clinical chemistry (lecture) - prelims
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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+ → yellow flame K+ → 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
P1 – distance travelled by the standard P2 – distance travelled by the unknown
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 ⎕
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
Beer’s Law conc. of light is
directly proportional to the absorbance inversely proportional to the logarithm of
transmittance if reading if (ABS) Absorbance:
If reading is % transmittance
Autoanalyzer
performs automated analysis of the clinical specimen
Parts of Autoanalyzer 1. Automatic Sampler
pipets approximate volume of the Sx 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 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
2. Discrete Analyzer
each specimen has its own dedication reaction tube/chamber
analysis is done simultaneously short time to finish
Designs of Autoanalyzer 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
Dry Slide Technology
one slide with 4 or 5 layers Four Layers Layer 1 – spreader - distribute specimen evenly Layer 2 – scavenger - destroy the interference in
the color reaction Layer 3 – reagent - specimens will act with
reagent → colored product Layer 4 – support - 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
Separation of Various Substances in One Mixture 1. Chromatography
isolate each amino acid in a mixture Stationary phase – paper Mobile phase – developing solvent
unknown should be compared to a known
standard with the same composition Electrophoresis
separate different protein substances in only one solution
means of electric current
o (-) anions at high pH migrate anode
o (+) cations at low pH migrate cathode
o at isoelectric point – neutral or zwitterions o isoelectric point of serum protein
pH 3.5 – 5.0 does not migrate in electric field
o rate migration dependent albumin – MW 40,000 (fastest) gamma globulin – MW 150,000
o pH used in sepa serum proteins = pH 8.6 o at this pH, the protein in anionic
gamma, beta, alpha2, alpha1, albumin
chromatographic paper strip
developing solvent
glass chamber
solvent front Distance travelled by
the Sx from the origin
(-)
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) always present – C , H , O always absent – N 3 Classes of Carbohydrates 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
Blood Glucose Level 1. Normal
normolycemia – 65-100 mg %
2. High hyperglycemia - >100 mg %
3. Low
hypoglycaemia - < 65 mg %
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
MECHANISM
insulin beta cells of pancreas lower glycolysis (insulin: glucose to the muscle)
glycogenesis (insulin: glucose to liver cells)
glucagon alpha cells of pancreas increase glycogenolysis
cortisol adrenal cortex increase gluconeogenesis
somatostatin delta cells maintain proper balance of
insulin/glucagon
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)
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. (2°Pp) 2 hours post prandial
collect specimen 2 hours after a meal
principle: blood glucose returns normal 2 hours after a meal
Screen Test is normal if FBG or 2°Pp is between
65-100 mg% If normal = (-) DM terminate! If abnormal = (?) DM – suspicious proceed to confirmatory test! CONFIRMATORY TESTS candidate: >100 mg% glucose 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
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
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
CRITERIA OF (NDDG) NATIONAL DIABETES DATA GROUP DECLARATION DIABETIS MELLITUS 1. FBS or 2°Pp
140 mg% - repeat - 140 mg% patient is (+) DM
2. OGTT 2 values out of 3 200 mg% = (+) DM
MONITORING TESTS
medicine prescribed – Do’s and Dont’s a. (HbA1C) glycated Hb glucose elevated + Hb of RBC → glucohemoglobin (reversible complex) 3 months
→ glycosylated Hb glycated Hb (unstable) (stable complex) (irreversible complex) normal value – 4-6% specimen – EDTA blood (purple/lavender top)
+ detergent – lyse RBCs Filter cell remnants hemolysate
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
Gestational Diabetes
manifested in pregnant women unclassified early warning
GLUCOSE METHOD best specimen – blood collected NaF, iodoacetate – prevents glycolysis NaF – inactivates the enzyme enolase Iodoacetate – inactivates glyceraldehydes-3-phosphate dehydrogenase 1. Copper Reduction Method
oldest method
a) Folin Wu b) Nelson-Somogyi
glucose is a very good reducing agent
glucose
Cu+2 Cu+ (cupric) (cuprous)
(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 3. Enzymatic a. (GOD) Glucose Oxidase Method glucose oxidase glucose + O2 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]
O2 + o-dianisidine oxy-orthodianisidine (colorless) (orange-brown) Journal - β-glucose – oxidized α-glucose – not oxidized
b. Hexokinase hexokinase
glucose + ATP G6P + ADP G6PD G6P + NAD PGA + NADH (phosphoglutonic (colored) acid) measured!
dehydrogenase – removes H+ G6PD – for RBC durability glucose oxidase:
mutarotase
α-glucose β-glucose Autoanalyzer Method Fe(CN)6
-3 + glucose Fe(CN)6-4
(serum) (colorless)
(NPN) Non-Protein Nitrogen contains nitrogen but not proteins
NPN vs PROTEINS a. NPN – low MW Proteins – high MW Urea – NPN (NH2)2CO
N - 2 x 14 = 28 H - 4 x 1 = 4 C - 1 x 12 = 12 O - 1 x 16 = 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. Urea – 45 – 50% 2. Amino Acids – 20% 3. Uric Acid – 20% 4. Creatinine – 5% 5. Creatine – 1-2% 6. Ammonia – 0.2%
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
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 doesn’t change abruptly
remains constant used to evaluate for the completeness of the
24 hour urine sample RR-male RR-female
higher muscle mass not always true
(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 CCr prescribe CCr before anti-hypertensive after medication medication if equal = normal MEASUREMENT OF CREATININE H2O
creatine creatinine dehydration dehydrating agent: conc. H2SO4
creatine – hydride of creatinine
a. Jaffe’s 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 (yellow crystals)
will last only for 6 hrs.
this should be freshly-prepared when in use because it easily decompose to picramic acid.
b. Lloyd’s Jaffe Reaction Lloyd’s 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
(OCT) ornithine cabamoyl transferase enzyme produced by liver ↓ OCT if liver is not functioning
OCT
NH3 urea urine liver kidney creatine precursor creatinine
anhydride of creatine
H2O 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 Neissler’s Reagent – K2HgI4
[OH-] K2HgI4 NH4 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 Berthelot’s 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