Download experiment six

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
Glycogenolysis
Gluconeogenesis
Stress hyperglycemia
Stress glucosuria
Insulin
Blood glucose
If exceeds renal
threshold of glucose
(8.9 mmol/L)
Glucosuria
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Relative balance of blood sugar source
and its outlet
food
Oxidation
and
decompo
sition
Digestion
and
absorption
CO2 + H2O
Hepatin synthesis
degradation
Glycogen
B
G
liver (muscle)glycogen
PPP et al
Other saccharides
gluconeogenesis
Fat, AA anabolism
Nonsaccharide
Fat, amino acids
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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
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Insulin
—— only hypoglycemic hormone in vivo
The mechanism of insulin action:
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
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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
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The regulation action
of adrenalin
Adrenalin, glucagon + acceptor
AC
（inactive ）
AC----adenylate cyclase
AC（active）
ATP
cAMP
Pi
Phosphorylase b kinase
PKA
PKA
(inactive)
(active)
Phosphorylase b kinase -P
Glycogen synthase Glycogen synthase-P
Pi
Phosphorylase b
Phosphoprotein
phophatase-1
–
PKA（active）
Pi
Phosphoprotein
phosphatase -1
–
Phosphorylase a-P
Phosphoprotein
phosphatase-1
–
Phosphoprotein phosphatase
inhibitor-P
Phosphoprotein phosphatase
inhibitor
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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 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
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【Protocol】
Take out 3 tubes, label and add reagents as
following table:
(ml)
Reagent
Sample
Standard
Blank
Serum
Standard of glucose
(5.55mmol/L)
Mixture solution of
enzyme and phenol
A505
0.02
－
－
0.02
－
－
3.0
3.0
3.0
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)
Before
injection
After
injection
No.2 rabbit (adrenalin )
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 prevent
the rabbit suffering low blood sugar shock？
2．To observe the symptoms of the rabbit
injected with adrenalin after the injection.
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2.Determination of Glucose in Urine
【Principle】
Glucose + Cu2+
OH-
Cu2O
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
No colour change（no sugar or infinitesimal in urine )
Display green, but no precipitation （sugar about 1g/L）
Judgement
－
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
( 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.
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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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