Clinical Chemistry

Review Handbook
 TABLE OF CONTENTS

page
Units of Measure
Quality Control 2
Parameters of Quality Control 2
Kinds of Quality Control 3
Variations 5
Statistics 8
Quality Control Chart 9
Analytical Methods 17
Instrumetation 32
Pipettes 34
Automation 29
Patient Preparation 36
Specimen Collection 39
Arterial Puncture 39
Venipuncture 41
Skin Puncture 45
Anticoagulants 52
Carbohydrates 54
Diabetes Mellitus 59
Gestational Diabetes Mellitus 56
Methods 59
Oral Glucose Tolerance Test 63
Glycosylated hemogiobin 65
Glycogen Storage Disease 67
CSF Glucose 67
Lipids and Lipoproteins 69
Phospholipids 69
Cholesterol 70
Triglycerides 73
Lipoproteins 77
Lipid and Lipoprotein Disorders 84
Fredrickson Classification 86
 Page

Proteins 88
Microalbuminuria 97
CSF Proteins 97
Kidney Function Test 100
Glomerular iltration Rate 100
Clearance 100
Cystatin C 103
Blood Urea Nitrogen 103
Creatinine 104
Blood Uric Acid 107
Tubular Function Test 110
Liver Function Test 112
Total Protein 112
Electrophoresis 113
Bilirubin 117
Ammonia 122
Enzymes 124
Classification of Enzymes 126
Alkaline Phosphatase 129
Acid Phosphatase 131
Transaminases 132
Amylase 134
Lipase 136
Lactate Dehydrogenase 137
Creatine Kinase 139
Aldolase 141
Gamma-glutamyl Transferase 141
Electrolytes 148
Anion Gap 156
Cystic Fibrosis 156
Iron 157
Blood Gases and pH Measurements 159
Vitamins, Trace Elements and Tumor Markers 166
Laboratory Mathematics 169
Conversion Factors 173
 Page

Reagent Water 174

Laboratory Safety 176
EndocrinoloEY 179
Pituitary Gland 181
Thyroid Gland 186
Parathyroid Gland 192
Adrenal Gland 194
Reproductive Hormones 203
Miscellaneous Hormones 206
Methods 207
Therapeutic Drug Monitoring 210
Toxdcoloey 222
Prohibited Drugs 227
Definition of Terms 233
References 245
Clinical Chemistry
it is basic science that utilizes the
a
speciaBty of chemistry to study human beings in various
stages of heaith and disease.
I t is applied science when analyses are performed on body fluids or
an
tissue specimens to
provide important information for the diagnosis or treatment of disease.

Units of Measure
In reportring the concentration of analytes in blood, urine, spinal fluid and other body fluids, the
units of should be strongiy considered.
measure
Measurements of analytes in a biochemistry
the systéme internationale.
laboratory may either use the conventional unit or

Systéme Iternationalee (SI)

t i s consists of
sevenindependent base units, and each unit is represented bya symbol.
It is used because
compounds react on a molar basis, and expression of amounts of substances
in such terms allows for a better
understanding of the relative proportion of compounds.
Three (3) classes of Sl units: Base, Derived and
Supplemental tnits
Seven Base Sl units
Measure Unit
Meter (m) Length
Kilogram (kg Mass
Second (s) Time
Mole (Mo Quantity of substance
Ampere (A) Electriccurrent
Kelvin (K) Thermodynamictemperaturee
Candela (cd) Luminous intensity
Notes to Remenber
Systéme internationale (SI) is recornmended because compounds react on a
expression of the concentration following that unit of measusrement molar basis, and
allows for a better
understanding of the relative proportion of compounds, hence, it has been
analytes be reported using moles of solute/volume of solution recommended that
(mmol/L).
Enzyrmatic activity is measured utilizing the international unit per liter (u/Lor U/l) or the katal
unit per liter (KU/L).
pH scale is retained for measurement of
Liter is used as the reference volume
hydrogen ion concentrations.
under systéme international.
 QUALITY ASSURANCE IN THE
CLINICAL CHEMISTRY LABORATORY
Quality Control
I t is a system of
ensuring accuracy
and grecision in the
laboratory by including quality control
reagents in every series of measurements.
t is a proess of ensuring-that analytical results are
correct by testing known samples that
resemble patient samples.
t involves the process of moaitoring the
characteristics of the analytical processes and detects
analytical errors- during testing, and ultimaltely preverit the reporting of inaccurate patient test
results.
I t is one component
of the quality assurance system, and is part of the performance monitoring
that occurs after a test has been established.

PARAMETERSOF
1. Sensittvity
QUALITY CONTROL:
I t is the ability of an analytical method to measure the smallest concentration of the analyte of
interest.
2. Specificity
It is the ability of an analytical method to measure only the analyte of interest.
3. AcCuraçy
It is the nearness or closeness of the
assayed value to the true or targetvalue.
I t is estimated using 3 types of studies: recovery, intereference and
patient sample comparison.
Recovery study determines how much of the analyte can be identified in the sarnple;
interference study determines if specific compounds affect the laboratory tests like
hemolysis,
turbidity and icteric; sample comparison study is used to assess presence of error (inaccuracy) in
actual patient sample.
4. Precision or reproduelbility
It is the ability of an analytical method to give repeated-results on the samie san1ple that agree
with one another.
5. Practicability
I tis the degree by which a methodisgasily repeated.
6. Rellability
I t is the ability of an analytical method to maintain accuracy and precision over an extended
period of time during which equipment, reagents and personnel may change.
7. Diagnostic sensitivity
I t i s the ability of the analytical method to detect the proportion of
individualswiththe
tindicates the ability of the test to generate more true-positiveresults and few false-negative
disease.
Screening tests require high sensitivityso that no case is missed.
Sensitivity(%) =100 x the numberofdiseasedindividualswith apositive test
Total number of diseased individuals tested

8. Diagnostic specificity
t is the ability of the analytical method to detect the proportion of individuals without the
disease.
It reflects the ability of the method to detect true-negatives with very few false-positives.
Confirmatory tests require high specificity to be certain ofthe diagnosis.

2
 Specificity (%) =100 x the number of individuals without the disease with anegative test
Total number of individuals tested without the disease

Note: 100% sensitivity and specificity indicate that the test or method detects every patient with the
disease and that the test is negative for every patient without the disease.

KINDS OE QUALITY CONTROL

1. Intralab Quality Control (Internal QC)
It involves the analyses of control
samples together with the patient-specimens.
It detects changesin
performance between the present operation and the "stable"-operation
t is important for the daily
monitoring of accuracy and precision of analytical methods
t detects both random and systematic errors in a daily basis.
It allows identification of
analytic erors within a one-week cycle.
2. Interlab Quality Control (Extemal QC)
t involves proficiency testing programs that periodically provide samples of unknown
concentrations to participating cinical laboratories.
It is important in maintaining long-term accuracy of the
analyticat methods.
It is also used to determine state-of-the-art interlaboratory performance.
The College of American Pathoiogists (CAP) proficiency program is the gold standard for clinical
laboratory external QC testing.

Conduct Aofseries
the ExternalOc Testing:
of unknown samples are sent to the laboratory from the reference laboratory or
authorized program provider.
Unknown samples must be tested by the laboratorians who regularly perform analysis of patient
specimensusing the same reagents and equipment for actual patient specimens, and the results
are submitted to the pragram provider, preferably as soon as every analysis is done.
Analysis of the unknown samples should be completed within the usual time as for the routine
samples.
Unknown samples should be treated like a patient specimen to determine the true essence of
accuracy.
Results of the proficiency testing must not be shared with other laboratories"during the testing
period" comparison studies can be made after the testing cycle to identify areas for
improvement.
Some proficiency tests are not quantitative but are qualitative, however for chemistry, it should
be quantitative.
f there is no available proficiency testing program for a certain analyte, it is required to
implementa non-proficiency test scheme.

