Evaluation of diuretic activity

Expt No- 3 Evaluation of diuretic activity I Sem M.Ph 27-8-2019, PESCP, PESU Page 1
Exp No- 3 Date- 27-8-2019
Evaluation of diuretic activity
Aim: To evaluate the effect of diuretics (frusemide, torsemide and metolazone) on the
output of urine in rats.
Requirements: Rats, metabolic cages, graduated measuring cylinder, oral feeding
cannula.
Drugs and solutions: Normal saline, frusemide, torsemide, metolazone.
Theory: Diuretics are compounds which increase the flow of urine. Loop diuretics act on
the Na+-K+-2Cl− symporter (co-transporter) in the thick ascending limb of the loop of
Henley to inhibit sodium, chloride and potassium reabsorption. Furosemide or
frusemide (Lasix) is a potent loop diuretic used in the treatment of oedematous states
associated with cardiac, renal and hepatic failure, and for the treatment of
hypertension. Torsemide is another potent loop diuretic.
The members of thiazide class of diuretics are derived from benzothiadiazine. They
control hypertension in part by inhibiting reabsorption of sodium (Na+) and chloride (Cl−)
ions from the distal convoluted tubules in the kidneys by blocking the thiazide-sensitive
Na+-Cl− symporter. E.g. Hydrochlorthiazide, metolazone.
Normal urine output in rats is very small (1-2ml/rat/day). Hence to get measurable
quantity the animals are first hydrated. The urine output is increased after the
administration of diuretics like urea and frusemide, etc. Increase in volume of urine is
measured with the help of measuring cylinder and compared with the normal urine
output.
Procedure- Albino rats (150-200g) are fasted (deprived of food and water) overnight
and normal saline (25ml/kg po) was administered orally with the help of oral gavaging
needle. These were divided into four groups of 6 animals each. To the group I only
normal saline was administered. To the group II along with normal saline, furosemide
(10mg/kg ip) administered parenterally. To the group III along with normal saline,
torsemide (10mg/kg po) was also administered. To the group IV along with normal
saline, metolazone (2mg/kg ip) administered. After the administration of drugs, animals

were placed in four different metabolic cages. Urine was collected in a measuring
cylinder. Time, when the first drop of urine was collected in a cylinder for each group is
noted down and the volume was recorded at intervals of 30 min for 3 h.
Observation table:
Preparation of drug solutions-
1.Normal saline- 900 mg NaCl dissolved in 100 ml water
2.Frusemide- Each ampoule contains- 10 mg/ml. stock solution-10mg/ml
3.Torsemide- Each tab contains-10mg torsemide. stock solution- 2 tab dissolved in 20 ml
water- 10mg/ml.
4. Metolazone- Each tab contains- 2.5 mg metolazone. Stock solution- Each tab
dissolved in 2.5 ml water- 1mg/ml solution.
Group I (NS) Dose- 25ml/kg BW po
SN Markings Wt in
gm
NS
in ml
Collected urine in ml
30min 60min 90min 120min
1 H 137 3.42 0.2 0.5 0.7 0.9
2 B 137 3.42 0.3 0.5 0.8 1.0
3 HB 138 3.45 0.2 0.4 0.7 0.8
4 TT 140 3.50 0.1 0.3 0.6 0.9
5 TB 141 3.52 0.2 0.5 0.7 1.0
6 UM 139 3.47 0.3 0.5 0.8 1.0
SD±MEAN 0.21±0.075 0.45±0.083 0.71±0.075 0.93±0.081

Group II (NS) Dose- 25ml/kg po; Furosemide (10mg/kg bw ip) Stock sol 10 mg/ml
(Ampoule -> 20mg/2ml)
SN Markings Wt in
gm
NS in
ml
Furosemide Collected urine in ml
mg Ml 30min 60min 90min 120min
1 H 167 4.17 1.67 0.16 1.6 3.8 4.6 4.8
2 B 120 3.00 1.20 0.12 1.5 3.2 4.2 4.2
3 HB 145 3.62 1.45 0.14 1.5 3.7 4.5 4.8
4 TT 138 3.45 1.38 0.13 1.5 3.3 4.4 4.6
5 TB 150 3.75 1.50 0.15 1.5 3.4 4.2 4.7
6 UM 160 4.00 1.60 0.16 1.3 3.2 4.3 4.2
SD±MEAN 1.48±0.098 3.43±0.258 4.36±0.163 4.55±0.28
Group III (NS) Dose- 25ml/kg po + Torsemide (10mg/kg bw po) Stock Soln. Stock-
10mg/ml Each tab- 10mg (2 tab dissolved in 2ml)
SN Markings Wt gm NS
ml
Torsemide Collected urine in ml
mg Ml 30min 60min 90min 120min
1 H 129 3.22 1.29 0.13 4.4 7.5 8.8 9.2
2 B 125 3.12 1.25 0.12 4.2 6.5 8.2 8.4
3 HB 130 3.25 1.30 0.13 4.3 7.4 8.7 9.1
4 TT 126 3.15 1.26 0.12 4.2 6.6 8.3 8.5
5 TB 130 3.25 1.30 0.13 4.3 7.4 8.4 8.6
6 UM 140 3.50 1.40 0.14 4.2 6.6 8.5 8.7
SD±MEAN 4.26±0.081 7±0.477 8.48±0.231 8.75±0.327

