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Principle: glucose + ATP ----hexokinase---> G6P + ADP

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

The amount of NADPH is directly


proportional to the amount of glucose

Specimen: Venous plasma glucose


Preservative: Sodium fluoride
GLUCOSE METHODS
Chemical Methods:
1. Copper Reduction Tests
Folin-Wu
uses tungstic acid to remove
protein from serum
measure sugar (FBS)
Nelson-Somogyi
Uses Zn(OH)2

Measure true glucose (FBG)


Neocuproine
2. O-toluidine (Dubowski Reaction)
Chemical color reaction
Most sensitive but carcinogenic
Reaction yields a Schiff base that
has a stable green color (read at
630 nm)
Enzymatic Methods:
1. Glucose Oxidase
Principle: glucose + O2 + H2O ----Glucose
oxidase----->gluconic acid + H2O2

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)

2. Hexokinase most specific glucose


method; considered as the reference
method

CLINICAL TESTS FOR GLUCOSE


1. RBS (Random Blood Sugar)/ Casual Blood
Glucose Test
Measures blood glucose regardless of
when you last ate.

Not used to diagnose DM

2. 2-Hour Post Prandial Blood Sugar (2HPPS)


Measures blood glucose exactly 2
hours after you start eating a meal.

Not used to diagnose DM

3. FBS (Fasting Blood Sugar)


Measures blood glucose after you have
not eaten for at least 8 hours.

It is often the first test done to check for


prediabetes and diabetes.

4. OGTT (Oral Glucose Tolerance Test)


Requirements:
Patient should be ambulatory
Fasting of 8-14 hours
Unrestricted diet of 150g
carbohydrate/day for 3 days prior to
testing
No smoking and drinking alcohol prior to
testing
Glucose Load:
75g (WHO standard glucose load)
100g
1.75g of glucose/kg body weight (children)
5. IVGTT (Intravenous Glucose Tolerance
Test)
0.5g glucose/kg body weight (given within
3 minutes) administered intravenously.
Fasting sample is also required.
The first blood collection is after 5 minutes
of IV glucose

LIPID MEASUREMENT

TRIGLYCERIDE

Fasting for 12 hours.


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If lipemic serum will be frozen at -20C


for assay at a later date, the specimen
should be warmed at 37C and mixed
before test.
Either plasma or serum
Interferences:
Ascorbic acid
Hemolysis
Bilirubin
Measurement of TAG (whether by chemical
or enzymatic means) involves the following
generalized processes:
a. hydrolysis of TAG to free glycerol
and FFA
b. measurement of glycerol present
A, Chemical Nonenzymatic methods:
1. Van Handel & Zilversmith
(Colorimetric Method)- (+) blue
compound
2. Hantzsch Condensation
(Fluorometric method)- (method of
choice)
end product: Diacetyl Lutidine
compound
3. Modified Van Handel and
Zilversmith- (CDC reference method)
step 1: chloroform extraction
step 2: silicic acid chromatography
step 3: saponification (alkaline
hydrolysis of TAG)
step 4: Glycerol + NaIO4
Formaldehyde + Formic acid
step 5: color reaction with
chromotropic acid (+) pink end color
B. Enzymatic methods

Relatively specific, rapid and easy to


use

Major interference:

Glycerol normally present in


plasma in conc. <0.163 mmol/L =
14mg/dL TAG

The analyses are performed directly in


plasma or serum, and are not subject to
interference by phospholipids or glucose.
1. Glycerol kinase method

