
(iii) Calculate the blood concentration of the distribution phase: Subtract the blood concentration of the elimination phase at the corresponding time from the actual blood concentration measured before the inflection point, and the difference is the blood concentration of the distribution phase. The logarithm of this concentration is used to perform a linear regression on time.
A linear regression of the logarithm of the concentration on time yields logA, α, which is then used to further solve the equation for A, tl/2, and the distribution phase C t = Ae-αt.
Write the total time equation for sodium sulfadiazine in the body: C = Ae-αt + B-e-βt. Calculate the theoretical blood concentration at each time according to this equation, and compare the difference with the measured value.
⑤ Calculate the kinetic parameters of sodium sulfadiazine in rabbits according to the pharmacokinetic equation. 