CLINICAL CHEMISTRY

INSTRUMENTATIONS
1. Transfer pipets
a. Volumetric- highest degree of accuracy
b. Ostwald- Folin- for highly viscous fluid; blow-out
c. Pasteur
d. Automatic: Air displacement, positive displacement (tips are not replaced);
dilutor/dispenser (can perform dispensing and sampling at the same time)

2. Measuring pipets
A. MOHR- no graduation to the tip
- self-draining

B. SEROLOGICAL- graduated to the tip; blow-out

CLEANING OF GLASSWARES

✓ Presoaking: soapy water or dilute bleach

✓ Cleaning solution: Acid dichromate (potassium dichromate + sulfuric acid), nitric acid
(substitute for H2SO4)
- Final rinsing: Use type 1 or type 2 water
- Washing of glass wares: Type lll

✓ Sterilization: Dry heat oven (>140 C or 160-180 C for 1.5-2 hours)

CENTRIFUGE
* RPM (Revolutions per minute or speed)
→ measured directly; checked quarterly (use tachometer or strobe light)
* RCF (Relative centrifugal force)
→ calculated parameter
→ determined by nomogram
→ RCF= RPM2 x r (radius of centrifuge) x 1.118 x 10(-5)

TYPES OF CENTRIFUGE
1. HORIZONTAL HEAD CENTRIFUGE (SWINGING BUCKET)

✓ Vertical: not in motion

✓ Horizontal: in motion
✓ Capable of speeds of up to 3000 RPM
- Recommended for SST
- High air friction and resistance -> result to increased heat generation

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2. FIXED ANGLE (ANGLE HEAD CENTRIFUGE or DESK/BENCH TYPE)

✓ Angle of tubes: 25-40 deg or 52 deg

✓ Rapid separation of the components
✓ Less air friction -> less heat generated
✓ Can reach up to 7000 RPM

3. ULTRACENTRIFUGE (REFRIGERATED BOX)

✓ Highest speed → generate tight sediments

✓ Tubes are held at a fixed angle
✓ Refrigeration serves as an advantage -> enhances separation of lipoproteins (CM, VLDL,
LDL, HDL)
✓ Reference method for lipoprotein analysis

MAINTENANCE OF CENTRIFUGE
* Weekly: disinfection using 10 % bleach
* Monthly: checking for unusual vibrations, braking mechanism and timer
* Quarterly: calibration using a tachometer or strobe light
→Aerosol hazard
→Capping of tubes: to prevent aerosols and evaporation of samples

QUALITY MANAGEMENT
Quality Assurance: complete system of creating and following procedures and policies to
aim for providing the most reliable patient laboratory results and to minimize errors in the
pre-analytical, analytical and post-analytical phases.
*Focusing on how we are going to meet the standards

Recall:
Total or over-all testing: Quality Assurance

Quality Control: aspect of quality assurance that is used to assess the analytical phase and
patient testing.
Quality Improvement: seeks to achieve new levels of performance; addresses chronic
problems in the laboratory
* We go beyond or exceed the standards

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 CLINICAL CHEMISTRY

LEAN SIX SIGMA

- strategy under quality improvement
PHASES
1. Define: describes the quality improvement issues
2. Measure: collects data to measure the process
-> determine the difference between the current process and the desired process
3. Analyze: searches for the root causes of inefficiencies
4. Improve: pilots process changes that seek to remove the identified the root problems
5. Control: continues to measure the process and ensures that changes are maintained
Example:
TAT is too long: 3 hours
TAT: 2 hours (target)
Root cause: 1 printer, CBC-manual, CC- Statfax (equipment)
Probable strategies or improvement:
Procurement of additional equipment; training of personnel
* Is there a change? Have we achieved?

3 MOST COMMON TEAM ROLES

1. BLACK BELTS: project coaches/leaders, dedicate 100% of their time
2. GREEN BELTS: project team members, dedicate 20 % of their time
3. BLUE BELTS: Project sponsors
a. Mid-level *
purple is not common
b. Senior-level sponsors

✓ DURATION: 6-8 months

✓ For smaller scale improvement projects

- headed by purple belts

- duration: 1 week

LEAN SIX SIGMA PRACTICAL APPLICATIONS

- detecting laboratory errors
1. Pre-analytical testing errors: 32-75 % 48¥
2. Analytical Testing errors: 4-32 %, 13-32 % (Henry’s)
3. Post-analytical errors: 9-55 %
Basic Standard (Henry’s)

✓ Six Sigma: performance improvement program

- Goal: improvement by eliminating process variations (defects)→ anything that does

not meet customer requirements
- DPMO: Defects per million per opportunity
- Strategy: DMAIC

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 CLINICAL CHEMISTRY

✓ Lean: Streamline an operation; a system that reduces waste (includes non-valued activities)
in the laboratory
- Strategies:
a. 5S (sort, set in order, shine, standardize, sustain)
b. PDCA (plan, do, act, check)

FACTORS AFFECTING TESTING

1. PRE-ANALYTICAL
a. Selection of assay relative ot patient need
b. Patient identification
c. Specimen collection
d. Specimen transport, preparation and storage
e. Monitoring of specimen condition

External quality assessment: Proficiency testing

2. ANALYTICAL
a. Assay validation and instrument selection
b. Sample identification
c. Laboratory staff competence
d. External and internal quality control

3. POST-ANALYTICAL
a. Accuracy in transcription and filing of results
b. Content and format of lab report, narrative report, reference interval and therapeutic range
c. Timeliness in communicating critical values, patient and physician satisfaction
d. Turnaround time, cost analysis

STATISTICAL TOOLS

✓ Measuring Central Tendency → true value: evaluates accuracy

a. Mean: average (most commonly used)

b. Median: middle value
c. Mode: most frequently obtained value
✓ Measuring dispersion of values → variability: precision

a. Range: simplest measure of dispersion; lowest-highest value

Example: 1-7 (the lower the difference between the lowest and highest value→ the more
precise)

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b. Standard deviation: Indicator of precision

Control limits
Upper: Mean + 1SD, +2SD, +3SD
Lower: Mean -1SD, -2SD, -3SD

∑(𝒙−𝒙)𝟐
Variance:
𝒏−𝟏
∑(𝒙−𝒙)𝟐
SD= √
𝒏−𝟏

c. Coefficient of variation: relative indicator of precision

- ideal CV: <5%
- the lower the CV → the higher precision

CV= (SD/mean) x 100

GAUSSIAN DISTRIBUTION CURVE

✓ Mean=median=mode

✓ +/-2SD (mostly used control limits)

* +/-1SD: 68.3 % (inside)

31.7% (outside)

* +/-2SD: 95.5 % (inside)

4.5 % (outside)

*+/-3SD: 99.7 % (inside)

0.3 % (outside)

STATISTICAL SIGNIFICANCE

-included in comparative statistics

- p value: 5% or 0.05 (most commonly used level of significance)
* p value </= 0.05 (statistical significance)
* p value >/= 0.05 (no statistical significance)

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 CLINICAL CHEMISTRY

✓ T-test
- significant difference between two means
- helps evaluate accuracy

✓ F-test Frecision
- significant difference between two variances or SD
- Helps evaluate precision

DIAGNOSTIC EFFICIENCY

TERMS:
✓ ACCURACY: closeness to the true value (when the values are accurate, values are
precise)
✓ PRECISION: closeness to a repeated value (not all precise values are accurate)
✓ RELIABILITY: ability of method to maintain accuracy and precision over an extended
period of time
✓ ANALYTICAL SENSITIVITY: ability to detect small quantities of the analyte
✓ ANALYTICAL SPECIFICITY: ability to detect only the analyte of which it is designed to
detect/measure