Interpretationof the Resuts.ofthe ProficencyTesting:

analyte has define performance criterla (example, +/-3SD for peer mean),
Each a
laboratories using the sane method are evaluated by comparing them with the group.
where

in external ac. difference of greater than 25D in the resuits indicates that a laboratory is not in
agreement with the rest of the laboratories included in the program.
f in case a cinical laboratory failed to identity or resolve an error or discrepancy in the test
process, the facitity is at risk of continous operation and may be recommended for dlosure.
Rationale of the External oc/Profciency Testing:
The ultimate goal of proficiency testing is to ensure our clinicians that patient results are
accurate
Proficlency testing allows each laboratory to compare and evaluate test
those laboratories that use the same methods results or outcomes with
(reagents and equipmert).
Data obtained from the proficlency testing can be used
to continuously
performance, and also serve as troubleshooting guide when investigating improve
analytic error. test

Obiectives of Quality Control

1. To check the stability of the machine.
2. To check the quality of
reagents.
3. To check technical (operator) errors.

Control
The
Solutions (QC Materals
accuracy of any assay depends on the control solutions, how
how they remain stable overtime.
they are originally constituted and
General chemistry assays used 2
levels of control solutions, while immunoassays used 3 levels.
To establish statistical quality control on a new
instrument or on new lot numbers of control
materials, the different levels of control material must be analyzed for 20 days.
For highly préclsed assays (wlth CV less than
1%) such as blood gases, analysis for 5 days is
adequate.

Control Limits (Control Values);

These are expected values represented by intervals of acceptable values with upper and lower
limits.
If the expected (control) values are within the desired controi limits, the clinícians are assured that
the test results are accurate and precise.
Control limits are calculated from the mean and standard deviation (SD).
The ideal control/reference limit is between +/-2sD.
Use of a single lot for an extended period allows reliable interpretative criteria to be established
which will permit efficlent identification of an assay problem.
When changlng to a new lot number, laboratorians use the newly calculated mean value as the
target mean butretain the previous SD value, but when more data are obtained, all values should be
averaged to get the best estimates of the mean and SD.
Determination ofthe mean and SD for the unassayed controls is also advisable because this process .

improves the performance characteristics of statistical control procedures.

Charecterstes of an IdealOC Material:

1. Resembles human sample.
2. Inexpensive and stablefor long periods.
3. No communicable diseases.
4. No matrbx effects/known matrix effects.
5. With knowrni analyte concentrations fassayed control).
6. Convenient packaging for easy dispensing and storage.
Notes to Renembek resembie human sampBe and be available for a minimum of one
Quality control materials should
sarme material have different concentrations
the
yeereemelot-number) different lot numbers of
which requires new estimates of the mean and standard deviation.
biohazard considerations,
control materlals are preferred but because of limited sources and
Human
bovinecotrotmaterials are used.
Bovine-based QC material is notr the choice forimmunochremistry, dye-bimding and certain bitirubn

aseys.
QC materlals thoutd be the same matrikas the specimens being tested, for example, measurement
of glucose in serum should have control solutions that are prepared from serum. wied human and
Mieti effec are resuts of improper-product menufacturing
nonhumen analyteadditves and altered protein componens
Control materials can be purchased with or without assayed values
Assayed contrels are more expensve but can be used as externalcheeks-fer-aseuraay.
Reconstitution of tyophilized control materials must be property done to avoid incorrect control

values.
Seeszedfrozen eoros do not require reconstitution but may have different characterizations
compared to actual patient specimens.

Comparison of Analytical Methods (Method Evaluation):

The test method must be compared always with a method of acceptable accuracy such as the
standard reference method (gold standard).
It is recommended by Westgard et al and Clinical Laboratory Improvement Amendments (CLIA)
that 40 to 100 samples be run by each method in duplicate on the same day over 8 to 20 days,

ideally within 4 hours, to determine its accuracy and precision.

If only 40 samples will be measured, daily analysis in duplicate of 2 to 5 specimens should be
followed for at least 8 days.
method and reference method) are
Duplicate analyses of each sample by each method (test
and in different order of
recommended, with the duplicate samples analyzed in different runs
analysis on the two runs (should be performed within 4 hours).
The rationale for performing repeated assays is to detect random errors that affect precision.
to rule out technical errors as
After analyses, samples with wide difference should be repeated
the source of variation.
is to determine if the total error
The most important characteristic of method evaluation
allowable error (E,).
(random and systematic error) is less than the

VARIATIONS
Areers encountered in the collection,preparation and measurementof samples,
including transcription and releasing of laboratoryresults.

Ivpes of Error
1. Random Error
.Itis present in all measurements; it is due to chance.
Itls a type oferror which varies from sample to sample.
measurements-variations in technique.
I t is the basis for varying differences between repeated
 .It is due to instrument, operator and environmental conditions (variations intechniques)such as
pipeting errer, mislabeling of samples, temperature fiuctuation, and improper mixing of sample
andreagent.

2. Systematie-Error
t i s anerorthat influences observations oenelstenthi-ene-d*eeton (constant difference).
I t is detected as ehe egeebas often related to calibration problems,
deterioration of reagents and control materlals, improperly made standard solutions,
contaminated solutions, unstable and inadequate reagent blanks, leaky ion selective electrode
(ISE), faliling instrumentation and poortly written procedures.
t is a measure of the agreement between the measured quantity and the true value.

a. Constant Error
it refers to a difference between the target value and the assayed value.
it is independent of sample concentration.
it exists when there is a continual difference between the comparative method and the
test method regardless of the concentration.

b.Proportional/Slope/Percent Error
it results in greater deviation from the target value due to higher sample concentration.
it exists when the difference between the test method and the comparative method
values is proportional to the analyte concentration.

3. Clerical Error
I t is the highest frequency of clerical errors occurs with the use of handwritten labels and
request forms.
most ommon
Notes to Remember
>Indicators of analytic performance: internal QC, proficiency testing, accreditation, quality
assurance monitoring and laboratory utilization
The first step in method evaluation is the precision study which estimates the random error.
T o study imprecision or random error, 2 control solutions are run twice a day in a 10- to 20-day
period-by testing multiple samples on different days, a better assessement of random error
over time is achieved.
The total imprecision analysis is the most accurate measure of performance that would affect
the laboratory values a dlinician might observe and reflects differences such as in the work of
technologists, pipetting and temperature fluctuations of the analyzers.

6
Pre-analyticalErmrors
1. Incorrectpatient identification
2. Improper patient
preparation
3. Incorrect
specimen collection
4. Mislabeled specimen
5. Incorrect order of draw
6. Incorrect used of tubes for blood collection
7. Incorrect anticoagulant to blood ratio
(short draw)
8. Improper mixing of blood and
9. Incorrect specimen
anticoagulant
preservation
10. Mishandled specimen
(transport and storage)
11. Incorrecty
interpreted/ordered
12. Incomplete centrtfugation
laboratory test
13. Incorrect data
log-in

AnalyticalLErrors:
1. Incorrectsample and reagent volume
2. Incorrect incubation of solution
3. Equipment/instrument malfunction
4. Improper calibration of
equipment/calibration error
Post-analvtical EronE
1. Unavailable or delayed laboratory results
2. Long turnaround time
3. Incomplete laboratory results
4. Wrong transcription of the patient's data and laboratory results
5. Missing laboratory results
6. Laboratory results submitted to the wrong physician/doctors who did not request for the lab test

Notes to Remember:
Preanalysis refers to all the activities that take place before testing, such as test ordering and
sample collection.
The most frequent preanalytic errors include improperly fiing-the-sample tube, placing
specimens in the wrong containers or preservatives, and selecting the incorrect test.
The analysis stage consists of the laboratory activities that actually produce a result, such as
running a sample on an automated analyzer. Postanalysis comprises patient reporting and result
interpretation.
>The length oftime elapsed between drawing and the separation of serum or plasma from the
cells can be a factor in analytical testing.
Nonlaboratory personnel were responsible for 29% of the errors with regard to laboratory
results.
Online computer input is the most error-free means of requesting laboratory tests.
Most laboratory errors occur in the preanalytic and pestenatytic stages.
In one study, error rates were reported to range from Q.05K ta0.61%, and the distribution of
errors among the testing stages was similar, with most (32%-75%) occurring in the preanalytiG
stage and far fewer (13%32%).in tbe anahticstage (Bonini et al, 2002 cited in Mc Pherson and
Pincus 2017).
Allowable Error (E)
t is determined for each test method and is expressed either in measurement units of the
analyte (mmol/L} or percentages.
Itis based on the quantity of error that will negatively affect clinical decisions.
The total error (random, proportional, constant and systematic error) must be less than the E, or
fixed imits for a method to be considered acceptable.
fthe total error is greater than E, corrections must be made to reduce the error or the method
be rejected
The determination of whether long-term precision is sufficient is based on the total imprecision
being less than one-third of the allowable error.

