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