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CLINICAL CHEMISTRY

(Prelims)
1. Gravimetric Method
Concentration of analytes in terms of W (g)
clinical microscopy
total lipid in 24 hour fecal sample
diagnosis of steatorrhea
high amount of fat in the stool
inability of human to digest fat
from food eaten
inadequate bile salts supply to
the small intestine
causes:
obstruction of biliary passage
to small intestine
gallstone; biliary stone
2. Volumetric Method (titrimetric method)
determine volume of desired analyte from
clinical specimen
known volume of clinical specimen is reacted
with standard solution until end point is
reached
o (change color of indicator)
clinical microscopy
total gastric acidity gastric juice
strongly acidic
pH of 1-2
to determine the [H+] concentration in gastric
juice, titrate std. base and gastric juice
Zollinger Ellison Syndrome
[H+] concentration
3. Instrumental Method
a. Colorimeter
concentration of analyte is determined by
basing it from the intensity of the colored
solution.
conc. analyte = color intensity
most common method

b. (EFP) Emission Flame Photometry


conc. of analyte is based on the intensity of the
color of the flame
measures Na+ and K+
Na+
K+

yellow flame
violet flame

(isolated by
400mm filter)

c. (ASS) Atomic Absorption Spectrophotometry


measures the amount of radiation absorbed by
the unknown
measures Ca+2 and Mg+2
hallow cathode tube
releases radiation absorbed by unknown
old method:
Ca+2

red flame
(may use EFP)
thick atom require high energy
Mg+2

no color flame (N/A for EFP)

large rich laboratories


EFP millions of pesos
not so rich laboratories
cannot afford EFP
cheaper/alternative method:
(ISE) ion-selective electrode
a) glass ISE - Na+
b) valinomycin ISE - K+
d. Nephelometry
measures the amount of light scattered
measures the conc. of immunoglobulin
(IgG, IgM, IgA, IgE)
e. Fluorometry
amount or degree of fluorescence
measures hormones
o substrate absorbs light w/ short
wavelength
o emits same light with higher
wavelength
f, Osmometer
measures the amount of solutes dissolved in
clinical specimen
molality specimen is not blood
osmolality blood specimen
solutes dissolved blood:
NaCl
glucose
(BUN) blood urea nitrogen

Colorimeter
1. Duboscq
color of the unknown solution is compared
with a white light

Beers Law
conc. of light is
directly proportional to the absorbance
inversely proportional to the logarithm of
transmittance
if reading if (ABS) Absorbance:

P1 distance travelled by the standard


P2 distance travelled by the unknown
If reading is % transmittance
2. Filter Photometer
utilize colored glass filter
o has inscribed number
400 nm/mm can isolate violet color
700 nm/mm can isolate red color
3. Spectrophotometer
uses a monochromator
a) quartz prism
b) defracting grating

Autoanalyzer
performs automated analysis of the clinical
specimen
Parts of Autoanalyzer
1. Automatic Sampler
pipets approximate volume of the Sx

Parts
1. Light Source
a. tungsten lamp
b. mercury lamp
2. Monochromator
to separate the incident light to 7 colors of the
rainbow
quartz prism
diffraction grating
3. Cuvet
holder of unknown colored solution
4. Photocell
coverts the color observed to an electric
current
5. Combined galvanometer and potentiometer
to read photocells
reading :
(ABS) Absorbance
% transmittance

2. Dialyzer
removes protein substances from the Sx
o causes turbidity that will mask
the color of the unknown
solution
o causes bubbles / foam
3. Peristaltic pump or proportioning pump
dispenses color reagents
4. Heating bath
promotes color formation of the end product
5. Reading devices
a. Spectrophotometer - records intensity of
color of the unknown solution
b. (EFP) Emission Flame Photometry intensity
of the colored flame
c. (AAS) Atomic Absorption Spectrophotomer
amount of radiation absorbed by the unknown
d. Nephelometer amount of light scattered by
the immunoglobulin
e. Fluorometer amt. or degree of fluorescence
6. Recorder
computes the test values
7, Printer
types the test results of the analysis