Group IV (NS) Dose- 25ml/kg po + Metolazone (2mg/kg bw ip) Stock soln: 1 mg/ml
Each tab- 2.5 mg (one tab dissolved in 2.5 ml  1 mg/ml)
SN Markings Wt
gm
NS
ml
Metolazone Collected urine in ml
mg ml 30min 60min 90min 120min
1 H 131 3.27 0.26 0.26 0.4 1.2 1.3 1.5
2 B 140 3.50 0.28 0.28 0.6 1.4 1.5 1.7
3 HB 135 3.25 0.27 0.27 0.4 1.3 1.3 1.6
4 TT 146 3.65 0.29 0.29 0.5 1.3 1.3 1.5
5 TB 140 3.50 0.28 0.28 0.6 1.5 1.4 1.7
6 UM 135 3.37 0.27 0.27 0.5 1.4 1.5 1.6
SD±MEAN 0.5±0.089 1.35±0.104 1.38±0.098 1.6±0.089
Statistical analysis-
Statistical Analysis (One way ANOVA followed by Dunnett compare all column
versus control column) Collected urine in ml after 30 min.
Dunnett's Multiple
Comparison Test Mean Diff. q
Significant? P <
0.05? Summary 95% CI of diff
Normal vehicle vs
Frusemide -1.267 25.33 Yes *** -1.394 to -1.140
Normal vehicle vs
Torsemide -4.050 81.00 Yes *** -4.177 to -3.923
Normal vehicle vs
Metolazone -0.2833 5.667 Yes *** -0.4104 to -0.1563
30
M
in
0
1
2
3
4
5
Normal vehicle
Frusemide
Torsemide
Metolazone
MINUTES
collectedurineinml

Dunnett's Multiple
Comparison Test Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff
Normal vehicle vs
Frusemide -2.983 18.48 Yes *** -3.393 to -2.573
Normal vehicle vs
Torsemide -6.550 40.58 Yes *** -6.960 to -6.140
Normal vehicle vs
60
M
in
0
2
4
6
8
Normal vehicle
Frusemide
Torsemide
Metolazone
MINUTES
collectedurineinml
Dunnett's Multiple
Normal vehicle vs
Frusemide -3.650 40.88 Yes *** -3.877 to -3.423
Normal vehicle vs
Torsemide -7.767 86.99 Yes *** -7.993 to -7.540
Normal vehicle vs

90
M
in
0
2
4
6
8
10
Normal vehicle
Frusemide
Torsemide
Metolazone
MINUTES
collectedurineinml
Dunnett's Multiple
Normal vehicle vs
Frusemide -3.617 27.97 Yes *** -3.945 to -3.288
Normal vehicle vs
Torsemide -7.817 60.45 Yes *** -8.145 to -7.488
Normal vehicle vs
120
M
in
0
2
4
6
8
10
Normal vehicle
Frusemide
Torsemide
Metolazone
MINUTES
collectedurineinml

Gp Group Name Collected urine in ml Diuretic
IndexAfter 30min After 60min After 90min After 120min
I Normal 0.21±0.075 0.45±0.083 0.71±0.075 0.93±0.081 _____
II Furosemide 1.48±0.098*** 3.43±0.258*** 4.36±0.163*** 4.55±0.28*** 4.89±3.45
III Torsemide 4.26±0.081*** 7±0.477*** 8.48±0.231*** 8.75±0.327*** 9.40±4.03
IV Metolazone 0.5±0.089*** 1.35±0.104*** 1.38±0.098*** 1.6±0.089*** 1.72±1.09
Results
30
M
in
60
M
in
90
M
in
120
M
in
0
2
4
6
8
10
Normal vehicle
Frusemide
Torsemide
Metolazone
MINUTES
collectedurineinml
Results and Discussion:
Furosemide is a loop diuretic which acts by inhibiting the luminal Na+-K+-2Cl− symporter
(co-transporter) in the thick ascending limb in the loop of Henle, by binding to the
chloride transport channel, thus causing sodium, chloride, and potassium loss in urine.
Torsemide is a potent loop diuretic which acts by the same mechanism as that of
furosemide. Metolazone belongs to the class of thiazide diuretics which acts by
inhibiting the Na+-Cl− symporter in the distal convoluted tubule of the kidney.
On comparing the results of all the three diuretics, it was found that Torsemide shown
highest diuretic activity whose efficacy is followed by that of Furosemide and this is
followed by Metolazone which has shown the least diuretic activity among the three
drugs.

Evaluation of diuretic activity

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