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
Beer’s 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
o
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. (2°Pp) 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 2°Pp is between 65-100 mg% If normal = (-) DM terminate! If abnormal = (?) DM – suspicious proceed to confirmatory test!
1. FBS or 2°Pp 140 mg%
CONFIRMATORY TESTS candidate: >100 mg% glucose
MONITORING TESTS medicine prescribed – Do’s and Dont’s
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)
β-glucose – oxidized α-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
α-glucose
β-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 doesn’t 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. 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
(OCT) ornithine cabamoyl transferase enzyme produced by liver ↓ OCT if liver is not functioning OCT
NH3
urea liver
kidney
creatine precursor
(yellow crystals)
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
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 Neissler’s 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 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