STATISTICS
I t is the science of gathering, analyzing, interpreting and presenting data.

1. Mean is a measure ofcentral tendency. It is associated with symmetrical or normal

distribution
Mean
n

2. Standard Deviation (SD)- is a measure of the dispersion of values from the mean. It helps
describe the ncrmal curve. A measure of the distribution range. It is
the most frequently used measure of variation

SD Zx-mean)}

n-1

3. Coefficient of Variation (CV) is a percentile expressionofthe mean; an index of precision

CV SD
x 100
Mean

4.Variance- is called the standard deviation squared; a measure of variability. It represents the
difference between each value and the average of the data
V (SD)

Teminologies:
1. Inferential Statistics are used to compare the means or standard deviations of two groups of data
2. F-test is used to determine whether there is a statistically significant difference between the
standard deviations of two groups of data
3. Median is the value ofthe observation that divides the observations into two groups, each
containing equal number of observations. t is the midpoint of a distribution; soh centile
4. Mode s the most frequent observation; it is used to describe data with two centers(bimodal).
S. Range-is the simplest expression of spread or distribution; it is the difference between the highest
and lowest score in a data
6. Standard Deviotion index (SDI)- is the difference between the value of a data point and the mean
value divided by the group's SD

8
7. t-test i s used to determine whether there is a statistically significant difference between the means

of two groups of data

Notes to Remember. a method has, and

Statistical analyses are used to determine the types and quantity of error that
clinical decisions.
to decide whether the test is still valid or unacceptable to make
Parametric te_ts: t-test and analysis of variance (ANOVA)
3 measures of spread or distribution: CV, SD and range
S D describes the distribution of all values around the mean.
S D and variance represent the average distance from the center of the data (mean) and every value
in the data set.
CV allows a laboratorian to compare SDs with different units.
The CV of analyzers described as having reproducible test results can be lower than 1%.
Degree offreedom (n-1) indicates the number of quantities free to vary.
ANOVA is used to analyze precision data to give estimates of the within-in run, between-run and
>
total imprecision.
is valid.
Method evaluation and statistical analysis are essential, but not sufficient to decide if a test
The acceptable range is 95% confidence limit which is equivaient to t 2SD.

QUALITY cONTROL CHART

It is used to observe values of control materials over time to determine reliability of the
analytical method.
It is utilized to observe and detect analytic errors such as inaccuracy and imprecision.

1. Gaussian Curve (Bell-Shaped Curve)

It occurs when the data set can be accurately described by the SD and the mean.
t is obtained by plotting the values from multiple analyses of a sample.
It is a population probability distribution that is symmetric about the mean.
It occurs when data elements are centered around the mean with most elements close to the
mean.
t focuses on the distribution of errors from the analytical method rather than the values from a
healthy or patient population.
The total area under the curve is 1.0 or 100%.

2. Cumulative Sum Graph (CUSUM)

t calculates the difference between QC results and the target means.
Common method: V-mask
It identifies consistent bias problems; it requires computer implementation.
This plot will give the eariest indication of systematic errors (trend) and can be used with the la
rule.
It is very sensitive to small, persistent errors that commonly occur in the modern, low
calibration-frequency analyzer.
Results are out of control when the slope exceeds 45 or a decision (t 2.7 SD) is exceeded.

9
3. Youden/Twin Plot
t is used to compare resuts obtained on a high and low control serum from different
laboratories.
t displays the results of the analyses by plotting the mean values for one specimen on the
ordinate (y-axis) and the other specimen on the abscissa (x-axis).
The points falling from a center but on the 45° line suggest a proportional error, and points
falling from the center but not on the 45 line suggest a constant error.

4. Shewhart Levey-Jennings Chart

t is the most widely used QC chart in the cinical laboratory.
t allows the laboratorians to apply mutiple rules without the aid of a computer.
* t i s a graphic representation of the acceptable limits of variation in the results of an analytical

method.
It easily identtfies random and systematic errors.

Erors whlch can beobserved in LJ chart:

a. Trend
I t i s formed by control values that either increase or decrease for six consecutive days.
Main cause: Deterioration of reagents

b. Shift
It is formed by control values that distribute themselves on one side or either side of the mean
for six consecutive days.
Shift in the reference range is due to transient instrument differences.
Main cause: Improper calibration of the instrument
.Outiiers
These are control values that are far from the main set of values.
These are highly deviating values.
These are caused by random or systematic errors.

5. Westgard Control Chart

I t recognizes that the use of simple upper and lower control limits is not enough to identify
analytical problems.
in Westgard, error detection rates can increase without increasing the false rejection rate.
Westgard used the term control rule to indicate if the analytical process is out of control.

10
 It ie2 obeerved when the last 2 control rosuta
either
(or reuts from the same run) exoeed
the mean 2SD; due to aystematic error.

34 07
The last four (or any four) consecutive control
results exceed either mean 1SD; due to A4ule
ystemaicerror. Hotion

1114 19_10
The range or difference between the highest
and lowest contral result within an analytical
runexceeds 4yi it is due to random error. FINRYNNT

ARMerton
rul

14 76110
6x We need to reject an analytical run when 6
us
consocutive control measurements fall on one vloktion
side of the mean. L

We need to reject an analytical run when

seven control measurements "trend" in the
same direction, ie., get progressively higher or
M
progresavely lower.
vlolstlon
3 48 7 8 10
We need to reject an analytical run when 8
consecutive control measurements fall on one
side of the mean.

Moliton
i
9 We need to reject an arnalytical run when 9
consecutive control measurements fal on one
side of the mean.
ru

10 It is obeerved when 10 consecuive results are

oa the same side of the mean; due to
aystematic error.

10,
vioiation
123 4 6 7 & 10
12 reject when 12 consocutive control
measurements fall on one side of the mean

2 , rule
violationn

133 45 7 0 11 12

11
 We need to reject an analytical run when 9
onsecutive control measurements fal on one
side of the mean.

10 It is obeerved when 10.consecutive rexulta are.

on: the same side of the mean; due to
ystematic error.
.
.
AA
Lil
ehtion
12. Teject hen 12 consecutive control
mesurements fall on one side of the mean

2ot3 1204 a 10 112

Wenced to reject an analytical run when 2 out
of 3 control meaurements exceed the same
neen pius 2s or mean mimus 2s control limit
i-{ vioaton

31 We need to reject an analytical run when 3

coneecutive control measurements exceed the
same mean plus ls or mean minus l8control
Mohion

Notes ta Remember.
In measuring.systematic error or inaccuracy, Westgard et al recommend that at least 40
samples, and preferably 100 samples be run by comparison-of-methods experiment (tes:
method andreference method).
The combination of the control rules used in confunction with a control chart has been called
the MultiruBe Shewhart procedure.
Multirules establsh driterfa for deciding whether an analytlc process Is out of control.
The sensitivity of the multirule procedure can be
increased to
detect smaller systenmatic errors
by increasing the numberofobservatlons considered.
Faise rejecttons can fappen because of the control limits design and not actualy ldentify a
problem wth the method.
Westgard rules" are generally used wth 2 or 4 control measurements per run, which means
they are appropriate when two different control materlals are measured 1 or 2 times per
materlal, whlch is the Casein many chemistry applications (https://www.westyard.com/mitrule.htm).
Some alternative control rules are more sultable when,three contrplmatarials are analyzed,
which Is common for applcatlons in hematology, coagulation, and Immunoassays.