* DIAGNOSTIC SENSITIVITY: proportion of individuals with the disease who test positively
with the test

Diagnostic sensitivity= 100 x # of diseased individual with a positive result

Total # of diseased individuals tested (TP + FN)

𝟏𝟎𝟎 𝒙 𝑻𝑷
Or 𝑫𝒊𝒂𝒈𝒏𝒐𝒔𝒕𝒊𝒄 𝒔𝒆𝒏𝒔𝒊𝒕𝒊𝒗𝒊𝒕𝒚 =
𝑻𝑷+𝑭𝑵

* DIAGNOSTIC SPECIFICITY: proportion of individuals without the disease who test

negatively with the test

Diagnostic specificity= 100 x # of individuals without the disease with a negative test
Total # of individual tested without the disease (TN + FP)
100 ✗ TN

TNF Or 𝑫𝒊𝒂𝒈𝒏𝒐𝒔𝒕𝒊𝒄 𝒔𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒊𝒕𝒚 =

𝟏𝟎𝟎 𝒙 𝑻𝑵
𝑻𝑵+𝑭𝑷

True positive: diseased individual with a positive result

True negative: not diseased individuals with a negative result
False positive: not diseased individuals with a positive result
False negative: diseased individuals with a negative result

* Pag diagnostic
:
TN FP/ Pag predictive
:
1- PAP
TP / FN TN/FN
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 CLINICAL CHEMISTRY

PREDICTIVE VALUES

Positive Predictive value: probability that a positive test indicates the presence of a disease
= 100 x TP/ TP + FP
Negative predictive value: probability that a negative test indicates the absence of a disease
= 100 x TN/ TN + FN

CALIBRATION FOR ACCURACY AND PRECISION

- part of internal quality control program

✓ Standard (calibrator): material of known concentration → calibration of instrument

* Universal standard (automation)→ auto-calibrator

✓ Control: material of known value that is analyzed with patient samples; used to determine
the acceptability of results→ resembles patient sample
Levey- Jennings (Shewhart Plot): controls values are recorded and plotted
- most commonly used
* x- axis (abscissa/horizontal): contains independent variables → not influenced
by other factors (example: time, age, gender)

* y-axis (ordinate/vertical): dependent variables (example: results such as

analyte concentration, enzyme activities, presence/absence of disease)

✓ Evaluate: Westgard
Shift: sudden/abrupt change in the values (6 or more values) →
values establishes a new distribution pattern above or below the
mean
- values distribute themselves on one side of the mean
- improper calibration

Trend: gradual deviation of control values that either increase or

decrease over a period of 6 consecutive days or more
→ at least 3 days (Turgeon’s)
→ deterioration of reagents

Outlier: control value that is far from the main set of values
→ control value that goes beyond +/- 2SD
→ 2 consecutive outliers→ reject (recalibrate/rerun controls)

Westgard Multi-rule: evaluate plotted control values

12s
1= # of values observed → one value that is outside 2SD
2S= location of the value observed→ exceeds, more than, greater than, outside

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 CLINICAL CHEMISTRY

a. Detection of random errors

12s: one control observation exceeds +/- 2SD
→ Warning or screening (require further investigation of QC data)
→ does not require immediate action

13s:one control observation exceeds +/- 3SD

→ REJECT → repeat the run

R4s: two consecutive values differ by more than 4SD

→ REJECT

b. Detection of systematic errors

22s: two consecutive control observations fall more than 2SD on the same side of
the mean

41s: four consecutive control observations are more than 1SD away from the
mean in the same
direction
→ warning or reject

10x: ten consecutive control observations are on the same side of the mean
→ most sensitive check of systematic error

* Bi-level QC: considered adequate (minimum) → normal range, abnormal range (elevated/
abnormally high)
* Tri-level QC: normal range, abnormally low, abnormally high

✓ Internal QC: performed by laboratory personnel using controls of known values and
comparing such values to established acceptable ranges
✓ External QC: performed by laboratory personnel using specimens sent by another
institution (NRL)

* Establishing a reference interval

- when there are no available analyte or method
- 120- 700 individuals
* Verifying a reference interval
- 20 individuals
RANDOM ERROR
✓ does not recur; no pattern (example: clerical error)
✓ Error with no trend and occurs unpredictably → due to chance
-Mislabeling of sample, pipetting error, improper mixing of samples and reagents,
voltage fluctuations, temperature fluctuations
✓ Remedy: rerun using the same reagents → same error should not be made again

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 CLINICAL CHEMISTRY

SYSTEMATIC ERROR
✓ recurring
✓ Error seen as a trend that occurs predictably
- improper calibration, deterioration of reagents, sample instability, changes in
standard and material, instrument drift

LABORATORY SAFETY

4: Extreme hazard No -
O

3: Serious hazard s -
l

2: Moderate hazard M - 2
1: Slight hazard S -
3
0: No or minimal hazard Ex -
4

YELLOW: REACTIVITY (STABILITY)

WHITE: SPECIFIC HAZARD
RED: FIRE (FLAMMABILITY)
BLUE: HEALTH HAZARD

✓ Handwashing: at least 15-20 seconds

✓ Universal precautions: all patients are considered carriers of blood-borne pathogens;
excludes urine and other body fluids that are not visibly contaminated by blood
✓ Body substance isolation: does not recommend handwashing unless there is visible
contamination
✓ Standard precaution: combination of UP and BSI

Recall:
1. Safety Controls (arranged from most effective to least)
Elimination
Substitution
Engineering controls
Administrative controls
PPE
2. When using gloves, what part of the chain of infection are we trying to
eliminate? Mode of transmission (when using PPE)

***Review: PASS, RACE, Classification of fire

Class E: liable to result in a detonation

Example: Arsenal fire

WASTE CONTAINERS:

Recall:
Empty pressurize containers/ aerosol containers (Red)

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 CLINICAL CHEMISTRY

RED: sharps and needles

YELLOW: infectious wastes
YELLOW WITH BLACK: chemical wastes
ORANGE: radioactive wastes
GREEN: Non-infectious wet wastes
BLACK: Non-infectious dry wastes

ANALYTICAL METHODS AND INSTRUMENTATION

* light- convertible to other forms of energy

*Wavelength- nm; inversely proportional to energy
*Energy
Light spectrum:
✓ Visible light: 400-700 nm
Violet: highest energy
Red: lowest energy
✓ UV light: <400 nm
✓ IR light: >700 nm

1. SPECTROPHOTOMETRY
- Beer’s law
-directly measure transmitted light
* Concentration:
✓ Absorbance is directly proportional to concentration
✓ Transmittance: inversely

Parts:
a. Light source: polychromatic light
* Visible to infrared region: Use tungsten-halogen, tungsten-iodide lamp
* UV region: mercury-arc, xenon, deuterium-discharge lamp

b. Entrance slit: allows entry of a narrow beam of radiant energy; minimizes stray light
-Polychromatic

c. Monochromator (wavelength selector):

-disperse polychromatic light into separate wavelengths
-isolate specific wavelength→ Monochromatic light

*Polychromatic light (reaches)

*Monochromatic light (exits)

d. Exit slit
- controls the width of light beam (bandpass)
-Monochromatic light

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e. Cuvette (Sample holder): holds the solution containing the analyte to be measured
-monochromatic light
2 types:
Round-end
Square-end

f. Detector: measures and converts the transmitted light into an equivalent amount of
electrical energy