Involves hydrolysis of TAG to free FA


and glycerol followed by the
phosphorylation of glycerol to
glycerophosphate

CHOLESTEROL

A. Chemical Nonenzymatic methods


(colorimetric assay)
Principle: Dehydration and oxidation
of cholesterol to form a colored compound
General steps in chemical
nonenzymatic methods:
1. Extraction release the cholesterol
from the lipoproteins. Ensures removal
of protein interference
a. Extraction using Bloors reagent
(3:1 EtOH-ether)
b. Zeolite reaction
2. Saponification- done using KOH to
hydrolyze the cholesterol esters since
only free cholesterol will be measured
3. Purification- done using Digitonin to
precipitate free cholesterol and
remove errors due to nonspecific
chromogen interference
4. Color development- may proceed
with Liebermann Burchardt or
Salkowski reaction
2

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
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step 3: colorimetric reaction using


Liebermann-Burchard reagent

2. Ultracentrifuge the fasting plasma (no


chylomicrons) without adjusting its density
(floating layer contains VLDL) and measure
the cholesterol of the infranatant (contains
HDL and LDL).

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

A. Polyanion precipitation *separation


step is done manually
step 1: beta-lipoCHONs are
precipitated using polyanion-divalent cations
such as: heparin sulfate-Mn2+, dextran
sulfate-Mg2+, sodium phosphotungstateMg2+and heparin-Ca2+
step 2: centrifugation*
step 3: a cholesterol assay is
performed on the supernate, w/c gives the
HDL conc.
B. Homogenous assays *automated
- reagents are added to form a stable
complex with non-HDL lipoCHONs preventing
them from participating in the reaction. A
second reagent measures the cholesterol
conc.
LIPID PROFILE
Steps in determining the lipid profile:
1. Determine first TAG, total cholesterol and
HDL cholesterol by chemical means.

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

level is lower than 400 mg/dL)


VLDL-C in mmol/L=

Plasma TAG
2.175

LDL-C = total cholesterol HDL VLDL


De Long equation:
VLDL (in mg.dL) =

Plasma TAG
6.5

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VLDL (in mmol/L) =

Plasma TAG
2.825

LDL= total cholesterol HDL VLDL

SITE OF ACTION: DCT (Distal


Convoluted Tubule)

Atrial Natriuretic Factor

blocks aldosterone

Hyponatremia
ATP III Classification of Lipids (NCEP)

Electrolytes

ions capable of carrying an electric


charge
classified as ANIONS and CATIONS

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

major extracellular cation (90-95%)


major determinant of plasma
osmolality
plasma concentration of sodium is
dependent on water intake and
excretion
affected by aldosterone and ANF
(Atrial Natriuretic Factor)
REFERENCE VALUES: 135-145 mmol/L

Aldosterone

main sodium retaining hormone


increases sodium, decreases
potassium
main mineralocorticoid

occurs when the sodium level is below


the normal lower limit

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

occurs when the sodium level is above


the normal upper limit

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:

SITE OF PRODUCTION: Adrenal


medulla
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1. Gastrointestinal loss - (vomiting,


diarrhea, malabsorption, intestinal
loss)
2. Renal loss - (diuretics, RTA,
hyperaldosteronism
3. Cellular shift - alkalosis
4. Decreased intake

Hyperkalemia
CAUSES:
1. Decreased renal excretion
2. Cellular shift - acidosis
3. Increased intake
4. Artifactual - errors in sample collection
(sample hemolysis)
DETERMINATION OF POTASSIUM

Enzymes are Classified into 6 groups:

SPECIMEN: serum, plasma and urine


ANTICOGULANT: heparin
METHOD: ISE (Ion selective Electrode)

Chloride

major anion in the extracellular fluid


chief counterion of sodium
chloride disorders are often a result of
the same causes that disturb Na levels
because Cl passively follows Na.
REFERENCE VALUES: 98-107 mmol/L

SPECIMEN: serum or plasma

ANTICOGULANT: lithum heparin

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

METHOD: ISE (Ion selective Electrode)