2 Categories of Autoanalyzer

Separation of Various Substances in One Mixture

1. Continuous Flow Analyzer


has only 1 reaction tube/chamber for all the
specimen
analysis are done one at a time
long time to finish

1. Chromatography
isolate each amino acid in a mixture
solvent front
Distance travelled by
the Sx from the origin

2. Discrete Analyzer
each specimen has its own dedication reaction
tube/chamber
analysis is done simultaneously
short time to finish

chromatographic paper strip


glass chamber

Designs of Autoanalyzer

developing solvent

1. Sequential Analyzer
1 analysis from 1 specimen at a time
2. Batch Analyzer
1 kind of test from many specimens at one
time.
3. Parallel Analyzer
many kinds of test from only 1 serum
4. Random Access Analyzer
modify order or sequence of analysis
prioritize
a. Wet Chem Analyzer
o reagents are in liquid form
b. Dry Chem Analyzer
o reagents are dry

Stationary phase paper


Mobile phase developing solvent

Electrophoresis
separate different protein substances in only
one solution
means of electric current
o

Dry Slide Technology


one slide with 4 or 5 layers

Four Layers
Layer 1 spreader
Layer 2 scavenger

o
o

Layer 3 reagent
Layer 4 support

- distribute specimen evenly


- destroy the interference in
the color reaction
- specimens will act with
reagent colored product
- so that the dry slide can
stand on its own

reflectance microscopy - measures colored product


Five Layers
Layer 1 spreader
Layer 2 scavenger
Layer 3 reagent
Layer 4 indicator*
Layer 5 support

unknown should be compared to a known


standard with the same composition

o
o
o

(-) anions
at high pH
migrate anode
(+) cations
at low pH
migrate cathode
at isoelectric point neutral or zwitterions
isoelectric point of serum protein
pH 3.5 5.0
does not migrate in electric field
rate migration dependent
albumin MW 40,000 (fastest)
gamma globulin MW 150,000
pH used in sepa serum proteins = pH 8.6
at this pH, the protein in anionic

gamma, beta, alpha2, alpha1, albumin


(-)
cathode

(+)
anode

covered by solid:
1. cellulose acetate
2. agarose
3. polyacrylamide gel
(PAGE) polyacrylamide gel electrophoresis
low MW = separated into 5
Spray dyes
1. Ponceau s.
2. Bromphenol blue
3. Silver stain
4. Coomasie brilliant blue
5. Amido black

Electrophoregram
the result is scanned by a densitometer
o measures
intensity of
color or band
o darkest band =
albumin
(most
abundant)

CARBOHYDRATES (CHO)
Blood Glucose Level
always present C , H , O
always absent N

1. Normal
normolycemia 65-100 mg %

3 Classes of Carbohydrates
2. High
1. Monosaccharide
1 saccharide unit
a. glucose (dextrose)
rotate polarized light to the right
b. fructose (levulose)
rotate polarized light to the left
c. galactose
cannot rotate light
2. Disaccharide
2 saccharide units
a. lactose : 1 mole glucose + 1 mole galactose
a. lactase
b. maltose : 1 mole glucose + 1 mole glucose
a. maltase
c. sucrose : 1 mole glucose + 1 mole fructose
a. sucrose
Disaccharases enzymes whose substrates are
disaccharides
(LT) Lactose intolerance
cannot digest lactose in milk
milk is not allowed
instead, give soya drink
3. Polysaccharide
3 or more saccharide units
a. starch
found in saliva
amylase enzyme to digest starch
o S-form amylase saliva
o P-form amylase - pancreas
b. cellulose
fount in plants (fruits/vegetables)
no nutritional value
no enzyme that can degrade
for normal functioning of the intestines
c. glycogen
found in liver of humans/animals
quick energy
o easily converted to glucose as
energy source
stored energy
o storage form of carbohydrates in
the body

hyperglycemia - >100 mg %

hypoglycaemia - < 65 mg %

3. Low

4. renal threshold
140 160 mg %
highest value of blood glucose afterwhich
glucose appears in the urine
glucosuria - > 160 mm %
5.panic value
blood glucose reaches 35 mg %
irreversible brain damage
RMT should inform the physician and the
nurse immediately
500 mg% organ failure occurs
Carbohydrate Processes
1. glycolysis in the muscles
breakdown of glucose into lactate + pyruvate
finally: CO2 + H20 + energy
2. glycogenesis in the liver
synthesis of glycogen from the glucose
3. glycogenolysis in the liver
breakdown of glycogen into glucose
4. gluconeogenesis in the liver
formation of glucose from non-carbohydrate
sources
examples: amino acid, fatty acids, glycerol