* Photomultiplier tube: most sensitive detector

g. Read-out device (meter): measures the magnitude of the current that is generated by the
detector

TYPES OF SPECTROPHOTOMETER
* Double beam: 2 cuvettes (1 for sample, 1 for control)

a. DOUBLE-BEAM-IN-SPACE
- all parts are duplicated except the light source
- 2 photodetectors

b. DOUBLE-BEAM-IN-TIME
- only the cuvettes are duplicated
- the detection is one at a time

2. FLUOROMETRY
-500-1000x more sensitive that spectrophotometry

Internal Components:

a. Light source: xenon lamp, hydrogen discharge lamp

b. Excitation filter: primary monochromator
→ receives light- increases energy level
→ produces excited light (high energy, shorter WV)
c. Cuvette holder: quartz cuvettes (commonly used)
* Analyte: absorbs excited light and gives off emitted light (fluorescence) → (lower energy,
longer WV)

d. Emission filter: secondary monochromator → detects fluorescence

e. Detector

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 CLINICAL CHEMISTRY
Recall:
1. Why is emission filter arranged @ 90 deg. from the excitation
filter? To avoid detecting light from the light source

2. Disadvantages of fluorometry
* Quenching: presence of molecules that absorbs or steal the
fluorescence→ decreased fluorescence
* Increased temperature→ result to more collisions → result to
heat generation instead of fluorescence

3. FLAME EMISSION PHOTOMETRY

- based on the characteristic emission of light by analytes which are easily excited when
exposed to sufficient heat energy

* Analytes with low oxidation state: Na, K, Li

✓ Light intensity → # of atoms→ concentration of analyte

✓ Color emission
Lithium: Red Light Red
Potassium: violet Point-of-view
Sodium: Yellow School Year
Rubidium: Red Red Red
Magnesium: Blue Megabyte
✓ Internal standards
Lithium
Cesium
Recall:
Dilution of serum for Na/ K FEP → 1:100 or 1:200

4. ATOMIC ABSORPTION SPECTROPHOTOMETRY

- measure light absorbed of atoms at a ground state (unexcited state of atoms) ~ concentration
of atom
- intended for analytes which are not easily excited by the flame

Recall:
Analytes that are not easily excited: Ca and Mg

Hollow Cathode lamp: light source

Flame: atomizer

5. NEPHELOMETRY
-measures light scattered
- detector is at a 90 or 30 deg. angle from the incident light
✓ Application:
- measurement of immune complexes
- cell counting procedures

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 CLINICAL CHEMISTRY

6. TURBIDIMETRY
- measures light blocked by particles
- detector is in line with the incident light
- Applications: Microbiology and hematology

AUTOMATION

✓ Single Channel: one test at a time

✓ Multi Channel: variety of tests at the same time
✓ Random Access: the different test reactions can be programmed to a variety of sequence
✓ Batch Analysis: group of samples are analyzed at the same time for the same test
✓ Sequential analysis: performing a set of tests in a particular order in each sample
✓ Open Reagent System: perform the reconstitution (example: AMS)
✓ Close Reagent system: no reconstitution

CONTINUOUS FLOW ANALYZERS

- specimen is aspirated through the sample probe into a continuous reagent stream →
pumped through a system of continuous coiled tubing
✓ Batch analysis: specimens are separated by air bubbles
✓ Primary source of error: CARRY-OVER (remnants of previous sample will affect the
succeeding sample)
→ Remedy: washing-out
* Tubing- reaction vessel (reagent + sample)

CENTRIFUGAL ANALYZERS
- uses a spinning rotor to generate centrifugal force to transfer and contain liquids in separate
cuvettes for analysis
-separate samples: acceleration and deceleration
✓ perform one test on multiple sample (batch analysis)

DISCRETE ANALYZERS
- each sample and the corresponding reagent is handled separately in its respective reaction
vessel
- runs multiple tests on one sample or one test on multiple samples
-most popular and versatile
- performs random access, batch and sequential analysis

SAMPLE COLLECTION AND PROCESSING

* Arterial Blood
✓ Blood gas analysis→ anticoagulant: heparin
* Capillary puncture

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✓ POCT
✓ NBS→ blood spot on filter paper, heel-stick, capillary puncture

Recall:
Arteriolized blood → capillary blood
(WARMING: 42 deg C → increases blood flow)

* Preferred site for Venipuncture: Antecubital veins

* Time of collection: morning
✓Basal state: early morning before the patient is physically active

FACTORS:

a. Diurnal variations:
Increase: ACTH, cortisol, iron, aldosterone
Decrease: ACP, growth hormone, PTH, TSH

b. Tobacco Smoking (common interference in Henry’s)

↑: catecholamines, cortisol, free fatty acids, lactate, inulin, epinephrine, GH, 5-HIAA,
WBC count, neutrophils, monocytes, carboxyhemoglobin, RBC count, Hb, Hct, MCV, IgE

↓: IgG, IgM, eosinophils, Vitamin b12

c. Alcoholism
↑: aminotransferases, lipoproteins, bilirubin, ketone bodies, TAG
↓: glucose, albumin, transferrin

d. Lipemia: increased lipid → TAG (>400 mg/dL)

→ interferes with amylase, urate, urea, CK, bilirubin, total protein

e. Bilirubin
* Icteric sample: 25.2 or 25 mg/dl

↑: ALP
- interferes with the following methods: HABA Method, assays using FeCL3, biuret
reaction
ANTISEPTIC TECHNIQUE

Recall:
✓Friction cleansing
✓Minimum PPE: tight fitting gloves (WHO)
Gloves, face mask, lab gown (Bishop’s)

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SPECIMEN HANDLING

✓ Protect from light: bilirubin and CK → falsely decreased

✓ Chilling: ammonia and blood gas

CARBOHYDRATES

✓CARBOHYDRATE METABOLISM
- oxidation of complex organic compounds (CHO, AA, lipids)→ energy
- Non-absorbable polymers (starch, glycogen)
→ digestion→ salivary amylase and pancreatic amylase

✓ GLUCOSE METABOLISM:

Glycolysis: metabolism of glucose to pyruvate or lactate or energy production (decrease

glucose)
Gluconeogenesis: formation of glucose from non-carbohydrate sources (increase glucose)
Glycogenesis: formation of glycogen→ glucose to glycogen (decrease glucose)
Glycogenolysis: breakdown of glycogen to glucose for energy (increase glucose)

Recall:
Purpose of glycogenesis? For storage

* Decreased blood glucose (Hypoglycemic agent):

Insulin: promote cellular uptake of glucose; increases glycogenesis and glycolysis

* Increase blood glucose (Hyperglycemic agents):

Glucagon: promote glycogenolysis and gluconeogenesis
Cortisol: gluconeogenesis
Growth hormone: inhibit action of insulin
Epinephrine: glycogenolysis
TH: glycogenolysis

SPECIMENS

✓ Fasting venous plasma- standard clinical specimen

✓ Capillary=arterial
✓ Venous: 5 mg/dl lower than arterial blood
✓ Whole blood: 10-15 % lower than serum or plasma

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* Anticoagulation
✓ NaF: more of antiglycolytic than anticoagulant
→ binds Mg → inhibits enolase
→ alone: 6-10 mg/ml of blood
→ with another anticoagulant: 2 mg/ml of blood

✓ K oxalate: main anticoagulant

✓ NaF + K oxalate → Prevent glycolysis 48-72 hours

* Time of collection: Morning

* Fasting: 8-10 hours
* Storage
✓ Room temperature: glycolysis rate at 7 mg/dl per hour
✓ 4 deg C: glycolysis rate at 2 mg/dl per hour
* Contamination
✓ 10 % contamination with 5 % dextrose: ↑ 500 mg/dl or more

ORAL GLUCOSE TOLERANCE TEST

* Patient preparation
✓ Normal to high carbohydrate intake for 3 days
✓ Fasting for 8-14 hours → best is 12 hours

* Glucose load: 75 g (WHO) taken within 5 minutes

* Sample is collected every 30 minutes for 2 hrs.