12
Seneral Intervretation of OC Results:
The acceptable reference limit is set at 2SD.
The upper and lower reference imits define a
specified percentage, usually 95%, of the values
for a certain group of population, hence, 5% of the
population will fall outside the reference
interval/limit in the absence of an abrormality or disease.
An anelytlcal methed is considered in éontrol when
there is symnetricaldictribuion
vakues around the mean and there are few-controlvalues of control
tron, If the outside-the 26-controllimits.
analytical test results are not within the 2SD confidence limit, run a newset
and repeat specimen testing. of controls
A eontrol
value-between 2s and 3s is a signof a potentialproblem.
Acontrol value outslde the 3s
would require corrective action.
Some laboratories use the 2 as a
warning
Not every rule violation indicates that an limitand
the 3 as an ereeii
analytic process is out control.
Continuous QC failure requires more trouble
shooting preparation of new reagents,
recalibration, instrument maintenance and repair, and contacting the
technical support or dealer/manufacturer for
After the likely causeservice.
of the problem has been identified and
corrected, a full set of control
materials should be retested.

Six Sigma (60)

It isperformance improvement program, in which the goal is to improve the process by
a

eliminating variations or errors: improved performance, improved quality, improved bottom

line, improved custormer satisfaction, and improved employee satisfaction.
It is a tool that can be used to reduce
laboratory errors, increase productivity and improve
quality in the clinical laboratory.
It measures the degree of variability
or error in products or services
through statististics and
quantitative parameters process defects or errors are analyzed, potential causes are identified,
and improvements are implemented.
Main Goal: To reduce the number of defects to near zero

InSixSigma, defects are

anything that does not meet customer requirements-laboratory result error, delay in
reporting, or a quality control problem; and
generally measured per million opportunities (DPMO).

Lean System
i t is a system for reducing waste ("nonvalued activities") especially in production or
manufacturing processes.
t was conceptualized to improve the automobile industry in terms of the quality and efficiency
of automobile production.
t utlizes the 55 (Sort, Set in order, Shine, Standardize, and Sustain), and PDCA (Plan, Do, Check,
and Act) systems to diminish costs by identifying daily work activities that do not directly add to
the delivery of laboratory services in the most efficient or cost-effective ways.
t focuses on work fow actions in performing specific tasks, procedures, or other activities
accomplished by critically reviewing each step in the process to detemine where ineficiencies
can be eliminated.

13
 A "Lean Clinical
Laboratory" utilizes fewer resources, reduces
costs, enhances productivity,
promotes staff
morale, and improves the quality of patient care
Pherson and Pincus, 2017). (Rutledge, 2010 cited by Mc
Example: relocating analytic equipment to an area
that would require fewer
improving turnaround time; consolidating test steps, thus
expense of panels to fewer instruments,
eliminating the
maintaining multiple instruments and supplies;
materials such as pipettes, culture accesabllity to instruments and
and minimize wasteful tablets, reagents, etc; and reallocating staff to maximize use
downtime
Teminologies:
1. Analytical Run
tis a set of control and patient specimens assayed, evaluated and
reported together.
2. Deta check
I t is the most commonly used patient based-QC technique.
It requires computerization of test data so that current results can be
compared with past
results.
It is the difference between two
consecutive measurements of the same
individual. analytes on the same

3. Interference experiments
These are used to measure systematic errors or inaccuracy caused by substances other than the
analyte.
Interferences: hemoglobin, lipids, bilirubin,
anticoagulants and presenvatives
4. Linear Range/Dynamic
Range
It is the concentration
range over which the measured concentration is
concentration without modification of the method. equal to the actual

5. Physiologic Limit
s sometimes referred to as absurd value.
I t helps detect sample contamination or
dilution, inadequate sample volume, inadequate
reagent volumes, sudden major problems with the method, or incorrect
transmission of the result.
or recording

6. Point Of Care Testing

(POCT)/Decentralized Testing
I t is a type of analytical testing
performed outside the confines of centrai laboratory, usually
by nonlaboratorian personnel (nurses, respiratory therapists, etc). the
Most commonly used POCT: use of portable whole blood
glucose meters for the management
of patients with diabetes melitus.

7. Quality Assurance (QA)

It can be envisioned as a
tripod with program development, assessment and monitoring, and
quality improvement forming the three legs.
is a systematic action necessary to provide adequate confidence that laboratory services will
satisfy the given medical needs for patient care.

14
 Philosophy of QA:Quality is important to customers.
Quality can be assessed and monitored.
Quality can be improved.
Quality's benefits exceed its costs.
Primary Goal of QA: To deliver quality services and products to customers.
8. Quality Patient Care
I t includes effective test request
forms, clear instruction for patient preparation and
handling, appropriate turn-around time for specimen processing, specimen
testing and result reporting,
appropriate reference ranges and intelligent result reports.
9. Recovery experiment
I t shows whether a method measures ali the
I t estinates inaccuracy or
analytes or only part of it.
systematic
error.

10. Predictive value

tdepends on sensitivity, specificity, and prevalence of the disease being test.
When a test cutoff
changes, its accuracy (sensitivity/specficity) andpredictive value also change.
Bayes' theorem (predictive value theory) describes the relationshipbetween posttest and
pretest probability of disease or no diseasebased on the sensitivity and specificity of the test.

a. Positive predictive value

I t is the probability that a positive test indicates disease ; it is the proportion of persons with a
positive test who truly have the disease.

b. Negative predictive value

i t is the probability that a negative test indicates absence of disease. It is the proportion of
persons with a negative test who are truly without disease.

11. Reference Limit/Reference Intervai/Reference Value

it is a value obtained by observation or measurement of a particular type of quantity on a
reference individual.
I t is a pair of medical decision points that extend the limits of test results for a certain healthy
population.
It is the range of values into which 95% of nondiseased individuals will fall - this definition
implies that 5% of nondiseased individuals can have laboratory results outslde the reference
range.
It is the usual values for a heaithy population that represents 959% central tendency.
It is usually established by the manufacturers of reagents or group of experts
It is mostly determined using nonparametric statistics (CLSI recommended method).
Therapeutic drug targets or values are ot derived from a healthy population, but rather require
unique physiologic conditions.

15
Factors To Be ConsideredWhen Establishing Reference Intervals:
1. The composition of the reference population as to age, gender, genetic and socioeconomic
factors must be carefuly evaBuated and determined (many laboratory data depend on age and
gender onl).
2. The quantity of reference individuals
3. The inclusion and exciusion criteria for the required reference population must be created.
4. The physiologic and environmental conditions of the reference population (employment, obesity,
lifestyle, habit, food and drug intake, etc).
5. Categorize the potential reference individuals based on the criteria set (questionnaire).
6. The specimen-collection procedure, including preparation prior to testing must be standardized.
7. The analytical method used.

Notes to Remember.
ldeally, the laboratory should have age and sex-stratified reference values on all populations
tested.
To derive reliable estimates of reference intervals, at least 120 individuals should be tested in
each age and gender categories.
Only 20 reference individuals need to be sampled for analysis on the test instrument if the
laboratorian determines that the test instrument and test subject population are similar to
those described in the manufacturer's package insert.
Forverification" of already existing and established reference intervalis, CLSI permits 20 subject
specimens/individuals.
The manufacturer's reference limits may be accepted provided not more than 10% of the tested
subjects fall outside the original reported limits; if greater than 10%, an additional 20 or more
subjects/samples should be analyzed.
The reference values vary slightly depending upon method and specimen type.
A critical or panic value is a laboratory resutthat may represent a life-threatening situation that
sometimes not readily identified. It should be communicated immediately to the clinicians for
appropriate medical interventions.

16
 ANALYTICAL METHODS

Light energy. wavelength.and.radiant energy Spectrum:

Energy- is transmitted via electromagnetic waves that are characterized by their frequency and
Wavelength
Wavelength is the distance between-twe-succassive-peaks and it is expressed in terms of
nanometer-{am
400-700nm visible spectrum
c400nm -ultraviolet region(UV)
700nm-infrared region (IR)
The relationship between
Where: ' PLANCK'S
wavelength and energy (E) is described by Planck'sformula:E=hy
f o R MULA h

E- is the energy
of aphoton in Joules or eV
h constant (6.626 x 10 erg sec)
v frequency

Fregueney is the
number-of vibrations of wave motion per-second
The lowerthe-wave-frequeney, the longer-thewavelength.
The wavelength is iaversely-relutech to frequency and eneeg; the shorter the wavelength, the
higher the frequency and energy and vice versa.