Hormonal Control

HORMONES

SOURCE

EFFECT GLUCOSE

insulin

beta cells of pancreas

lower

glucagon

alpha cells of pancreas

increase

glycolysis
(insulin: glucose to the muscle)
glycogenesis
(insulin: glucose to liver cells)
glycogenolysis

adrenal cortex

increase

gluconeogenesis

delta cells

maintain proper
balance of
insulin/glucagon

cortisol
somatostatin

Insulinoma
tumor in pancreas
no. of beta cells = insulin = glucose
lab finding: low blood glucose
(DM) Diabetes Mellitus
Pancreatic damage
Slow production of insulin by the liver
Blood insulin deficient
Glucose is not utilized as the main source of
energy
lab finding: glucose level
Patterns of Blood Glucose Level
a. 30 minutes after meal
fastest increase of glucose level
b. 1 hour after meal
peak glucose level in the blood stream
c. after 1 hour of meal
glucose level of blood starts to go down
d. after 2 hours of meal
blood glucose returns to original level (prior
to the meal)

MECHANISM

MANAGEMENT (DM) DIABETES MELLITUS


1. (FBS) Fasting Blood Sugar (80 120 mg%)
2. (FBG) Fasting Blood Glucose (65 - 100 mg%)
sugar
higher value
glucose + other sugars + saccharoids
o

substances
with sugar-like
characteristics

glucose
lower value
true blood glucose
3 SYMPTOMS (P-Triad)
1. Polyuria
excessive urine excretion
3L volume of urine
2. Polydipsia
excessive thirst
3. Polyphagia
excessive hunger
SCREEN TEST
very sensitive thirst
to see if (+) or (-) to DM
measures minute concentration of glucose
always yield a (+) result for presence of
diabetes
a. (FBG) fasting blood glucose
fasting for 8 hours (overnight fasting)
b. (2Pp) 2 hours post prandial
collect specimen 2 hours after a meal

principle: blood glucose returns normal 2


hours after a meal

CRITERIA OF (NDDG) NATIONAL DIABETES DATA


GROUP DECLARATION DIABETIS MELLITUS

Screen Test is normal if FBG or 2Pp is between


65-100 mg%
If normal = (-) DM
terminate!
If abnormal = (?) DM suspicious
proceed to confirmatory test!

1. FBS or 2Pp
140 mg%

CONFIRMATORY TESTS
candidate: >100 mg% glucose

MONITORING TESTS
medicine prescribed Dos and Donts

1. (OGTT) oral glucose tolerance test


oral route
ingest glucose
more common
2. (IGTT) intravenous glucose tolerance test
inject to veins
5 mL of 5% glucose
invasive procedure
seldom used
for unconscious patients

a. (HbA1C) glycated Hb

GUIDELINES OF OGTT
a) patients should have 3 days preparation
(CHO) carbohydrates intake daily
should have an average of 150g/day
b) overnight fasting a night prior to the test
c) no physical exertion allowed

- repeat 140 mg%


patient is (+) DM

2. OGTT
2 values out of 3
200 mg% = (+) DM

glucose elevated + Hb of RBC glucohemoglobin


(reversible complex)
3 months

glycosylated Hb
(unstable)

normal value 4-6%


specimen EDTA blood (purple/lavender top)
+ detergent lyse RBCs
Filter
cell remnants

hemolysate

collect the fasting blood sample to check the


blood glucose level
if value is 65 100 mg% - STOP!
If value is > 100 mg% - glucose challenge
140 mg% - do not proceed to
glucose challenge
to the candidates
undergo glucose load challenge
o adult 75g glucose
o pregnant 100g glucose
o child depends on weight
1.75glucose/kg body weight

take note the time


finish in 1 minute
collect 3 blood samples
o after 30 minutes
o after 1 hour
o after 2 hour

glycated Hb
(stable complex)
(irreversible complex)

subject the hemolysate to column


chromatography
subject to HbA1C determination

b. (FS) fructosamine
(HbA1C) glycated Hb
stable complex of Hb + glucose
once in 3 months
(FS) fructosamine
stable complex of albumin + glucose
once a week
albumin halflife of 21 days
specimen hemolysate
subject to column chromatography