GLYCOSYLATED HEMOGLOBIN

- most accurate test to establish hyperglycemia

- long-term monitoring
- glucose + amino group of Hgb → ketoamine (HBA1c)
- index of average blood glucose level for the past 2-3 months
✓ Sample: EDTA whole blood
✓ Rate of formation of glycosylated hgb is
✓ > 6.5 % is indicative of DM

* Glycosylated albumin (fructosamine)

- short-term monitoring (2-3 weeks)

METHODS
(for blood glucose determination)

*Chemical methods, condensation methods, enzyme methods→ encompass the reaction to

quantify glucose→ spectrophotometric → chromogenic product

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*POCT→ still based on the first 3 methods

→ usually potentiometry

a. Chemical Methods (reducing property: glucose, fructose, lactose, galactose and maltose)

1. Alkaline Copper Reduction

✓ Folin-Wu: phosphomolybdic→ phosphomolybdenum/ molybdynum oxide (blue)
✓ Nelson-Somogyi: arsenomolybdic acid→ arsenomolybdenum (blue or greenish-blue)
✓ Neocuproine (Campbelland King method): cuprous + neocuproine (2,9-dimethyl-
1,10-phenanthroline hydrochloride) → Yellow or yellow-orange

2. Alkaline Ferric Reduction

✓ Johnson
✓ Folin/Prussian Blue
✓ Hagedorn-Jensen

b. Condensation Method
1. With aromatic amines

✓ Ortho-toluidine Method by Dubowski:

Glucose + orthotoluidine→ N-glycosamine (green)
*Interferences: galactose, mannose and aldopentose→ capable of forming shift bases

2. With phenols
✓ glucose→ water + hydroxymethylfurfural (HMF)
✓ HMF + anthrone → green-colored compound

c. Enzymatic Methods
✓ Glucose oxidase method → measured at 480-520 nm
a. Glucose + O2 + H2O→ gluconic acid + H2O2
b. H2O2 + reduced chromogen → oxidized chromogen + H2O

* Glucose is falsely decreased with the presence of uric acid, bilirubin and
ascorbic acid (increased)
* Glucose alone can only detect beta-D-glucose→ represents only 65% of glucose
Remedy: Mutarotase→ allows the conversion of alpha-D-glucose into beta-D-glucose

✓ Hexokinase method
→ more accurate with less interferences; not affected by uric acid and ascorbic
acid
→ Interferences: decreased in the case of hemolysis and high bilirubin
→ Reference method

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 CLINICAL CHEMISTRY

a. Glucose + ATP → glucose-6-PO4 + ADP (primary enzyme is hexokinase)

b. Indicator reaction (secondary reaction):

Glucose-6-PO4 _ NADP→ NADPH + H + 6-phosphogluconate (measured at 340
nm)
Coupling enzyme: G6PD

Recall:
Effect of hemolysis to glucose oxidase? Falsely increased (hemoglobin has a
pseudoperoxidase activity→ it can allow the indicator reaction to happen)

d. POCT→ bedside testing

- Glucose: highest volume of POCT in health care institutions
- Recommended for Type 1 diabetes
- Others: Electrolytes, blood gas analysis, hematology

CRITERIA FOR DIAGNOSIS OF DIABETES MELLITUS

✓ Fasting blood glucose ≥ 126 mg/dL
✓ Random blood glucose ≥ 200 mg/dL
✓ 2-hour post prandial ≥ 200 mg/dL during OGTT
✓ Glycosylated Hb ≥ 6.5 % (48 mmol/mol)
RBG + Classic symptom hyperglycemia
→ monitor insulin shock
* Any of these tests are diagnostic if confirmed by repeat testing in a subsequent day

TYPES OF DIABETES MELLITUS

TYPE I TYPE II
Previous name Insulin-dependent DM Insulin-independent DM
Pathogenesis Autoimmune Lifestyle related
Risk factor Genetic (HLA-DR/DQ) Genetic, obesity, sedentary
lifestyle, race/ethnicity
Onset Juvenile Adult
Frequency 5-10 % 90-95 %
Ketoacidosis Prone Not prone
C-peptide Low Normal
Medication Insulin injections Oral hypoglycemic agents

LIPIDS

✓ LIPIDS: Soluble in non-polar organic solvents (ex: chloroform and ether) and insoluble in
polar solvents (ex: water)
a. Cholesterol
- not readily catabolized and does not serve as a source of fuel
- converted to form bile acids (for the digestion of lipids), steroid hormones and
vitamin D3

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- hydrophilic portion + hydrophobic portion

2 forms:
1. Free cholesterol: amphiphilic lipid
2. Cholesteryl ester: hydrophobic → formation is facilitated by LCAT
(Lecithin cholesterol acyltransferase)

b. TAG
- hydrophobic
- 3 fatty acids + glycerol
- least reliable indicator of coronary heart disease
✓ > 400 mg/dl → lipemia

c. Phospholipids
- hydrophobic (composed of FA) + hydrophilic (determines the name of
phospholipid) head group

d. Fatty Acids
- most are bound to albumin

✓ LIPOPROTEINS (carriers of lipids): Water-soluble macromolecules that consists of varying

proportions of protein, cholesterol, TAG, and phospholipid

a. VLDL: major carrier of endogenous (hepatic derived) TAG

- Produced by the liver
- causes turbid appearance of plasma during fasting → fasting hyperlipidemia
- pre-beta lipoprotein
✓ 45-60 % TAG
✓ Apo B100

b. LDL: metabolite of VLDL → carries cholesterol via the circulation

- produced by the lipolysis of VLDL
- 45-55 % cholesteryl ester
✓ Apo B100
✓ Has a direct relationship to atherosclerosis and CHD risk
✓ Most atherogenic → carries cholesterol to the different part of the body
→ BAD CHOLESTEROL
- promotes plaques of cholesterol to the blood vessels

* FRIEDEWALD EQUATION→ only reliable if the TAG is < 400 mg/dl

For SI unit (mmol/L)

LDL = (total cholesterol) – (HDL- cholesterol) – (Plasma TAG) / 2.175

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 CLINICAL CHEMISTRY

If using mg/dl: TG/5

* DE LONG EQUATION→ if the TAG is ≥ 400 mg/dl

LDL= TC- HDL- TAG/ 6,5 (mg/dl)

or LDL= TC- HDL- TAG/ 2.85 (mmol/L)

c. HDL: carry cholesterol from peripheral cells back to the liver → reverse cholesterol
transport → GOOD CHOLESTEROL
- cleans cells of excess cholesterol
- Produce by the liver and intestine
- Apo A1
- Has inverse relationship to atherosclerosis and CHD risk

d. Chylomicrons: packaged with absorbed dietary lipids

- Main cargo: exogenous (usually comes from the diet) TAG
- least dense; largest
- produced by the intestine
✓ 80-95 % TAG
✓ Apo B48 → primary structural protein in chylomicron
→ readily reflects light: milky appearance of plasma or serum
✓ Causes turbid or milky appearance of plasma
✓ Accumulate as a floating creamy layer
✓ Postprandial turbidity

