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G-6-Phospate + NADP
----G6PD----> NADPH + H+ + 6phosphogluconate
GLUCOSE MEASUREMENT
Purpose:
1. Diagnose and manage diseases affecting
glucose metabolism
2. To monitor the effects of certain
medications
Methods of Detection:
Coupled peroxidase reaction
H2O2 + reduced chromogen
----peroxidase----> oxidized chromogen +
H2O
Amount of color produced by the oxidized
chromogen is directly proportional to
the amount of glucose.
Polarographic
Measures rate of O2 consumption
(directly proportional to the amount
of glucose)
LIPID MEASUREMENT
TRIGLYCERIDE
Major interference:
CHOLESTEROL
types of reaction:
a. Liebermann Burchardt Reactionuses sulfuric acid and acetic anhydride
to produce unstable green
cholestadienyl monosulfunic acid;
color stabilized by sodium sulphate
b. Salkowski or Zak Reaction- uses
sulfuric acid and ferric ions to produce
a stable red to red-violet
cholestadienyl disulfonic acid
Methods:
1. Modified Abell-Kendall method(standard reference method)
step 1: hydrolysis with alcoholic KOH
step 2: extraction with petroleum ether
step 3: colorimetric reaction using
Liebermann-Burchard reagent
2. Abell, Levy and Brodie Method- (CDC
reference method)
step 1: hydrolysis with alcoholic KOH
step 2: hexane extraction
2
B. Enzymatic method
1. Cholesterol oxidase method- most
common method; measures the
amount of hydrogen peroxide
Cholesterol: mg/dL to mmol is 0.02586
produced; color reaction is measured Triglyceride: mg/dL to mmol is 0.011
at 500nm; cholesterol oxidase also
Triglyceride Total
HDLreacts with other sterols
Cholesterol Cholest
erol
LIPOPROTEIN QUANTITATION
<1
Normal
<2
desirabl
<4
low
METHODS:
50
00 e
0
1. Ultracentrifugal methods150
Borderli
200
Borderli
>9 high
reference method
2. Electrophoretic methods
ne high ne high 0
3. Polyanion precipitation
199
239
methods
200 high
>2 high
- polyanions and divalent 40
cations are used to
499
separate unwanted
>5 Very
lipoprotein fractions
00 high
- Most commonly used
polyanions are heparin
sulfate, dextran sulfate,
phosphotungstate and
divalent cations such as
Ca2+, Mg2+ and Mn2+
HIGH-DENSITY LIPOPROTEIN
LDLCholesterol
<1 optimal
00
100 Near
optimal/abo
129 ve optimal
130 Borderline
high
159
160 high
189
>1 Very high
90
3. Calculate LDL cholesterol and VLDL
cholesterol as follows:
LDL = infranatant cholesterol HDL
VLDL= total cholesterol infranatant
cholesterol
If Ultracentrifugation is not available, do the
ff:
1. Measure TAG, total chol and HDL directly.
2. Use the Friedewald or the DeLong
equation to solve the other lipoprotein
fractions.