Oxidoreductases - they catalyze

Active Site - is the region within an


enzyme that fits the shape of molecules
called substrate
Substrate - substance that binds to the
active site
Zymogen proenzyme - inactive
enzyme that converted to active enzyme
by another enzyme
Isoenzyme - enzymes which have given
specificity but different amino acid
sequences, thus exhibit different physical
properties
Haloenzyme - refers to the enzyme with
its cofactor
Apoenzyme - refers to the protein portion
of the holoenzyme. In the absence of the
appropriate cofactor, the apoenzyme
typically does not show biologic activity
Activator - a substance which modifies a
catalyzed reaction in a positive manner
Inhibitors - chemicals that reduce the
rate of enzymatic reactions

enzymes are proteins that functions as


biological catalyst and are neither
consumed nor permanently altered during
a chemical reaction
few are ribonucleic acids called ribozymes

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Two Theories have been proposed to explain the


specificity of enzyme action:

Lock and Key theory - The enzyme


active site is complementary in
conformation to the substrate, so that
enzyme and substrate recognize one
another.

CK2: CK-MB found in heart, skeletal,


fibers
CK3: CK-MM found in skeletal muscle

Macro-CK: largely comprises CK-BB complexed


with immunoglobulin; used to describe lipoprotein
complexes
CK-Mi (Mitochondrial CK): detected in cases of
extensive tissue damage; appears to be indicator
of severe illness

Measurement of CK:

Induced fit theory - The enzyme changes

shape on binding substrate, so that the


conformation of substrate and enzyme is
only complementary after binding.

Electrophoresis (immunoinhibition)
method of choice
Mass Immunoassay most commonly
used; uses CONAN monoclonal antibody
specific

Forward: Optimal pH 9.0

Tanzer & gilvarg


Pyruvate kinase-LDH-NADH

Reverse: Optimal pH 6.8


Factors affecting enzyme reaction:

Temperature
pH
Substrate concentration

CK (Creatine Kinase) E.C. 2.7.3.2


Class: Transferases

very sensitive indicator of ACUTE


MYOCARDIAL INFARCTION with highest
elevation seen in Duchenes Muscular
Dystrophy
Uses Magnesium as Cofactor

Oliver & Rosalki


Hexokinase-glucose-6-phosphate

ALP (Alkaline Phosphatase)


E.C.3.1.3.1
Class:

involve in the cleavage of phosphate


containing compound alkaline pH(9.010.0)
facilitate movement of substance across
cell membrane

Isoenzymes:
CK Isoenzymes: (B-brain, M-muscle)

CK1: CK-BB found predominantly in


brain and smooth muscle

Liver Isoenzyme (ALP-1)


Bone Isoenzyme (ALP-2)
Placental Isoenzyme
Intestinal Isoenzyme (AlP-3)

From fastest to slowest:

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L,B,P,I (Land Bank of the Philippine Island)

SUBSTRATE: p-nitrophenylphosphate

From most Heat-Stable to most Heat-Labile:

END PRODUCT: p-nitrophenol or


yellownitrophenoxide ion

P,I,L,B (Pangako, Ikaw Lang, Babe)

Measurement of ALP:

Electrophoresis - most useful single


technique for ALP isoenzyme analysis
Heat Stability - ALP is measured before
and after the serum at 56C for 10mins.

AST (Aspartate
Aminotransferase)/SGOT (Serum
Glutamic Oxaloacetic
transaminase) EC.2.6.1.1
Class: Transferase

ALP Isoenzyme in Cancer Patients:

Regan - Lung Cancer


Nagao - Adenocarcinoma
Kasahara - Hepatoma
Real Love Never Abandon, Keep Holding
on

widely distributed in tissue, highest in


cardiac tissue, liver, muscle
highest elevation of AST can be found in
Acute Viral Hepatitis

Most Heat-stable Isoenzyme: Regan Isoenzyme


2 Isoenzymes:
Selective Chemical Inhibition:

Phenylalanine - inhibits Intestinal and


placental ALP, Regan ALP, Nagao ALP
Levamisole Reagent inhibits Liver and
Bone ALP
3M Urea - inhibits Bone ALP
L-Leucine - inhibits Nagao ALP