TWO TYPES OF (DM) DIABETES MELLITUS


1. Type 1 (IDDM)
Insulin-Dependent Diabetes Mellitus
insulin level deficient
insulin = glucose
pancreas is damaged (hypoinsulinism)
o producer of insulin
10% of diabetic population
juvenile onset
o symptoms manifest before 20 years
old
o average 9 years old
o non-obese/lean
more serious type of diabetes
common ketosis
mode of treatment: insulin injection
2. Type 2 (NIDDM)
Non Insulin-Dependent Diabetes Mellitus
pancreas is healthy
major problem low # of insulin receptors at
surface cells
90% of diabetic population
adult-onset diabetes
o >40 years old
o Obese
seldom ketosis
mode of treatment: diet and exercise

(PMA) phosphomolybdic acid

Mo blue product measured


(AMA) arsenomolybdic acid

Copper Reduction Method is stopped!


Folin Wu measures (FBS)
glucose + saccharoid
Nelson-Somogyi measures (FBG)
true blood glucose
requires (PFR) protein free filtrate
2. O-toluidine Method (Dubowski Method)
HA
(acetic acid)

glucose serum + toluidine


(blue) water bath
glycosylamine + Schiff base
(green)
glycosylamine measured by spectrophotometer

Gestational Diabetes
manifested in pregnant women
unclassified
early warning

3. Enzymatic

GLUCOSE METHOD
best specimen blood collected

glucose + O2

a. (GOD) Glucose Oxidase Method


glucose oxidase

NaF, iodoacetate prevents glycolysis


NaF inactivates the enzyme enolase
Iodoacetate inactivates glyceraldehydes-3phosphate dehydrogenase
1. Copper Reduction Method
oldest method
a) Folin Wu
b) Nelson-Somogyi
glucose is a very good reducing agent

gluconic acid + H2O2


( hydrogen
peroxide )
H2O2 tested by Trinders Reaction
GOD Trinders
H2O2 + colorless organic dye colored dye
measured!
GOD Clark
peroxidase

H2O2 + o-dianisidine

H2O + O2

O2 measured by Clark electrode


GOD-ODS
[O]

glucose

Cu+2

Cu+

(cupric)

(cuprous)

O2 + o-dianisidine
(colorless)
Journal -

oxy-orthodianisidine
(orange-brown)

-glucose oxidized
-glucose not oxidized

b. Hexokinase
hexokinase

glucose + ATP

G6P + ADP
G6PD

G6P + NAD

PGA

+ NADH

(phosphoglutonic
acid)

(colored)
measured!

dehydrogenase removes H+
G6PD for RBC durability
glucose oxidase:
mutarotase

-glucose

-glucose

Autoanalyzer Method
Fe(CN)6-3 + glucose
(serum)

Fe(CN)6-4
(colorless)

(NPN) Non-Protein Nitrogen


contains nitrogen but not proteins
NPN vs PROTEINS
a. NPN low MW
Proteins high MW
Urea NPN
(NH2)2CO
NHCO-

2 x 14
4x1
1 x 12
1 x 16

= 28
=4
= 12
= 16
60 MW

urea 60
BUN 28

albumin
smallest protein
40,000 50,000 MW
IgG MW 150,000 g/n
IgM MW 900,000 g/n
b. NPN - crystal in nature
Proteins colloids
COMPONENTS OF NPN

1.
2.
3.
4.
5.
6.

