Glucose testing methods Flashcards
Metabolism of glucose (4)
- Converted to glycogen via glycogenesis & stored ( mainly in liver & muscle )
- Metabolized to CO2, H2O & energy via Tricarboxylic acid cycle, electron transport chain & Oxidative Phosphorylation
- Converted to ketoacids, amino acids & proteins ( transmission)
- Converted to fat & stored in adipose tissue as triglycerides ( via acetyl CoA)
Glycolysis
Metabolism of glucose to pyruvate or lactate for production of energy ( important for muscle tissue )
Glycogenolysis
Breakdown of glycogen to glucose for energy
Glycogenesis
Conversion of glucose to glycogen for storage
- glucose can be used by the liver & other cells for energy or stored as glycogen for later use
Gluconeogeneisis
Formation of glucose from non carbohydrate sources
Ex. Conversion of amino acids by the liver
Lipogenesis
Conversion of carbohydrates to fatty acids
Lipolysis
Decomposition of fats
- if glucose supply is low the liver can use glycogen ( lasts 12-16hrs) & other substrates to raise blood glucose concentration
- glycerol from triglycerides
- lactic acid from skin & muscle
- amino acids
Hormonal control of glucose
Increase blood glucose
- glucagon
- Adrenalin ( Epinephrine)
- glucocorticoids ( cortisol & cortisone )
- growth hormone
- adrenocorticotropic hormone ( ACTH)
- thyroxine
- somatostatin
Decreases blood glucose
- insulin
Glucagon
Primary hormone responsible for increasing glucose
Produced by alpha cells of islets of Langerhans in the pancreas
Released during stress & fasting states
Promotes glycogenolysis & gluconeogensis
Epinephrine
Aka adrenaline
Produced by adrenal medulla
Increases glucose production by:
- inhibiting insulin secretion
- increasing glycogenolysis ( breakdown of glycogen)
- promoting lipolysis ( breakdown of fat )
Glucocorticoids
Primarily cortisol
Secreted by adrenal cortex when stimulated by ACTH
Increases plasma glucose by
- decreasing intestinal entry into cell
- increasing gluconeogenesis, liver glycogen & lipolysis
Growth hormone
Produced by anterior pituitary gland
Increases glucose production by:
- decreasing entry of glucose into the cells
- increasing glycolysis ( metabolism of glucose to pyruvate / lactate)
Adrenocorticotropic Hormone ( ACTH)
Produced by anterior pituitary gland
Increases release of cortisol
Increases plasma glucose by:
- converting liver glycogen to glucose
- promoting gluconeogenesis
Thyroxine
Produced by thyroid gland
Increases glucose production by :
- increasing glycogenolysis, gluconeogenesis & intestinal absorption of glucose
Somatosatin
Produced by the alpha- cells of the islets of Langerhans in the pancreas
Increases plasma glucose by :
- inhibiting insulin, glucagon & growth hormone
Insulin
Primary hormone responsible for decreasing glucose
Produced by beta - cells of islets of Langerhans
In the pancreas
Promotes the storage of glucose as glycogen
Promotes the formation of lipids from glucose
Increases the permeability of glucose to cell
Theory of insulin action
Beta cells detect an increase in glucose, they release insulin
The insulin causes glucose to move into the cells ( muscle & adipose tissue ) via transport receptors Pg.98
Sodium dependent glucose transporters
Promote uptake of glucose form lumen of the small bowel
Promote reabsorption of glucose from urine I’m the kidney
Facilitative glucose transporters ( GLUTs)
Located on the surface of all cells
Designated GLUT1- GLUT14
Eleven mediate glucose transport
Divided into 3 subfamilies
- class l ( GLUT1-4)
- class ll ( GLUT 5,7,9,11)
- class lll ( GLUT 6,8,10,12,14)
GLUT 1&3
GLUT 1& 3 provide the glucose requirements of the brain
GLUT 2 are found in
hepatocytes
B-cells of the pancreas
basolateral membranes of intimal & renal cells
allows the movement of glucose into & out of these cells
GLUT 4
catalyzes a rate-limiting step for glucose uptake and metabolism in skeletal muscle
-skeletal muscle is the major organ of glucose consumption
when insulin concentration in the blood are low, GLUT 4 is localized in intracellular compartments & is inactive
insulin is released after a meal & stimulate the translocation of GLUT 4 to the plasma membrane
-this promotes uptake of glucose into skeletal muscle & fat