Notes to Remember:
Nominatwavelength represents the wavelength in nanometers at peak transmittance.
A slight error in wavelength adjustments can introduce a significant error in absorbance
readings.
Wavelength-aceracy is the wavelength indicated on the contro-diatis the actual wavelength of
light passed by the monochromator.
Disymlum or hoimitur oxide fiter is used to checkwavelength-eeeuraor{wavelength calibration).
Neutral density fiters and dlchromate solution verify absorbance accuracý on linearitý.

L COLORIMETRY
Photoetectrie Colortmetry
The primary analytical utility of spectrophotometery or filter-phetometry is the isolation of
discreet portions of the spectrum for purposes of measurement.
a. Spectrophotometric measurenent-is measurement of light intensity in a narrower wavelength.
b. Photometric measurement is measurement of ightintensity. ()

A. Spectrophotometry
I t involves measurement of the light transmitted by a solution to determine the concentration
of the light-absorbing substances in the solution.

Single beam spectrophotometer

It is the simplest type of absorption spectrometer.
tis make one measurement at a time
designed to at one
specified wavelength.
The absorption maximum of the analyte must be known in advance when a single-beam
instrument is used.

17
 Doubte-beam spectrophotometer
lsinstrument that splits the monechromatic
an
light into two components one beam passes
-

om: vari aion throught the sample, and the other through a reference solution or blank.
in tight t * HThe additionalbeam corrects
for variation in-ight
in this type of source intensity.
spectrophotometer, the absorbance of the sample can be recorded directly as the
electricat output of the sampter beam.
2Types of Double-beam Spectrophotometer: pate $a inyang daa
a. double-beam in spacg- with 2 photodetectors, for the
b. double-beam in
sample beam and reference beam
time wth one photodetector and
alternately passes themonochromatic llght
through the sample cuvet and thenreference cuvet using a chopper or
94. lang wy Kaioiyn, rotating sector mimor

6 basic components of single or double-beam confiquration spectrophotomete stable source of

radiant energy, filter that isolates a specific
region of the electromagnetic spectrum, sample hoider,
radiation detector, signal processor and readout device
Parts of the Spectrophotometer:
1. Light/Radiant source
I t provides pelyehromaticlight and must generate sufficient radiant energy or power to measure
the analyte of interest.
An intense beam of light is directed through the monochromator and the
sample.
To give accurate absorbance measurements throughout its absorbance range, its response to
change in light intensity must be linear.

2 Types:
a.Continuum souree -emits radiation that changes in intensity; widely used in the laboratory.
Example: tungsten, deuterium and xenon lamps
Tungsten light bulbis the commonly used light source in the visible and near infrared region
utDeuterium lamp is routinely used to provide UV radiation in analytic spectrometers.
Be Xenon discharge lamp produces a continuous source of radiation, which covers both the UV and
the visible range.

b. Line-seuce- emitslimitedradiation and wavelength.

Examples: mercury and sediumvapor lamps in spectrophotometers (Uand visibteregions),
and the hellow cathode lamp{AAS)
Line sources that emit a few discrete lines find wide use in atomic absorption, molecular, and
fluorescent spectroscopy.
Light Amplification by Stimulated Emission of Radiation (LASER) is also used as light sources for
spectrophotometry.

Facters for choosing.a lightsource: Fange, spectral distribution within the range, the sourcefradiant
Foduction, stabitity of the radiant energy and temperature

Alternatives for tunasten bulb: 4. Xenon lamp - UV

1. Mercury arc (visible and UV)
2. Deuterium lamp (165nm)- UV 5. Merst glower -IR
6. Globar (silicone carbide) - IR
3. Hydrogen lamp -UV

18
2. Entrance Stit
it reinimizes unwanted-or-stray-light and
monochromator system.
prevents, the entrance of scattered light into the
Strey ightrefers to any wavelengths outside the band transmitted by the monechromator; it
does not originate from the polychromatic light source; it causes
absorbance
error.
Straytighttimits the maximun-abserbance that a spectrophotometer can achieve.
Straylight is the most common cause of less-oflinearity at high»analte.concentration.
3. Monochromator
It isolates speeifie or
individual-wavelength of iight.
Kinds of Monochromators
a. Prisms
These are wedge-shaped pieces ofelass, auartzor sodium.chloride
These can be retated.allowing only the desired wavelength to pass through an extslt.
A
narrowight-focused on a prism is refracted as it enters the more dense glass.
b. Diffractiongratings mo ommon.parakel grcoves 1sits, & benas a 1hey pas.

These are the most commonly used; better resolution than prism.
These are made by cuttinggrooves (parallet grooves) orslit3 into an atuminized-surface of a flat
piece.ofcrownglass wavelengths are bent as they pass asharp cormer.
c. Filters uhes mogntsum fluotide(diecsric)
These are simple, least expensive, not precise but useful.
These are made by placing semi-transparent silver films on both sides of a dielectric such as
magnesium fluoride.
Filters produce monochromatic light based on the principle of constructive interference of
waves light waves enter one side of the filter and are reflected at the second surface.
Filters usually pass a wide band of radiant enerey and have a low transmittance of the selected
wavelength.

d. Hologrophic gratings

4. Exit Slit
I t controls the width of_jight beam (bandpass) allows only a narrow fraction of the spectrum
to reach the sample cuvette.
Bundpass is the tetatrenge of wavelengths transmitted.
Accurate absorbance measurement requires a bandpass less than 1/5 the natural bandpass of

the spectrophotomete.
The degree of wavelength isolation is a function of the type of device used and the width of
entrance and exit slits.
that is, the narrower the
SpectraB purity of the spectrophotometer is reflected by the bandpass,
bandpass, the greater the resolution.

5.Cuvet
I i t is also called absorption cell/analytical cell/sample cell.
t holds the solution whose concentration is to be measured.

19
Kinds of Cuvets:
1. Alumina silica glasS- most commonly used {can be used in
2. Quartz/plastic used for measurement 350-2000nm)
of solution requiring visible and ultraviolet spectra
3. Borosilicate glass
4. Soft glass

Notes to Remember:
Cuvets with scratches on their optical surface scatter
Sikea-cuvattes transmit light effectively at
light and should be discarded.
Alkaline solutions should not be left
wavelengths2220nm
standing in cuvets for prolonged periods because alkali
slowly dissolves glass producing etching.
The path length of
cenvetsis 1-cm, although much smaller path lengths are used in
automatedsystems.
To
inerease sensitvity, some cuvets are designed to have petrtengths of 10 cm, increasing the
absorbance for a given solution by a factor of 10.

6. Phetedetector
I t detects and cevertstransmitted
1ight into photoelestrie energy.
It detects the amount of light that
passes through the sample in the cuvet.
Kinds of Detectors:
a. Berrierayer-eetfPhetocellyPhotovoltoiccet#
I t is the simpliest
detector least expensive; temperature-sensitive.
I t is used in filter photometers with a wide
bandpass.
It is a basic phototransducer that is used for
detecting and measuring radiation in the
visible region.
I t is composed of
selenium on a plate of iron covered with transparent layer of silver.
It requires external voltage source but utilized internal
electron transfer for current
production- low internal resistance.
This cell typically has a maximum sensitivity
at about 550 nm and the response falls offto
about 10% of the maximum at 350 and 750 nm.

b. Phototube
It contains cathode and anode enclosed in a glass case.
It has a photosensitive material that gives off electron
when light energy strikes it
I t requires an external voltage for operation.

c. Photomultiplier tube (PMT)

It is the most commonly used
detector measures visihle and tiV reoinne
t has excellent sensitivity and has a rapid response
The response of the PMT begins when
detects very low levels of light.
incoming photons strike a photocathode. These tubes are
limited to measuring low power radiation because intense
the photoelectric surface.
light causes irreversible damage to
It should never be exposed to room
light because it will burn out.

20
 d. Phetodiode
t is netassensitive-as PMT but with excettenttinearity.
tmeasures light at a mutitude ofwavelengths-detects lessamount-ofight.
thas a lower dynamic range and higher noise compared to PMT.
tis most useful as a simutaneous multichannel detector.

7. Meter or read-out device

It displays
qutputof the detection system.
Example: galvanometer/ammeter/light-emiting diode (LED) display

Beer's Law
t states that the concentration
of the unknown substance is directly proportional to the
absorbed light (absorbance or optical density) and inversely proportional to the amount of
transmitted light (% Transmittance).
It mathematically establishes the relationship between concentration and absorbance.
Absorbance (A)
is the amount of light absorbed.
is proportional to the inverse log of transmittance.
it is mathematically derived from %T.