Friedewald equation:
VLDL-C in mg/dL=
Plasma TAG
5
(If TAG
Plasma TAG
2.175
Plasma TAG
6.5
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
Plasma TAG
2.825
blocks aldosterone
Hyponatremia
ATP III Classification of Lipids (NCEP)
Electrolytes
FUNTIONS:
volume and osmotic regulation (Na, Cl,
K)
myocardial rhythm and contractility
(K, Mg, Ca)
cofactors (Mg, Ca, Z)
regulation of ATPase Ion Pump (Mg)
acid-base balance (Bicarbonate, K, Cl)
neuromuscular excitability (K, Ca, Mg)
glycolysis (Mg, Phosphate)
Sodium
Aldosterone
CAUSES:
Overhydration - dilutional effects
Diuretics - icrease water excretion, not
reabsorbing Na
SIADH (Syndrome of Inappropriate
Anti-Diuretic Hormone) - cause by
tumors releasing ADHwater
retention
Adrenal failure - hypoaldosteronism
Diabetes Mellitus
Hypernatremia
CAUSES:
1. Dehydration
2. Diabetes Insipidus - characterized by
polyuria, low specific gravityinhibit
water intake
DETERMINATION OF SODIUM
SPECIMEN: serum, plasma and urine
ANTICOGULANT: lithum heparin, ammonium
heparin and lithium oxalate
METHOD: ISE (Ion selective Electrode)
Potassium
major INTRAcellularcation
REFERENCE VALUES: 3.5-5.2mmol/L
Hypokalemia
CAUSES:
Hyperkalemia
CAUSES:
1. Decreased renal excretion
2. Cellular shift - acidosis
3. Increased intake
4. Artifactual - errors in sample collection
(sample hemolysis)
DETERMINATION OF POTASSIUM
Chloride
ENZYMES
oxidation-reduction reaction
o (Ex. Lactate Dehydrogenase)
Transferases - they catalyze the transfer
of functional group
o (Ex. Amino or phosphate group)
Hydrolases - they catalyze the cleavage
of bonds by addition of water
o (Ex. Amylase)
Lyases - they catalyze the breaking of
various chemical bonds without
hydrolysis
o (Ex. Pyruvate Decarboxylase)
Isomerases - they catalyze the
arrangement of atoms within a molecule
o (Ex. Phosphohexose isomerase)
Ligases - they catalyze the formation of
bonds between the two molecules
o (Ex. Pyruvate carboxylase)
Enzyme Structure:
DETERMINATION OF CHLORIDE
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
Measurement of CK:
Electrophoresis (immunoinhibition)
method of choice
Mass Immunoassay most commonly
used; uses CONAN monoclonal antibody
specific
Temperature
pH
Substrate concentration
Isoenzymes:
CK Isoenzymes: (B-brain, M-muscle)
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
SUBSTRATE: p-nitrophenylphosphate
Measurement of ALP:
AST (Aspartate
Aminotransferase)/SGOT (Serum
Glutamic Oxaloacetic
transaminase) EC.2.6.1.1
Class: Transferase
Method:
Karmen Method
o pH 7.3-7.8
o uses an absorbance at 340nm
ALP Methods:
Bodansky
Shinowara
Jones
Reinhart
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
1.
2.
3.
4.
Metabolic Acidosis
Respiratory Acidosis
Metabolic Alkalosis
Respiratory Alkalosis
Acidemia /Acidosis
low pH
excessive amounts of acid in the
blood
Acids are produced naturally in the
body as a product of metabolism
and other specific body processes
Respiratory Acidosis
lowered pH
decreased respiratory rate
(hypoventilation)
Metabolic Acidosis
Arterial Puncture:
Alkalemia/Alkalosis
high pH
refers to the condition of excessive
bicarbonate ions (bases) in the
blood
Respiratory Alkalosis
increased pH
increased respiratory rate
(hyperventilation)
Acid-Base Regulation:
Metabolic Alkalosis
Blood pH
Blood Gas Analysis
-
Arterial pH = 7.40
Venous pH = 7.35
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
Pre-Analytical Considerations:
Measurements:
Spectophotometric (Cooximeter)
Measures the various hemoglobin
species
O2Hb, HHb, COHb, MetHb
2. Blood Gas Analyzers: pH, pCO2, and
pO2
Use electrodes
(macroelectrochemical or
microelectrochemical sensors)
pO2 amperometric (amount of
current flow is indication of the
oxygen present)
pH and pCO2 potentiometric
(change in voltage indicates the
activity of analyte)
pH measured by a glass
membrane sensitive to H+ is
placed around an internal Ag-AgCl
electrode (measuring electrode)
Reference electrode:
Calomel (Hg-HgCl) or AgAgCl half cell
pCO2 measured by modified pH
electrode, Severinghaus
electrode
Change in activity of H+ is
measured by pH electrode
HCO3 calculated value from the
HH equation
pO2 measured by a Clarke
electrode
Allens Test
Procedure:
1. Prepare the arterial blood gas syringe
according to establish procedures.
2. The needle should pierce the skin in an
angle at approximately 45-60 degrees.
3. The pulsations of blood into the syringe
confirm that it will fill by arterial pressure
alone.
4. If the plunger is pulled back and air is
aspirated, immediately withdraw the
syringe.
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES
7. Apply pressure on the puncture site for 25 minutes using a sterile gauze.
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ABAD, AMOSIN, ANGELES, BASCO, DE ASIS, ESTANDARTE, INUMERABLES