* Apolipoproteins- capable of binding lipids

- backbone of lipoprotein

CHEMICAL COMPOSITION OF PLASMA LIPOPROTEINS

Lipoprotein Protein Cholesterol Cholesteryl TAG Phospholipids
(determine the ester
density)
Chylomicrons 1-2 1-3 2-4 80-95 3-6
VLDL 6-10 4-8 16-22 45-65 15-20
LDL 18-22 6-8 45-50 4-8 18-24
HDL 45-55 3-5 15-20 2-7 26-32

OTHER LIPOPROTEINS

a. Beta- VLDL (floating beta-lipoprotein

- Type 3 hyperlipoproteinemia
- Density range similar to VLDL
- Electrophoretic migration similar to LDL

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b. Lp (a): sinking pre-beta lipoprotein

- Coronary disease and stroke
- independent risk factor for atherosclerosis
- Density similar to LDL
- Electrophoretic migration similar to VLDL
c. LpX
- obstructive biliary disease
- familial LCAT deficiency

MAJOR: CM, VLDL, LDL, HDL

MINOR: IDL, Lp (a) → not necessary cause pathologic conditions
ABNORMAL: LpX, Beta- VLDL → mostly indicates pathological conditions

METHODS FOR LIPOPROTEIN

* Ideal fasting: 12 hours

* TC and HDL: can be measured in non-fasting patients
* TG and LDL: fasting becomes a requirement

Recall:
Not performed in non-fasting individuals?
LDL
HDL
TC
TG (because it comes from the diet→ greatly affected by the diet)

a. Standing plasma test

- detect presence of chylomicron and VLDL
- 2 ml plasma→ ref (4 deg C) overnight and undisturbed
- Overnight refrigeration of plasma
→ Floating creamy layer: CHYLOMICRON
→ Floating creamy layer + turbid plasma: CHYLOMICRON AND VLDL
→ Absence of creamy layer and turbidity: absence of both
→ Turbidity: VLDL

*There may be a pathologic condition that is manifested by chylomicron even if the patient is
fasting

b. Ultracentrifugation – reference for lipoprotein assay

- Chylomicrons: <0.96
- VLDL: 1.006- 1.063
- HDL: 1.006- 1.21

* Buoyant density: ability of substance to float

INCREASING: HDL→ LDL→ VLDL→ CM

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 CLINICAL CHEMISTRY

* Density: heaviness of substance → ability of substance to sink

INCREASING: CM→ VLDL→ LDL→ HDL

c. Electrophoresis: for proteins

✓ Support medium: Polyacrylamide gel, agarose gel (most commonly used)

✓ Visualization: Oil Red O, Fat Red 7B, Sudan Black B
- Chylomicrons: does not migrate
- LDL: migrates the slowest→ migrates to the beta region
- VLDL: migrates to the pre-beta
- HDL: migrates the fastest → migrates to the alpha region

✓ Quantification: densitometry

TOTAL CHOLESTEROL
Methods Procedure
Zlatkis, Pearson, Watson’s One-step (colorimetry)
Bloor’s, Carr-Drekter Two-step (Extraction + colorimetry)
Abell- Kendall (reference Three-step (extraction, saponification, colorimetry)
method)
Schoenheimer-Sperry, Four-step (Extraction, saponification, purification, colorimetry)
Sperry-Webb

✓ Colorimetric Reactions
a. Liebermann-Burdchardt
- cholestadienyl nonsulfonic acid (emerald green)
b. Salkowski
- cholestadienyl disulfonic acid (red)

✓ Enzymatic methods
* Cholesterol esterase (cholesterol + H2o → cholesterol + FA)
* Cholesterol oxidase (cholesterol+ O2 → cholestenone + H2O

TRIGLYCERIDES

- Conversion factor: 0.00113

- HAntzsch-Lutidine Reaction (most common)
- Van Handel Zilversmit (reference method: modified Van Handel Zilversmit)
Formaldehyde + H2SO4+ chromotropic acid→ pink chromopore

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ATP lll Classification for TAG, HDL, LDL AND TC VALUES

TAG LDL
✓ < 150: normal ✓ < 100: optimal
✓ 150-199: borderline ✓ 100-129: near optimal/ above
✓ 200-499: high optimal
✓ ≥ 500: obese ✓ 130- 159: borderline high
✓ 160- 189: high
✓ ≥ 190: very high

HDL TOTAL CHOLESTEROL

✓ < 40: low ✓ < 200: desirable
✓ ≥ 60: high ✓ 200-239: borderline high
✓ ≥240: high

Recommended cut-off points for serum Guidelines for Acceptable

cholesterol Measurement Error
Age Moderate High Risk Analyte Total Bias CV
Risk Error
2-19 > 170 mg/dl > 185 mg/dl Cholesterol ≤ 9% ≤3% ≤3%
20-29 > 200 mg/dl > 220 mg/dl Triglyceride ≤ 15 % ≤5% ≤5%
30-39 > 220 mg/dl > 240 mg/dl HDL ≤ 13 % ≤5% ≤4%
40 and over > 240 mg/dl > 260 mg/dl LDL ≤ 12 % ≤4% ≤4%

TANGIER DISEASE
- defect in ABCA1 gene (chromosome 9)
- Ineffective transfer of cholesterol to Apo A1 → no HDL (complete absence)

SITOSTEROLEMIA
- extremely rare autosomal recessive → plant sterol (phytosterols) accumulate in
plasma

BASSEN-KORNZWEIG SYNDROME (ABETALIPOPROTEINEMIA)

- absence of beta-lipoprotein; undetectable plasma apo B containing lipoproteins

PROTEIN

- building blocks: amino acids (linked via peptide bonds)

- made-up of 16 % nitrogen → unique to protein
- primary, secondary, tertiary structure
- water-soluble
- Importance: energy production, water distribution or balance, proper fluid balance, buffers,
transporters, structural function, enzymes

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Plasma Protein fractions

→ based on electrophoretic mobility
→ electric charge densities (separated via serum-protein electrophoresis)
* Support medium: cellulose acetate (alkaline buffer: pH 8.6) → proteins exist
as ANION
Acidic: cations
Alkaline: anions

Review: Cathode: negatively charged electrode→ attracts cations

Anode: positively charged electrode→ attracts anions

Migration pattern of plasma protein: Cathode→ anode

Most anodal: albumin

Least anodal: gamma-globulins

a. Albumin
- negative APR
- binds bilirubin, steroids and fatty acids
- major contributor to oncotic pressure
- clinically significant if decreased (except dehydration→ increased albumin)

b. Pre-albumin (Transthyretin)
- more anodal than albumin
- indicator of malnutrition → increased in poor nutrition
- binds thyroid hormones and retinol-binding protein

c. Alpha 1- globulins

i. Alpha 1-antitrypsin: increased in Alzheimer’s disease

ii. Alpha 1-acid glycoprotein
iii. Alpha 1-antichymotrypsin
iv. Alpha-fetoprotein: can be associated with Trisomy 21
v. Gc-globulin (Group specific component globulin): transport vitamin D
vi. Inter-alpha-trypsin inhibitor
vii. Thyroxine binding globulin
viii. HDL
d. Alpha 2-globulins
✓ Alpha 2 macroglobulin: Increased in nephrotic syndrome→ 10 x increase
✓ Ceruloplasmin: copper carrying protein with enzymatic activities; marker for Wilson’s
disease
✓ Haptoglobin: carrier of hemoglobin in the circulation→ binds free hemoglobin to
prevent loss via urine→ decreased in intravascular hemolysis