A abc = 2-log%T
where: A Absorbance
a
=molar absorptivity; absorptivity of the compound under standard conditions
b length of light thrcugh the solution
c concentration of absorbing molecules/solution

Unknown solution: Au_xC

As

Percent Transmittance
is the ratio of the radiant energy transmitted (T) divided by the radiant
energy
incident on the sample ().
%T k x100
o
where: transmitted lightthru the sample
l = intensity of light striking the sample
The %T measured by commercial spectrophotometers is the ratio of the sample transmitted
beam divided by the blank transmitted beam.
Inactuai practice, the light transmitted by a blank is substituted for
l
%T =sample beam signal x 100
blank beam signal

21
 cOLORS AND COMPLIMENTARY COLORS OF THE VISIBLE SPECTRUM

Wavelength Color Absorbed Complementary Color

350-430 Violet Yellow-blue
431-475 Blue Yellow
476-495 Green-blue Orange
496-505 Blue-green Red
506-5555 Green Purple
556-575 Yellow-green Violet
576-600 Yellow Blue
601-650 Orange Green-blue
651-700 Red Blue-grec
Note: If a solution absorbs light of a certain color (2nd column), the observed colorof the solution is the
complementary color.

Notes to Remember
The amount of light absorbed at a particular wavelength depends on molecular andion types
present and may vary with concentration, pH or temperature.
The light path must be kept constant to have absorbance proportional to concentration.
Deviations from Beer's law may be caused by changes in instrument functions or chemical
reactions.
Instrument-deviation is commonly a result of the finite band-pass-ofthe filter or
moneehremator.
Turbidity readings on a spectrophotometer are greater in the blue region than in the red region
of the spectrum.
A n absorbanee check is performed using glass filters or sotutions that have known absorbance
vaues for a specificwavelength the operator simply measures the absorbance of each soBution
at a specified wavelength and compares the results with the stated values.
The linearty of a spectrophotometer can be determined using optical filters or solutions that
have known absorbance values for a given wavelength.

Blanking Technique
Blanking techniquemeans the blank contains serum but without the reagent to complete the
assay.
Reagent blank corrects absorbance caused by thee color of thee reagents - the absorbance of
reagents is automatically subtracted from each of unknown reading.
Sample blank measures absorbance of the sample and reagent in the absence of the
endproduct, and corrects the measurement for optical interference (like hemoglobin) absorbing
the wavelength of measurement.
A blanking process may not be effective in some cases of turbidity, and ultracentrifugation may
be necessary to clear the serum or plasma of chylomicrons.
Lipids interferemainly by increasing light scatter (turbidity).
To correct for artifactual absorbance readings, "blanking" procedures or dual-wavelength
methods may be used.
B. Flame Emission Photometry (FEP)
i t measures the light emitted by a single atom burned in a flame.
Principle: Excitation of electrons from lower to higher enerEy state.
Light source: Flame (also serves as the cuvette)
Method: Indirect Intermal Standard Method
Internal standard: Lithium/Cesium-corrects variations in flame and atomizer characteristics
I t is used for the measurement of excited ions (sodium and
potassium).
Flickering light indicates changes in the fuel reading of the instrument.

CAtomic
t
Absorption Spectrophotometry (AAS
measures the light absorbed by atoms dissociated by heat.
Principle: Element is not excited by merely dissociated from its chemical bond and place in an
unionized, unexcited, ground state.
Light source: Hollow-cathode lamp
Interferences: chemical, matrix (differences in viscosity) and ionization
It is used for measurement of unexcited trace metals (calcium and magnesium).
tis more sensitivethan FEP; it is accurate, precise and very specific.
Internal standard is not needed changes in aspiration have litte effect on the number of
ground state atoms.
An atomizer (nebulizer/graphite furnace) is used to convert ions to atoms; a chopper is used to
modulate the light source.
Lanthanurn or strontium chloride is added to samples to form stable complexes with phosphate.

I vOLUMETRIC (Titrimetric)
Principle: The unknown sample is made to react with a known solution in the presence of an
indicator.
Examples: Schales and Schales method (Chloride test)
EDTATitration method (Calcium test)

L . TURBIDIMETRY
and bacterial suspensions.
For measuring abundantdárge particles (proteins)
blocked (reduction of light) by a particulate matter
Principle: it determines the amount of light
in a turbid solution.
concentration particle
and size.
t depends on specimen
reduction of light is due to particle formation.
The measurement of
measured using visible photometers or
requiring quantitatlon by turbidimetry
are
Solutions
visible spectrophotometers.
to detect bacterial growth in broth cultures;
U s e : protein measurements (CSF and urine);
formation
antimicrobial test (broth method); and to detect clot

23
. NEPHELOMETRY
For measuring the amount of
antigen-antibody complexes (proteins).
Principle: It determines the amount of scattered light by a particulate matter suspended in a
turbid solution.
Light scattering depends on wavelength and particle size.
Lightscattered by particles is measured at an angle, typically 15-90 degrees tothe beam incident
on the cuvet.
Most antigen-antibody complexes have a diameter of 250-1500
nm, and the wavelengthsused
are 320-650 nm, thus light is scattered forward
The detector (PM tube) output is
(Raylelgh-Debye type).
proportional to concentration.
Components of nephelometer light source (mercury-arc lamp, atungsten-filament lamp, light emitting
diode, anda laser), collimator, monochromator, sample cuvet, stray
light trap, and photodetector

V. ELECTROPHORESIS
Is the migration of charged
particles in an electric field.
It separates proteins on the basis of their electric
charge densities.
The acidic and basic amino acids determine the net
charge on a protein, hernce its
lectrophoretic mobility.
During electrophoresis, proteins are negatively charged (anions) and they move towards the anode.

Components of electrophoresis: Electrical power, Support medium, Buffer, Sample and Detector
Buffer: Barbital (pH 8.6)

Teminologies
1. Amphoteric- has anet charge that can be either
positive or negative depending on pH conditions
2. Electroendosmesis/Endosmosis is themevement of buffer ions and
solvent relative to the fixed support
-

3. lontepheresis is the
-

migrationt ofsmettchargedions
4. Zone electrophoress i s the migration of
charged macromolecules
Factors Afecting Rate of Migration: Ctncic ( ) ^ anode (1)
1. Net electric charge of the molecule
2. Size and shape of the molecule Sreps
3. Electric field strength igraon
. nying
4. Nature of the supporting medium
5. Temperature of operation
.Fixiy
Jtoniny
Supportina Media:CM
1. Celtslese acetate- separates by melecular size
2 Agarose gel-separates byelectricateharge; it does notbind protein
3. Polyacrylamide Gel separates on the basis
of charge and molecular size;separates proteins into 20
PEC isoe nymes fractions; used to study isoenzymes
Filter peg

oerny tttM Deosiomeer meaNn tN densiny tne thitkeëiy a the a

tiecro endeTmonu me moucmtn} ons rtianu o f pied MP

bah tv th cMh2 d
CaMTs ®me o8 he protins
24
Stainsfor Vsalzation of Fractions(Bands):
1. Amido black
2. PonceauS
3. Oil Red 0
4. Sudan Black
5. Fat Red 78
6. Coomassie Blue
7. Gold/Siver stain very sensitive even to nanogram quantities of proteins

Densttometry
I t measures the absortancesfetein -coneentretiomofthedyeand protefrartion.
It scans and quantitates elacteepheraticpattan.
uingectopnonis , folous ekumphorcis
Notes to Remember and wersely proportional to
lectropheratic-mebky is diracthy-proportiornal to net eharge
motecutar size and viseosity ofthesupporting-medium
A particle without a net charge will not migrate, and it remains in the point of application.
The ions carry the applied electric current and allow the buffer to maintain a constant pH during
gratelectrophoresis.
T h e ionic strength of the buffer determines the amount of current and the movement of the
proteins for a fixed voltage i f ionic strength is low, more current is carried by the charge
if ionic strength is high, less current is
proteins which move a longer distance (fast mobility);
carried by the proteins which move a shorter distance (slow mobility).
Increasing the strength of the field (increase current) also increases migration.
is the
The more the pH of the buffer differs from the isoelectric point (pl), the greater
faster it will in the electric field.
magnitude of the net charge of that protein and the move
the fact that they are
At pH 8.6, the gamma globuiins move toward the cathode, despite
negatively charged endosmosis.
an electrical field is determined
T h e actual distance traveled by a particular protein migrating in
feature of the protein itself and the
by the combined magnitudes of the electromotive force (a
of the support medium).
pH) and the electroosmotic force (a function primarily
After electrophoresis, the gel is treated with a mild fixative, such as aceticacid, that precipitates
They are then stained, and the gel is
the proteins at the positions to which they have migrated.
dried and cleared of excess stain.