Cell Cytoplasm (predominant from


occurring in serum)
Mitochondria

Method:

Karmen Method
o pH 7.3-7.8
o uses an absorbance at 340nm

ALP Methods:

Bodansky
Shinowara
Jones
Reinhart

ARTERIAL BLOOD GAS ANALYSIS


Acid-Base Equation:

CO2 + H2O H2CO3 HCO3- + H+


SUBSTRATE: Beta-glycerophosphate
END PRODUCT: Inorganic phosphate+
Glycerol

CO2 = Respiratory component (Acid)


HCO3- = Blood/renal component (Base)

ALP METHOD: Bessy-Lowry-Brock, Bowers


McComb
Imbalances:

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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

unexplained tachypnea, dyspnea (esp. in


pxs with cardiopulmonary dse.)
unexplained restlessness and anxiety in
bed patients
drowsiness and confusion in patients
receiving oxygen therapy
assessment of surgical risk
before and during prolonged oxygen
therapy and during ventilator support of
patients
progression of cardiopulmonary disease

Respiratory Acidosis

lowered pH
decreased respiratory rate
(hypoventilation)

Metabolic Acidosis

results from failure of the kidneys


to perform excretory functions
(removing H+) that build up in
excess

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

More difficult to perform than venous


puncture
High risk of bleeding (arterial pressure)
Development of hematoma
Radial, brachial and femoral arteries

Failure of the kidneys to remove


excess HCO3 ions in the blood

Three mechanisms to maintain pH


Respiratory (CO2)
Buffer (in the blood: carbonic
acid/bicarbonate, phosphate
buffers, Hgb)
Renal (HCO3-)

Blood pH
Blood Gas Analysis
-

Laboratory test of blood where the purpose is


primarily to measure:
Ventilation is the rate at which gas
enters or leaves the lungs
Oxygenation process by which
concentration of O2 increases within the
tissue

EVALUATING ACID-BASE STATUS

ABG studies may be helpful to diagnose and


treat the following:

Arterial pH = 7.40
Venous pH = 7.35

Seesaw inverse values i.e. if pCO2 is


above its normal value, then pH is below
its normal value, the reverse might also
be the case
Swinging if HCO3 is above its normal
value, then pH is also above its normal
value

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Pre-Analytical Considerations:

1. Collect arterial blood anaerobically using


heparin as anticoagulant (avoid exposure
to ambient air, and avoid bubbles in the
syringe)
if exposed to air: CO2 and
pCO2 , pH , pO2
2. Tightly cap and transport on ice-water or
crushed ice
if delayed testing at room
temp: CO2 and pCO2 , pH ,
pO2

To determine whether the ulnar artery can


provide collateral circulation to the hand
after the radial artery puncture

Measurements:

1. Actual Percent Oxyhemoglobin

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

Must be performed before radial arterial


puncture

Modified Allen Test


1. Have the patient make a fist and occlude
both the ulnar and radial arteries by
compressing two fingers over each artery
2. Have the patient open his fist and observe
if the hand is bleached of blood
3. Release the pressure on the ulnar artery
only, and observe if blood return is present
Adequate perfusion is a positive test
indicating that arterial blood may be
drawn from the radial artery. BLOOD
SHOULD NOT BE TAKEN IF THE TEST
IS NEGATIVE.
Arterial Puncture Technique

The artery to be punctured is identified


by its pulsations.
The nonanesthetized arterial puncture
provides an accurate measurement of
resting pH and pCO2, however theoretical
errors of resulting hyperventilation
because of the pain is possible.
The amount of anticoagulant should be
0.05ml of liquid heparin per ml of
blood

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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5. After the blood is aspirated, mix it with


heparin.
6. Place in ice water or other coolants to
maintain the temperature of 1-5 degrees
Celsius.

7. Apply pressure on the puncture site for 25 minutes using a sterile gauze.

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