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e. Beta-globulin
✓ LDL, HDL
✓ Transferrin: binds free iron (ferric iron); negative APR → decreased in cases of
inflammation
✓ Hemopexin: binds free heme
✓ Beta 2-microglobulin: component of light chain of HLA class 1
✓ Complement
✓C-reactive protein: APR, cardiac and inflammatory marker

f. Gamma-globulins
- Immunoglobulins

* Serum-protein electrophoresis: primarily used for monoclonal gammopathies

* Abnormal Total protein/ albumin→ SPE

Steps:

1. Separation of protein fractions

-Net negative charge (pH 8.6)→ migrate towards the anode
-Five bands: (cathode) gamma, beta, alpha 2, alpha 1, albumin (anode)

2. Fixation of protein fractions

→ immobilization via denaturation or precipitation → use acetic acid (precipitates proteins at
which they have migrated

3. Staining
- Ponceau S, Amido black, Coomasie blue
- qualitative identification: visual inspection of the bands → Width of the bands

4. Quantitation
→ densitometric scanners
→ relative value (%): multiply with total protein concentration → absolute concentration of the
protein fractions

ABNORMAL ELECTROPHORETIC PATTERNS

a. Nephrotic syndrome

- protein-lossing condition
- excretion of low molecular protein
- significant decrease in albumin
- no decrease in alpha 2 (relative increase in alpha 2-macroglobulin→ too large to be excreted
as part of the urine)

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b. Cirrhosis
- there is shadowing in gamma region to beta region→ BRIDGING EFFECT (significant
increase in IgA)

c. Emphysema
- significant decrease in alpha 1 region → deficiency in alpha 1-antitrypsin (major plasma
protein that comprises alpha 1 region → approx. 90 %)

d. Multiple myeloma
- involves monoclonal gammopathy (antibodies are produced in a single clone)
- there is a spike increase in the gamma region

e. Chronic inflammation
- polyclonal gammopathy
- diffuse increase

f. Acute inflammation

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 CLINICAL CHEMISTRY

TOTAL PROTEIN METHODS

Method Principle
Kjeldal (Reference for total protein) Protein digestion→ measure nitrogen content

Refractometry Based on refractive index of solute in serum

Biuret Cuprous ions bind with peptide bonds
(minimum of 2)→ violet

Alkaline environment
Dye-binding Ability of protein to bind to certain dyes
Turbidimetric Precipitation of proteins

ALBUMIN METHODS
Method Principle
Salt precipitation Globulins are precipitated, albumin remains
in the supernatant→ Biuret reaction
Methyl Orange Dye-binding; not specific
HABA
BCG (Bromcresol green) Dye-binding; sensitive but overestimate low
value
BCP (most recommended dye) Dye-binding; specific, sensitive and precise
Electrophoresis Most accurate

NON-PROTEIN NITROGEN COMPOUNDS

Urea→ amino acids→ uric acid→ creatinine→ creatine→ ammonia (least predominant)
Recall:
Predominant NPN? UREA

a. UREA: end-product of protein

✓ Azotemia (pre-renal, renal, post-renal)

- increased urea level in blood
a. Pre-renal: decreased perfusion/blood flow to the kidneys→ normal renal function
Example: congestive heart failure, shock, hemorrhage, dehydration, septicemia,
hypovolemia
b. Renal: abnormal renal function
c. Post-renal: obstruction

✓ Uremia
- increased urea with renal failure

✓ BUN: Conversion to Urea= BUN x 2.14

- 1st metabolite to increase in kidney disease
-correlated to concentration of creatinine
- BUN to Creatinine (10-20:1)

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 CLINICAL CHEMISTRY

-Avoid used of fluoride or citrate: tends to inactivate urease

- Refrigerate if delay of processing is anticipated

ENZYMATIC METHODS FOR UREA (Indirect methods)

Urea+ H2O→ 2NH4 + CO3

✓ GLDH (Glutamate Dehydrogenase) coupled: measures rate of NADH disappearance

NH4 + 2-oxoglutarate+ NADH→ glutamate + NAD= H2O
✓ Indicator dye: measures color change of pH indicator
✓ Berthelot: NH4 + NaOCL + Phenol→ Indophenol + NaCl + H2O
- sensitive to interferences

CHEMICAL METHODS (Direct methods)

✓ Diacetyl monoxime (DAM)

DAM + H2O → DIACETYL + HONH2
Urea+ diacetyl→ diazine (yellow) + water

✓ o-phthaldehyde (OPA)

b. URIC ACID: end-product of purine metabolism

-increase in cell turnover→ nucleic acids→ purine→ UA

✓ Chemical method
Uric acid+ phosphotungstic acid→ allantoin+ tungsten blue

✓ Enzymatic Methods: uricase is needed

Uric acid + O2 + H2O→ allantoin + CO2 + H2O

→ spectrophotometric @ 293 nm
→ coupled with catalase/peroxidase

Recall:
1. NPN increased in patients undergoing chemotherapy? URIC ACID
2. Lesch-Nyhan Syndrome? URIC ACID

c. CREATININE: end-product of muscle metabolism

- arginine, glycine, methionine→ creatine→ phosphocreatine→ creatinine
-index of over-all renal function
- completeness of 24-hour urine collection

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✓ Jaffe Reaction (Alkaline picrate method)

- Creatinine+ picric acid→ red-orange chromogen

a. Direct Jaffe method (End-point Jaffe): get 1 absorbance only; many interferences
(non-specific)
- Adsorbent technique: Fuller’s earth (aluminum magnesium silicate) or Lloyd’s
reagent (sodium aluminum silicate)

b. Kinetic Jaffe: Two absorbance reading

- serum + alkaline picrate→ rate of change is measured

Recall:
Fuller’s earth and Lloyd’s reagent

✓ 2,5-dinitrobenzoic acid (DNBA) + creatinine→ purple product

d. AMMONIA: by-product of amino acid deamination

- venous (EDTA) or arterial (heparin)→ freshly drawn to prevent false increase
- ammonia rises rapidly due to in-vitro deamination of amino acid
- Chilled/ iced→ spun (0-4 deg C within 20 minutes)→ separated→ tested immediately
* No hemolysis: RBC is 2-3x more ammonia than plasma

- Liver: parenchymal cells consume ammonia to produce urea → increased amount can
damage liver
- Neurotoxic substance→ encelopathy
- CSF can be used → glutamate is measured
- Blood: ammonia
- Reye’s Syndrome

Recall:
Analyte measure in comma using CSF? Glutamate

ENZYMOLOGY

ENZYMES
- specific biological proteins that catalyze biochemical reactions by lowering the activation
energy

✓ Cofactor: non-protein molecule needed for enzyme activity

Activator: inorganic cofactors (Mg, Cl and other electrolytes)
Coenzyme: organic cofactors → second substrates (NAD, NADH, vitamins
✓ Inhibitors:
Competitive: inhibitor binds to the active site
Non-competitive inhibitor: binds to the allosteric site

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Uncompetitive inhibitor: binds to enzyme substrate complex

✓ First-Order Kinetics: reaction rate is directly proportional to substrate concentration
✓ Zero-order Kinetics: dependent upon the enzyme; reaction rate depends on enzyme
concentration