measurement of blood glucose
serum, plasma, whole blood, urine, & CSF can be analyzed
serum & plasma should be separated from cells within 1 hour of collection
if delayed analysis, collect in sodium fluoride tube ( grey)
this will prevent glycolysis
fasting blood glucose tests should be performed in the morning after an 8 - 10 hour fast
- higher in the morning than the afternoon ( diurnal)
- diabetes may be missed in patients tested in afternoon
glucose results based on sample type
serum& plasma values will be higher than whole blood values
venous whole blood ( lowest)
capillary/arterial
serum/plasma ( highest )
whole blood concentrations are approx. 10-12% lower than plasma
methods for glucose testing
oxidation/reduction reaction methods
aromatic amines methods
enzymatic methods
- glucose oxidase
- hexokinase
electrode methods
oxidation/reduction reaction- Copper Reduction
Cupric copper is reduced to cuprous copper by a reducing sugar ( glucose )
used in urinalysis ( clinitest)
( blue) (red)
- cuprous copper + reducing sugar ———> cuprous copper
- cuprous copper + Phosphomolybdic acid———————————>phosphomolybdous acid ( blue )
blue color measured using spectrophotometer and is related ti the sugar concentration
recorded as 1+ 2+ 3+ 4+
positive for all reducing sugars
used for determining genetic disorders
-ex. galactosemia
urine dipstick neg ( no glucose in urine)
urine flintiest pos ( galactose detected )
glucose oxidase
most specific enzyme for glucose
only reacts with ß- D glucose
method:
glucose + O2—>Gluconic acid + H2O2
H2O2 + chromagen —> oxidized chromagen + H2O
chromagen is colorless until oxidized
conc of glucose is proportional to color
what are 2 step reaction called
Trinder Reactions
chromagens in glucose oxidase
chromogens are colourless chemical compounds that can be converted to a dye or color compound through a chemical reaction
o-dianisidine ( brown)
o-tolidine( blue)
4-aminophenizone( red) **
two other common chromogens:
3-methyl-2-benzothiazolinone hydrazone
N,N-Dimethylalaniine
alpha & beta glucose
& what converts one to the other
Blood glucose :
alpha-D glucose ( 36%)
beta -D glucose ( 64%)
glucose oxidase can only cause oxidation of the Beta for,
Mutarotase can be added to convert alpha to beta forms
extend incubation time will allow spontaneous conversion
interfering substances in glucose oxidase
in 2nd step other substances in the sample can be oxidized instead of the chromagen
- results in a decrease of color & a false decrease in glucose reading
other reducing substances :
uric acid
bilirubin
ascorbic acid ( vitamin C)
glutathione ( anti-oxidant)
a false increase in glucose conc. can be caused by strong oxidizing substances ( bleach )
glucose oxidase -CSF
CSF glucose is a STAT ( freeze if cant do ASAP & freeze after)
CSF levels will be ~60% of blood glucose levels ( plasma )
reference range : 2.2-3.9 mol/L
decreased :
- bacterial meningitis
- fungal infection
increased :
- hyperglycaemia
- traumatic tap
whats in the glucose reagent
BUFFER
NOT GLUCOSE
what is the reference method for glucose
Hexokinase
- more accurate than glucose oxidase
- less interferences ( not affected by uric acid & ascorbic acid )
- can be performed on serum, plasma , urine, CSF &serous fluid
what causes a false decrease in hexokinase results
grosss hemolysis & very high bilirubin
steps of hexokinase
Glucose + ATP –> glucose- 6-phosphate + ADP
G-6-P + NADP+ —-> 6- phosphogluconate + NADPH + H+
NADP+ no absorbance at 340 nm
NADPH absorbance at 340 nm
increase in absorbance is read @ 340 nm
enzymatic methods- Glucose dehydrogenase
Glucose + NAD+ —-> Gluconolactone + NAD + H+
increased absorbance at 340 nm( form NADH) is measured
NADH generated is proportional to glucose conc
not widely used
polarographic electrode methode ( oxygen electrode)
measures the rate of oxygen consumption ( depletion ) after sample is added to a solution containing glucose oxidase
glucose + O2 –> gluconic acid + H2O2
H2O2 needs to be eliminated through two side reactions with catalase & molybdate ( to prevent the reaction from reversing )
suitable for serum, plasma & CSF
NOT WHOLE BLOOD ( blood cells consume oxygen)