Precautions: be denser on one side of the gel than

If the electrodes are not properly aligned, the current may
on the other; proteins will migrate
farther on the side with more current.
into the buffer.
felectrophoresis prooceeds too long, the proteins may migrate off the gel
the proteins will not move from
If there is a break in the electrical circuit and no current passes,
the point of application.
at the center of the gel migrate
Frequently, gels show the "smile artifact," in which samples
farther than those at the edges.

25
Isoelectric Focusing
t separates molecules by migration through a pH gradient.
It is ideal for separating proteins of identical sizes but with different net charges.
pH gradient is created by adding acid to the anodic area of the electrolyte cell and adding hase
to the cathode area.
Proteins move in the electric field until they reach a pH equal to their isoelectric point.
Supporting media: agarose gel, polyacrylamide gel and cellulose acetate.

Advantages
1. The ability to resolve mixture of proteins.
2. To detect isoenzymes of ACP, CK and ALPin serum.
3. To identify genetic variants of
proteins such as alpha-1-antityrpsin.
4. To detect CSF oligoclonal
banding.
Capillary Electrophoresis
In this method, sample molecules are
separated by electro-osmotic fiow (EOF).
I t utilizes nanoliter quantities of
specimens.
Positively charged ions in the specimen emerge early at the capillary outlet because the EOF and
the ion movement are in the same direction.
Negatively charged ions in the specimen move towards the capillary outlet but at a slower rate.
Uses: separation, quantitation and determination of
moleciular weights of proteins and
peptides; analysis of PCR products; analysis of organic and inorganic substances and drugs.

VI CHROMATOGRAPHY
I t involves separation of
soluble components in a solution by specific differences in physical-
chemical characteristics of the different constituents.

Bases of Separation:
1. Rate of Diffusion
2. Solubility of the solute
3. Nature of the solvent
4. Sample volatility/solubility
5. Distribution between 2
liquid phases
6. Molecular Size (molecular
sieving)
7. Hydrophobicity of the molecule
8. lonic attraction
9. Differential distribution between two
immiscible liquids
10. Selective separation of substances
11. Differences in
adsorption and
desorption of solutes

26
2Fomsof
A. Planar
Chromatosraphy
1. Paper Chromatography
I t is used for fractionation of sugar and amino acid.
Sorbent (stationary phase) - Whatman paper

2. Thin Layer Chromatography (TLC) Hlber paper

t is a semiquantitatve drug screening test.
Sample components are identified by comparison with standards on the same plate.
Extraction of the drug is pH dependent-the pH must be adjusted to reduce the solubility of
the drug In the aqueous phase.
When all drug spots including the standards have migrated with the solvent front, it is cause
by incorrect aqueous to nonaqueous solvent mixture.
Biological samples such as blood, urine and gastric fluid can be used for the test.
Sorbent: thin plastic plates impregnated with a layerofslicagel or alumina.
Each drug has a characteristic Re value and it must match the R value of the drug standard.

Retention factor (R) value- Is the relative distance of migration from the point application.
R distance leading edge ofcomponent moves
total distance solvent front moves

B. Column
1. Gas Chromatography (GC)
It is used for separation of steroids, barbiturates, blood, alcohol and lipids.
it is useful for compounds that are naturally volatile or can be easily converted into a volatile
form.
f the molecule of interest is not volatile enough for direct injection, it is necessary to derivatize
it into a more volatile form.
Samples (urine or blood) are introduced into the GC column using a hypodermic syringe or an
automated sampler.
The specimens are vaporized and swept onto the column.
Flame ionization is used as a detector for GLC.
Elution order of volatiles is based on their boiling point.
ta- the retention time of the soiute in GC or HPLC
Mobile phase: nitrogen, helium, hydrogen and argon (inert gases)

2Types:
a. Gas Solid Chromatography (GSC)
surfaces.
Separation occurs based on differences in absorption at the solid phase
b. Gas Liquid Chromatography (GLC)
mobile phase and
Separation occurs by differences in solute partitioning between the gaseous
the liquid stationary phase.

Mass Spectroscopy {MS)

it is based on the fragmentation and ionization of molecules using a sultable source of energy.
it can also detect structural informatlon and determination of molecular weight.
GC.
before a compound can be detected and quantified by Ms, it must be separated by

27
Gas Chromatography-Mass
Spectroscopy (GC-MS)
Itis the gold standard for
I t is also used
drug testing.
for xenobiotics,
anabolic steroids and pesticides.
I n this method,
quantitative measurement of drug can be
monitoring performed by selective ion
It electron beam to split
uses an
the drug emerging from the column into its component ions-
drugs are detected by means of the presence of
degradation of the anahlytes. decomposition fragments which arise after
The position of
the parent molecule-ion and degradation products give rise to
patterns which will provide the final identity of the fingerprint
drug of interest.
Every drug has its own fingerprint pattern which is
compared to a computer library of known
fragmentation patterns.
Tandem mass
spectroscopy (MS/MS can detect 20 inborn errors of metabolism froma
single blood spot.
2. Liquid Chromatography
It is based on the distribution of solutes
between a liquid mobile phase and a stationary phase.
HPLC is the most widely used liquid
chromatography.
a. High Performance Liquid Chromatography (HPLC)
I t uses pressure for
fast separations, controlled temperature, in-line detectors and
gradient
elution technique.
Use: fractionation of drugs, hormones, lipids,
carbohydrates and proteins; separation and
quantitation of various hemoglobins associated with specific diseases (e.g., thalassemia); rapid
HbAe test
In reverse
(within 5 minutes).
phase HPLC,the mobile phase is more polar than stationary phase.
b. Liquid Chromatography-Mass Spectroscopy (LcMS)
It is for detecting nonvolatile substances in body fluids.
I t is utilized to confim positive results from screening of illicited drugs - it isa complementary
method to GC-MS.
t is also used in therapeutic drug montoring, toxicology and studies of drug metabolites.
It requires interface methods to convert nonvolatile to volatile compounds.
Interface methods: Electrospray (ES) and Atmospherlc Pressure Chemical lonization (APCI)

Note: An internal standard is used in HPLC and GC methods to compensate for variation in extraction
and injection.

Separation Mechanisms Usedin Liquid Chromatorgraph

1.Gel/Gel Permeatlon/Gel Filtratlon/Sixe Exclusion /Moiecular Sieve Chromatography
I t separates molecules based on differences in their size and shape.
As solutes travel through the gel, large molecules remain in the mobile phase areeluted rapidly
from the column.
a. Hydrophilic gel (Gel Ftration)
i t is for separation ofenzymes, antbodies and proteins
-example: dextran and agarose

28
b. Hydrophobic gel (Gel Permeation)
it is for separation of triglyceride and fatty acid
-

example: sephadex

2. lon Exchange Chromatography

The mechanism in this type of
chromatography is the exchange of sample ions and mobile-
phase ions with the charged group of the stationary phase.
i s for separation of
amino acids, proteins and nucleic acids.
Separation of nucleic acids and protelns depends primarily on the sign and ionic
charge density.
3.Partition Chromatography (Liquid-Liquid
Chromatography
Separation of compounds is based on their partition between a liquid mobile phase and a liquid
stationary phase coated on a solid support.
Is for separation of therapeutic
drugs and their metabolites.
4. Affinity Chromatography
t uses immobilized biochemical ligands as the stationary phase to separate a few solutes from
other unretained solutes.
This type of separation uses the so-called
lock-and-key binding that is widelypresent in biologic
systems.
I s for separation of lipoproteins, carbohydrates and glycated hemoglobins; antibodies.
5. Adsorptlon Chromatography (Liquid-Solid Chromatography)
Separation is based on the differences (competition) between the adsorption and desorption of
solutes at the surface of a solid particle.
The compounds are adsorbed to a solid support such as silica or alumina.