ENZYME CLASSIFICATION AND NOMENCLATURE

* amino: aminotransferase, transaminase
* phosphate group: kinase

1. OXIDOREDUCTASE: catalyze oxidation-reduction reactions

- LDH, G-6-PDH
* dehydrogenase

2. TRANSFERASE: catalyze the transfer of a group

- AST, ALT, CK, GGT

3. HYDROLASE: catalyze hydrolysis of various bonds

- ACP, ALP, AMY, CHS
-enzymes of digestions

4. LYASE: catalyze the removal of groups

- Aldolase, pyruvate decarboxylase, glutamate decarboxylase

5. ISOMERASE: catalyze the interconversion of isomers

- glucose phosphate isomerase, ribose phosphate isomerase

6. LIGASE: catalyze the joining of two substrate molecules

- glutathione synthetase

***Review: EC NUMERICAL CODE

1ST digit: enzyme class
2nd digit: enzyme subclass
3rd digit: sub-subclass
4th digit: serial number specific to the enzyme

ASPARTATE AMINOTRANSFERASE (SGOT)

✓ used in conjunction with ALT → Hepatocellular damage
✓ catalyzes the reversible reaction between aspartate and alpha-ketoglutarate to form
oxaloacetate and glutamate
✓ Karmen method: coupled enzymatic reaction
✓ Coupling enzyme: malate dehydrogenase

Hepatocellular damage: AST + ALT

Hepatobiliary: ALT + GGT
ALP (abnormal)→ GGT (normal): Bone disease
GGT (abnormal): Liver disease

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ALANINE AMINOTRANSFERASE (SGPT)

✓ catalyzes the reaction between alanine and alpha-ketoglutarate to form glutamate and
pyruvate
✓ Highest elevation: acute liver hepatitis
* De Ritis ratio (AST: ALT ratio)
> 1: non-viral (alcohol)
< 1: viral hepatitis
✓ Enzymatic method: Wacker method → coupling enzyme: Lactate dehydrogenase
✓ Reitman-Frankel Method: reaction with 2,4-dinitrophenyl hydrazine

LACTATE DEHYDROGENASE

H subunit; M subunit
LD1: HHHH
LD2: HHHM
LD2: HHMM
LD4: HMMM
LD5: MMMM
Acute Myocardial Infarction: LD1>LD2

Isoenzymes catalyze the same reaction; increase specificity of enzymes

✓ Catalyzes the reversible conversion of lactate and NAD into pyruvate and NADH
✓ Electrophoretic mobility: LD5→ LD4→ LD3→ LD2→ LD1
✓ Concentration in serum: LD2→ LD1→ LD3→ LD4→ LD5
✓ Highest elevation: pernicious anemia (megaloblastic)
✓ Wacker: forward reaction (lactate to pyruvate)
✓ Wrobleuski La Due: reverse reaction (pyruvate to lactate)

Recall:
LD5 → cold labile (specimen should not be frozen)

CREATININE KINASE
✓ Catalyzes the transfer of phosphate to creatine
✓ Electrophoretic mobility: CK3→ CK2→ CK1
✓ Highest elevation: Duchenne’s muscular dystrophy
✓ Specimen consideration: no to hemolysis and should be protected from light
*Adenylate kinase (present in RBC): catalyzes the same reaction as CK → falsely
increased
* CK is inactivated by light→ falsely decreased

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✓ Tanzer-Gilvarg: forward reaction

Coupling enzyme: pyruvate kinase and lactate dehydrogenase
✓ Oliver-Rosalki: reverse reaction
Coupling enzyme: Hexokinase and G6PD
✓ Isoenzymes
* CK1: CK-BB
* CK2: CK-MB→ cardiac muscle
* CK3: CK-MM→ skeletal muscles

ALKALINE PHOSPHATASE

✓ Catalyzes the hydrolysis of phosphomonoesters into alcohol and phosphate at an alkaline

pH (pH 9-10)
✓ Normal isoenzymes: intestinal, placental, bone, liver
✓ Carcinoplacental isoenzymes: Regan, Nagao, Kasahara
✓ Electrophoretic mobility (increasing): intestinal→ placental→ bone→ liver
✓ Heat denaturation: placental (most heat stable) → intestinal→ liver→ bone
*Shinowara-Jones-Reinhart: beta-glycerophosphate
*King-Armstrong: phenylphosphate
*Bessey-Lowry-Brock: p-nitrophenyl phosphate
*Bowers-McComb: p-nitrophenyl phosphate
✓ Used in the evaluation of hepatobiliary and bone disorders
✓ Highest elevation: Paget’s disease

ACIP PHOSPHATASE
✓ same reaction with ALP but in an acid pH
✓ Highest concentrations: prostate gland RBC
✓ Electrophoretic separation: Erythrocytic ACP→ Prostatic ACP
Bodansky: beta-glycerophosphate
Gutman, King-Armstrong: phenylphosphate
Hudson: p-nitrophenylphosphate
Babson and Reed: alpha-naphthyl

GAMMA GLUTAMYL TRANSFERASE

✓ Involved in the transfer of the glutamyl residue from gamma glutamyl peptides
✓ most sensitive: hepatobiliary disease
✓ used for the evaluation of obstructive jaundice, biliary obstruction and liver cancer
✓ First abnormal liver function test in heavy drinkers

Other enzymes

✓ Cholinesterase: significant when decreased

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✓ Amylase:
Amyloclastic:
-starch-iodine (dark-blue)
-conversion of starch substrate with iodine→ starch will be degraded
- the lighter the color, the more amylase

Saccharogenic:
-classic reference; uses Somogyi units
- reducing property of sugars

Chromogenic:
-increase in color intensity

Continuous monitoring
-coupled enzyme systems

✓ Lipase:
- in conjunction with amylase→ markers for acute pancreatitis
- elevated up to 7 days

CARDIAC MARKERS

Enzymes used as cardiac markers:

1. AST
2. LDH
3. CK: readily detected in plasma after MI
4. CK-MB → increases more rapidly than CK

Cardiac proteins as cardiac markers:

1. Myoglobin: more sensitive than CK-MB only during the first hours→ not reliable long-term
marker (returns to normal rapidly)

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2. Troponin l: most sensitive indicator of cardiac injury; remain elevated for a longer period of
time (within 6 days)

3. Troponin T: remains elevated longer than Troponin l (higher sensitivity after 7 days)

ENDOCRINOLOGY

Exocrine gland: gland with ducts

Example: sweat glands, sebaceous glands, pancreas (secretes
enzymes of digestion)

Endocrine glands: ductless glands→ release secretions into the blood streams
Examples: pituitary, adrenal, kidneys, pancreas (hormones for
glucose metabolism)

Two mechanisms of how the endocrine works

a. Negative feedback

✓ Stimulus: releasing hormone (hypothalamus)→ stimulating hormone (anterior

pituitary gland)→ target → release specific hormone

✓ An increase in the hormone will cancel out the release of the hormone itself
✓ Example: PTH→ Stimulus: low blood Ca→ PTH (elevate blood Ca)→ decreased
PTH

b. Positive feedback
✓Stimulus: release of a hormone→ target→ increase in the stimulus
✓ Oxytocin: uterine contraction→ increase oxytocin→ increase uterine
contraction
: only hormone that works via positive feedback

a. HYPOTHALAMUS
-lower portion of the brain above the pituitary gland
✓ Control gland of the entire endocrine system
✓ Releasing hormones→ hypophysiotropic hormones (releasing hormones):
-Thyroid releasing hormone: TSH and prolactin
-Gonadotropin releasing hormone: Luteinizing hormone and follicle-stimulating
hormone
-Somatostatin: inhibits growth hormone and TSH
-Corticotropic releasing hormone: ACTH and GHRH
-Growth hormone releasing hormone: Growth hormone

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Anterior pituitary gland→ release stimulating hormones

Posterior Pituitary gland: stores and releases ADH and oxytocin to the circulation

Hormones produced by hypothalamus

1. ADH (Arginine vasopressin): water reabsorption
Stimulus: hyperosmolar plasma
Target: DCT and collecting duct

2. Oxytocin: uterine contractions and ejection of milk for females and aids in erection for
males

b. PITUITARY GLAND

✓ Anterior pituitary gland (Adenohypophysis):

- TSH, ACTH
-GH
-FSH Master gland: pituitary gland
-LH
-Prolactin

✓ Posterior pituitary gland (Neurophysis): does not produce hormones

-ADH
-Oxytocin

Recall:
2 types of hormone released by anterior pituitary gland?

