EXPERIMENT SIX

1. Influence of Adrenalin and Insulin on Glucose Concentration in Serum

2. Determination of Glucose in Urine

2012-09

Change of Carbohydrate Metabolism

Hyperglycemia catecholamine

glucagon growth hormone corticosteroid

Insulin

GluconeogenesisGlycogenolysis

Blood glucoseIf exceeds renal thre

shold of glucose (8.9 mmol/L)

GlucosuriaStress hyperglycemia Str

ess glucosuria2

B

G

food Digestion and absorption

Glycogen degradation

Nonsaccharide

gluconeogenesis

Oxidation and decomposition

CO2 + H2O

Hepatin synthesis liver (muscle)glycogen

PPP et alOther saccharides

Fat, AA anabolism

Fat, amino acids

Relative balance of blood sugar source and its outlet

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Regulation of blood glucose

1. Hormone regulation ： glycemic hormone----adrenalin ， glucagon ，

glucocorticoid hypoglycemic hormone----insulin2. Organ regulation ： liver ， kidney ， muscle,

et al3. Integrative regulation ： Nerve, humoral co

mposition commonly participate in the process

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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:

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Adrenalin ——a hormone to elevate strongly blood sugar

Mechanism of adrenalin action

To accelerate glycogen degradation result

ed from the cascade activation of phosphoryla

ses through the continuous actions of accepte

rs on hepatic or muscle cell membrane, cAMP,

protein kinases

Mainly exert effect under stress state

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AC（ inactive ）

AC （ active ）

Adrenalin, glucagon + acceptor

ATP cAMP

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 Pi Phosphoproteinphophatase-1

Phosphoproteinphosphatase-1

–

–

–Phosphoprotein phosphatase inhibitor-P

Phosphoprotein phosphatase inhibitor

PKA （ active ）

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The regulation action of adrenalin

AC----adenylate cyclase

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

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【 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

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【 Principle 】

Glucose is oxidized by glucose oxidase to form gluconic acid and peroxide of hydrogen (H2O2), and then H2O2 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.

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【 Principle 】

The whole reactions are showed following:

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【 Protocol 】1 ． Select two rabbits, weigh, register the weight2 ． Pumping and collecting vein blood before inject

ion of hormone 3 ． Injecting hormone insulin ： subcutaneous injection ， dosage 0.75

U/kg body weight ； adrenalin ： subcutaneous injection ， dosage 0.

2mg/kg body weight register the injection time, respectively4 ． Pumping and collecting vein blood again after i

njection of hormone 30 min 5 ． Separate serum and measure blood glucose c

oncentration, respectively

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【 Protocol 】Take out 3 tubes, label and add reagents as followi

ng table: (ml)

Reagent Sample Standard Blank

Serum 0.02 － －Standard of glucose (5.55mmol/L)

－ 0.02 －

Mixture solution of enzyme and phenol

3.0 3.0 3.0

A505

Mixing, place at 37 water bath for 20 min, take the tubes ℃out, cool down to room temperature, and measure A505.

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【 Data record 】

Items No.1 rabbit (insulin) No.2 rabbit (adrenalin )

Before injection

After injection

Before injection

After injection

Standard A505

Sample A505

BS (mmol/L)

Difference before and after injection

Percentage change in blood glucose (%)

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【 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 preve

nt the rabbit suffering low blood sugar shock ？2 ． To observe the symptoms of the rabbit inj

ected with adrenalin after the injection.

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2.Determination of Glucose in Urine

【 Principle 】

Glucose + Cu2+ Cu2OOH-

Brick-red precipitate

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【 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

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【 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 ）

++++

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【 Notice 】1 ． If the patient took slather reducing agent ( dru

gs), 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.

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【 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.

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【 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?

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