Urea 45 50%
Amino Acids 20%
Uric Acid 20%
Creatinine 5%
Creatine 1-2%
Ammonia 0.2%

1. Urea
waste product of protein metabolism
excreted through urine
carnivorous rich in protein - urea
90% urea is excreted in bloodstream
2. Creatinine
waste product of muscle metabolism
99% creatinine excreted in bloodstream
excreted through urine
more reliable for KFT
o 99% excreted by kidney
o Not influenced by protein diet
meat = correspondently
increase
urea
but
not
creatinine
o muscle mass origins of retaining
doesnt change abruptly
remains constant
used to evaluate for the completeness of the
24 hour urine sample
RR-male
higher muscle mass
not always true

RR-female

(CCR) Corrected Creatinine


U urine creatinine (mg/dL)
V volume of 24 hour urine (mg% or mg/100)

P plasma/serum creatinine
1.73 average surface area
SA surface area of patient
3L polyuria = DM

Measured in lab
1. Urea - KFT
2. Creatinine - KFT
3. Uric Acid - gout
4. Ammonia hepatic Coma
immediately
tests for patient who will die/ seriously
illed
STAT!
avoid delay
NH3 - determination of glutamine

CCr
before
medication

prescribe
anti-hypertensive

if equal = normal

MEASUREMENT OF CREATININE
H2O

creatine

creatinine
dehydration

dehydrating agent: conc. H2SO4


creatine hydride of creatinine

CCr
after
medication

a. Jaffes Reaction
not specific for creatinine.
may also measure other substances.
o reducing substances (ascorbic acid,
Vitamin C, glucose, uric acid)
creatinine in serum + alkaline picrate creatinine
(color rgt.)
picrate
unstable
(orange-red
compound)
Picric acid + 10% NaOH

(OCT) ornithine cabamoyl transferase


enzyme produced by liver
OCT if liver is not functioning
OCT

NH3

urea
liver

kidney

creatine precursor

(yellow crystals)

b. Lloyds Jaffe Reaction


Lloyds reagent (Na Al silicate)
o Remove interferents
end color orange-red compound
creatinine = orange-red
normal serum = yellow

3. Uric Acid
end product of purine or nucleic acid
metabolism
after chemotherapy = greatly elevated UA
if elevated
o has the tendency to be deposited at
joints
o fluid dries UA crystals (tophi)
o friction
o inflammation
o pain
2 KINDS OF URIC ACID
a. exogenous
from food (beans, peanuts, mongo, etc.)
b. endogenous
manufactured by body from purine
metabolism
4. (NH3) Ammonia
from bacterial breakdown of urea
lowest concentration of all NPN
liver converts all NH3 to urea which is
excreted through urine
measured to monitor hepatic coma
liver not functioning
NH3
o special test
o unscheduled
o for seriously ill patients
normal NH3 normal liver
elevated NH3 defective liver

creatinine
anhydride of
creatine
H2O

will last only for 6 hrs.


this should be freshly-prepared when in use
because it easily decompose to picramic acid.

urine

5. Amino Acids
not measured in chemistry
measured in clinical microscopy
Proteins
separated by electrophoresis
most common
hormone
serum
proteins in nature
enzyme

UREA DETERMINATION
1. Direct Method
measures urea
Rosenthal Method
Fearon Method
DAM (diacetyl monoxide) Method
urea + DAM

yellow product

2. Indirect Method
measures BUN by Kjeldahl-Nessler Method
a. digest N
NH4+
b. Neisslerization
Neisslers Reagent K2HgI4
[OH-]

NH4

K2HgI4

NH3

NH2Hg2I3
(yellow)
(diamino mercuric iodide)

obtained value is BUN so convert it to urea


3. Enzymatic Method
Urease-Berthelot Method
urease

urea in serum

CO2 + NH3
measured using
Berthelots Rxn

liberated NH3 + phenol hypochlorite


(color reagent )
catalyst

blue indophenol
Na nitroprusside

measured by spectrophotometry

Interferent - NH3
Disadvantage urease is inactivated by NaF

avoid delayed analysis of NH3 conc. in blood


delay leads to deamination of glutamine
end product of deamination is ammonia

Urea output
not correction due to muscle mass
muscle mass is not related to urea output
Creatinine Output
correction due to muscle mass
muscle mass = creatinine output