1. Tropic hormones: actions are specific for another

endocrine gland→ stimulating hormones
Example: TSH, ACTH, FSH, LH

2. Direct effectors: act directly on peripheral tissue

Example: GH, Prolactin (milk production)

c. THYROID GLAND
-Follicular cells produce thyroid hormones/metabolic hormones (T3 and T4)

Protein Carriers:
Thyroid-binding globulin (70-75% of the total thyroid hormone)
Thyroxine-binding pre-albumin (20%)
Thyroid-binding albumin (10%)

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Hormones involved:
TRH (hypothalamus) → TSH (released by anterior pituitary gland)→ Thyroid gland (T3 and T4)

Primary: organ defective is the target gland

Secondary: organ defective is the anterior pituitary gland

✓ Hypothyroidism (myxedema in adults or cretinism/congenital hypothyroidism in infants)

a. Primary: decrease T3/T4; increase TSH
b. Secondary: decrease T3/T4; decrease TSH

Clinical manifestations:
* Obese/fat- because of low level of T3/T4→ low metabolic rate→ weight gain
* Inactivity
* Decrease in appetite
* Cold intolerance

✓ Hyperthyroidism
a. Primary: increase T3/T4; decrease TSH
b. Secondary: increase T3/T4; increase TSH

Clinical manifestations:
* Thin/slim- increased metabolic rate
* Weight loss
* Hyperactivity
* Increase in appetite
* Heat intolerance

✓ Autoimmune disorders
Hashimoto’s: anti-microsomal Ab, anti-thyroglobulin Ab, anti-thyroid peroxidase Ab
(Thyroid peroxidase Ab)
-hypothyroid state

Grave’s: anti-TSH receptor Ab→ this antibody mimics the TSH (function in stimulating
thyroid hormone production)
-hyperthyroid state
d. PARATHYROID GLAND
- calcium homeostasis

Substances for maintaining normal Ca homeostasis:

1. Parathyroid hormone: increase in blood Ca levels

Stimulus: low blood Ca
-promote bone resorption/ bone dissolution/bone destruction

2. Calcitriol: increase blood Ca levels

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→ Vitamin D is the inactive form

→ directly involved in intestinal absorption of Ca
3. Calcitonin: decrease in blood calcium levels

Recall:

1. Which of the following is not a hormone that increases

blood calcium level?
a. PTH
b. Calcitriol
c. Calcitonin→ produced by the parafollicular cells of the
thyroid gland or c cells
d. Vitamin D

2. A marker for medullary thyroid carcinoma? Calcitonin

e. ADRENAL GLANDS

✓ Cortex (outer layer)

a. Zona glomerulosa: produces mineralocorticoids
* Aldosterone
→ a mineralocorticoid that is involved in reabsorption of sodium

b. Zona fasciculata: produces glucocorticoids

* Cortisol
→ a glucocorticoid that is also known as the stress hormone but not the
first to be produced (respond within minutes)
→ a hyperglycemic agent that increases blood glucose cells

c. Zona reticularis: produces weak androgens (sulfates DHEA or

dehydroepiandrosterone)

✓ Medulla (inner layer)

a. Catecholamines
→ epinephrine and norepinephrine
→ first responders to stress (within seconds)

Recall:
Metabolite of epinephrine and norepinephrine: VMA (vanillylmandelic acid)

f. PANCREAS
→ has exocrine (secretes enzymes of digestion: pancreatic lipase and pancreatic
amylase for the diagnosis of acute pancreatitis) and endocrine functions

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Hormones produced:
a. Glucagon: produced by alpha cells
b. Insulin: beta cells
c. Somatostatin: delta cells

SEX HORMONES

a. Testosterone: most potent male sex hormone

b. Estrogen
✓ Estrone: significant in menopausal women
✓ Estradiol: most potent female sex hormone
✓ Estriol: main source is placenta (together with progesterone)→ significantly increased
in 2nd and 3rd trimester of pregnancy

c. hCG: detected during the 1st trimester of pregnancy

- maintain vascularization of uterine wall
- Pregnancy, Trisomy 21, malignancies
- Ectopic pregnancy, Trisomy 18, Trisomy 13

ACID-BASE BALANCE

DEFINITION OF TERMS

✓ According to Arrhenius

ACID: substance that can yield hydrogen ion or hydronium ion (H3O+) when dissolved in
water
BASE: substance that can yield hydroxyl ion (OH-) when dissolved in water
BUFFER: consists of a weak acid and its salt or conjugate base; weak acid or weak base and
its salt

Henderson-Hasselbach Equation→ computation of pH

pH= 6.1 (pK) + log (HCO3/pCO2 x 0.03)

HCO3→ concentration of bicarbonate

pCO2→ partial pressure of CO2

Note:
Bicarbonate: numerator (indicates kidney function→ modifies concentration of HCO3)

Lecture by: Ma’am Peewee Compiled by: Ara

 CLINICAL CHEMISTRY

Partial pressure CO2/ carbonic acid: denominator (represents lung function→ modifies
pCO2)

Imbalances:
Normal values
✓ pH 7.35-7.45: normal pH of the body
✓ pCO2: 35-45 mmHg
✓ HCO3: 22-26 mmol/L

a. Alkalosis (pH >7.45)

Increase pH= increase bicarbonate, decrease in CO2

b. Acidosis (pH <7.35)

Decrease pH= increase Co2, decrease bicarbonate

✓ Metabolic/ Non-respiratory AB imbalance

Affected organ: kidney
Abnormal concentration: HCO3
Compensatory organ: Lungs

a. Metabolic Alkalosis: increase HCO3

Response: conserve CO2→ hypoventilation

b. Metabolic Acidosis: decrease HCO3

Response: expel CO2→ hyperventilation

✓ Respiratory AB Imbalance
Affected organ: lungs
Abnormal concentration: pCO2
Compensatory organ: kidneys

a. Respiratory alkalosis: decrease pCO2

Response: decreased reabsorption HCO3 (excretion)

b. Respiratory acidosis: increase pCO2

Response: increase reabsorption of HCO3
Compensation:
a. LUNGS: immediate, short-term and incomplete
b. KIDNEYS: slower (2-4 days), long-term and complete

✓ Full compensation: pH is normal (the other 2 parameters are abnormal)

✓ Partial Compensation: pH is approaching normal
* All parameters are abnormal
✓ No compensation: pH is abnormal

Lecture by: Ma’am Peewee Compiled by: Ara

 CLINICAL CHEMISTRY

* Normal: other parameter; normal: pH plus one parameter

Measurement:
ABG Analysis
→ pH: electrode is glass
→ pCO2: Severinghaus
→ pO2: Clarke
→ HCO3: calculated parameter

Lecture by: Ma’am Peewee Compiled by: Ara