1. EXPERIMENT SIX 1. Influence of Adrenalin and Insulin on Glucose Concentration in Serum 2. Determination of Glucose in Urine 2012-09

2. Change of Carbohydrate Metabolism Hyperglycemia catecholamine glucagon growth hormone corticosteroid Insulin Gluconeogenesis Glycogenolysis Blood glucose If exceeds renal threshold of glucose (8.9 mmol/L) Glucosuria Stress hyperglycemia Stress glucosuria 2

3. BGBG food Digestion and absorption Glycogen degradation Nonsaccharide gluconeogenesis Oxidation and decompo sition CO 2 + H 2 O Hepatin synthesis liver (muscle)glycogen PPP et al Other saccharides Fat, AA anabolism Fat, amino acids Relative balance of blood sugar source and its outlet 3

4. Regulation of blood glucose 1. Hormone regulation ： glycemic hormone----adrenalin ， glucagon ， glucocorticoid hypoglycemic hormone----insulin 2. Organ regulation ： liver ， kidney ， muscle, et al 3. Integrative regulation ： Nerve, humoral composition commonly participate in the process 4

5. Insulin ① to accelerate glucose transfer into Extrahepatic cells ② to accelerate glycogen synthesis, inhibition of glycogenolysis ③ to accelerate the aerobic oxidation of glucose ④ to inhibit hepatic gluconeogenesis ⑤ to reduce fat mobilization —— only hypoglycemic hormone in vivo The mechanism of insulin action: 5

6. Adrenalin ——a hormone to elevate strongly blood sugar Mechanism of adrenalin action To accelerate glycogen degradation resulted from the cascade activation of phosphorylases through the continuous actions of accepters on hepatic or muscle cell membrane, cAMP, protein kinases Mainly exert effect under stress state 6

7. AC （ inactive ） AC （ active ） Adrenalin, glucagon + acceptor ATPcAMP PKA (inactive) Phosphorylase b kinase Glycogen synthase Glycogen synthase-P PKA (active) Phosphorylase b Phosphorylase a-P Phosphorylase b kinase -P Pi Phosphoprotein phosphatase -1 Pi Phosphoprotein phophatase-1 Phosphoprotein phosphatase-1 – – – Phosphoprotein phosphatase inhibitor-P Phosphoprotein phosphatase inhibitor PKA （ active ） 7 The regulation action of adrenalin AC----adenylate cyclase

8. Aims To understand the actions of adrenalin and insulin on the regulation of glucose metabolism, respectively To know how to measure the glucose concentration in serum and its significances To learn the method about the determination of glucose in urine 8

9. 【 Method 】 To select two rabbits as experiment animals To give the rabbit adrenalin or insulin by subcutaneous injection respectively, To collect the vein blood and separate the serum before or after injection, respectively To measure the concentration of blood glucose in serum, and compare the change of blood glucose level before or after injection of hormone To confirm the effect of adrenalin and insulin on blood glucose level 9

10. 【 Principle 】 Glucose is oxidized by glucose oxidase to form gluconic acid and peroxide of hydrogen (H 2 O 2 ), and then H 2 O 2 react with phenol and 4-aminoantipyene to form a red complex. The glucose concentration can be calculated by measuring absorbance of the red complex at 505nm. 10

11. 【 Principle 】 The whole reactions are showed following: 11

12. 【 Protocol 】 1 ． Select two rabbits, weigh, register the weight 2 ． Pumping and collecting vein blood before injection of hormone 3 ． Injecting hormone insulin ： subcutaneous injection ， dosage 0.75U/kg body weight ； adrenalin ： subcutaneous injection ， dosage 0.2mg/kg body weight register the injection time, respectively 4 ． Pumping and collecting vein blood again after injection of hormone 30 min 5 ． Separate serum and measure blood glucose concentration, respectively 12

13. 13

14. 【 Protocol 】 Take out 3 tubes, label and add reagents as following table: (ml) ReagentSampleStandardBlank Serum0.02 －－ Standard of glucose (5.55mmol/L) － 0.02 － Mixture solution of enzyme and phenol 3.0 A 505 Mixing, place at 37 ℃ water bath for 20 min, take the tubes out, cool down to room temperature, and measure A 505. 14

15. 【 Data record 】 Items No.1 rabbit (insulin)No.2 rabbit (adrenalin ) Before injection After injection Before injection After injection Standard A 505 Sample A 505 BS (mmol/L) Difference before and after injection Percentage change in blood glucose (%) 15

16. 【 Notice 】 After pumping blood from the rabbit injected with insulin, immediately inject 10 ml of 250g/L glucose solution by Intra-abdominal or subcutaneous injection, to prevent the rabbit sufferring Low blood sugar shock (insulin shock ) 【 Discussion 】 1 ． Whether are there any other way to prevent the rabbit suffering low blood sugar shock ？ 2 ． To observe the symptoms of the rabbit injected with adrenalin after the injection. 16

17. 2.Determination of Glucose in Urine 【 Principle 】 Glucose + Cu 2+ Cu 2 O OH - Brick-red precipitate 17

18. 【 Protocol 】 1 ． Add 1ml of urine sugar reagent into a test tube, heating to boiling 2 ． Add 3~4 drops of clear urine, heating to boiling again 3 ． Observe the phenomenon, and evaluate the result 18

19. 【 Result 】 Phenomenon Judgement No colour change （ no sugar or infinitesimal in urine ) － Display green, but no precipitation （ sugar about 1g/L ） Tiny Display a little of green precipitation ( sugar about 1~5g/L ） + Boiling 1min and present a little yellow green precipitation (sugar about 5~14g/L) ++ Boiling 10~15s immediately present yellowish brown precipitation (sugar about 15~20g/L) +++ Boiling and immediately display yellow precipitation and turn to be brick-red precipitation (sugar about >20g/L ） ++++ 19

20. 【 Notice 】 1 ． If the patient took slather reducing agent ( drugs), such as streptomycin or vitamin C, false positive result maybe display. It should be measured after to stop administration of medicine a few days later 2 ． The protein in urine would interfere in the reaction. It can be done after boiling urine in acid condition and filtrating it 3 ． Uric acid salt would interfere in the reaction because of its weak reducing. Remain the urine in refrigerator until precipitation of uric acid and take the top layer clean solution for the assay. 20

21. 【 Normal reference extent 】 In normal body urine, glucose tiny (less than 0.1~0.3 gram per day, the result should be negative ( － ). 【 Clinical significance 】 Pathological sugar urine: Mainly seen in urine whose patient suffers diabetes. In addition, hyperthyroidism, certain kidney disease, intracranial pressure, cerebral concussion, etc. sugar could also be seen in the patient urine. 21

22. 【 Practice and Thinking 】 1. What is the significance to assay the sugar in urine for the diagnosis, treatment of diabetes? 2.When urine test for sugar is negative, is it means that the diabetes has been cured? 22