VIL FLUOROMETRY/MOLECULAR LUMINESCENCE

SPECTROPHOTOMETRY
t measures the amount of light intensity present over a zero background.
Principle: It determines the amount of light emitted by a molecuie after excitation by
electromagnetic radlation.
Light source: mercury arc or xenon lamp
Light detector: Photomultiplier tube or phototube
Use: porphyrins, magnesium, calcium and catecholamines
It uses 2 monochromators (either filters, prisms or gratings) the wavelength that is best
absorbed by the solution to be measured is selected by the primary fiter; the incident light is
prevented from striking the photodetector by the secondaryfiter.
I s about 1000x more sensitive than spectrophotometer-emitted radiation is measured directly.
it is affected by quenching pH and temperature changes, chemical contaminants, UV light
changes.

29
VIL CHEMILUMINESsCENCE
I t differs from fluorescence and phosphorescence in that the emission of light is created from a
emical or electrochemical reaction, and not from absorption of
electromagnetic energBY
Principle: The chemical reaction yields an electronically excited compound that emits light as it
returns to its ground state, or that transfers its energy to another compound, which
then produces emission.
Use: Immunoassays
Photodetector: Photomutiplier tube (luminometer)
It is more sensitive than fluorescence.
In this method, no exctation radiation is required and no monochromators are needed because
the chemiluminescenoe arises from one species.
It involves the oxidation of an organic compound (e-g, dioxetane, luminol, acridinium ester) by
an oxidant (hydrogen peroxide, hypochlorite, or oxygen). These oxidation reactions may occur in
the presence of catalysts, such as enzymes (alkaline phosphatase, horseradish peroxidase, or
microperoxidase), metal ions (Cu2+ or Fe3+ phthalocyanine complex), and hemin.
The excited products formed in the oxidation reaction produce chemiluminescence on return to
the singlet state.
A typical signal from a chemiluminescent compound rises rapldly with time and reaches a
maximum when reagent and analyte are completely mixed.

DX. OSMOMETRY
t is the measurement of the osmolality of an aqueous solution such as serum, plasma, or urine.
Principle:t is based on measuring changes in the colligative properties of solutions that occur
owing to variations in particle concentration.
Osmotic particles: glucose, urea nitrogen and sodium
Colligotive properties of the solution: osmotic pressure, boiling point, freezing point, and vapor
pressure
When active osmotic particles are added to a solution, osmolality increases and four other
properties of the solution are also affected.
As the osmolality of a solution increases the following reactions occur: osmotic pressure
increases; boiling point is elevated; freezing point is depressed; andthe vapor pressure is also
depressed

Freezing-polnt depression osmometry

I s the most commonly used method for measuring the changes in colligative properties of a
solution.
It is based on the principle that addition of solute molecules lowers the temperature at which a
solution freezes.
A 10 mOsm/kg solution has a freezing point depression of 0.00186° C when compared with
pure solvent (usually water).
Blood plasma, with an osmolality of about 285 mOsm/kg, has a freezing point of about -0.53* C.

30
X. ELECTROCHEMISTRY TECHNIQUES
The measurement of current or votage generated by the activity of a specific ion.

1. Potentiometry
It is the measurement of
electrical potential due to the activity of free ions change in
-

voltage indicates activity of each analyte.

It is also the measurement of differences în voltage (potential) at a constant current.
It follows the Nernst equation.
Concentration of ions in a solution can becalculated from the measured
potential difference
between the two electrodes.
Reference electrodes: calomel and silver-silver chloride
Use: pH and pCOh tests

lon Selecthve Electrode (ISE)

t is an electrochemical transducer
capable of responding to one given ion.
t is very sensitlve and selective for the ion it
measures it measures the activity of one ion
-

much more than other ions present in the sample.

Its ionic selectivity depends on the
membrane/barrier composition used.
ISE analyzers measure the electrolyte dissolved in the fluid
phase of the sample in mmol/L off
plasma water.
t is a sensitive method but not
specific i t does not discriminate between ions in causing
voltagedifferences between the measuring electrode and the standard electrode.
ISE analyzers using undiluted samples are not
subject to pseudohyponatremia caused by
hyperlipimec samples.
2 Tvpes of ISE:
1. Direct ISE (without sample dilution)
2. Indirect ISE (with sample dilution)

ISEMembrane:
Glass aluminum silicate (sodium), valinomycin gel (potassium) organic liquid membrane ion
exchangers (calcium and lithium), gas and enzyme electrodes

Interference
Protein coating the ISE membrane would cause a response error - if interference is due to
excess protein, an alternative mode of analysis such ISE in undiluted specimen can be
employed to yield correct electrolyte activity (i.e., equivalent of osmolality).

Causes of Malfunctions ofISE:

Defective ISE membrane, buildup of countervoltages from liquid junction potentials at the salt
bridge and buildup of proteins at the electrodes

31
2. Coulometry
Itis the measurement of the
amount of electricity (in coulombs) at a fixed potentia.
It is an electrochemical titration in which the titrant is
endpoint is detected by amperometry. electrochemically generated and the
It follows the
Faradays law.
Use: chloride test (CSF,serum and
sweat)
Interferenc: bromide, cyanide and cysteine
3. Amperometry
t is the measurement of the current
flow produced by an oxidation-reaction.
Use: pO. glucose, chlorlde and peroxldase determinations
Polarography
It is the measurement of differences in
current at a constant voltage
It follows the lkovic
equation.
4. Voltammetry
The measurement of current after which a
I t allows sample to be
potential is applied to an electrochemical cel.
preconcentrated,
thus utilizing minimal analyte.
Anodic stripping votametry-for lead and iron testing

32
 INSTRUMENTATION

IYpes of Glasgwarei
1. Borosilicate glass (pyrex and kima
it is used for heating and sterilization purposes; most commonly used.
t is characterized by a high degree of thermal resistance, has low alkali content, and is free from
the magneslumlime- zinc group of elements, heavy metals, arsenic, and antimony.
strain point: 515" C (Pyrex)
2. Boron-free glossware/soft gloss
t has high resistance to alkali.
its thermal resistance is less as compared to borosilicate glass.
3. Corex (Corning)
is a special lumina-slicate glass that has been strengthened chemiclly than thermally; six
times stronger than borosilicate.
4. Vycor (Corning)
it is utilized for high thermal, drastic heat shock and extreme chemical treament with acids
(except hydrofluoric) and dilute alkali; it can be heated to 900"C.
5. Flint glass
t i s made up of soda-lime glass and a mixture of calcium, silicon and sodium oxides.
it has poor resistance to high temperature easy to melt and used to make disposable
glasswares.

Pipet Cassiication
L.Calibration Marks/Destgn:
1. To Deliver (TD) - it delivers the exact amount it holds into a container
2. To Contain (TC) - It holds the particular volume but does not dispense the exact volume

. Drainage Characteristies:
1. Blowout-it has a continuous etched rings on top of the pipet; exact volume is obtainedwhen the last
drop is blown out
2. Self-draining absence of etched rings; liquid is allowed to drain by gravity

II. Types:
1. Transfer Pipet
Volumetric Pipet for nonviscous fluid; self-draining; small amount left in the tip should not be
blown out
Ostwald Folin for viscous fluid; with etched ring
Pasteur Pipet transfers fluids without consideration of a specific
volume
Automatic macro- or mlcropipettes

2. Graduated or Measuring Pipet

Serological Pipet- with graduations to the tip; blowout pipet
self-
Mohr Pipet- without graduations to the tip; calibrated between 2 marks;
draining pipet
Bacteriologic Pipet
Ball, Kolmer and Kahn Pipet

33
 Micropipetes(< 1ml) TC pipets -

1. Sahii-Helige pipet
2. Lang-Levy Plpet
3. RBC and WBC pipets
4. Kirk and Overflow Pipet

Mechanical or Automatic Pipets

a. Air Displacement Ptpet
I t relies on piston for
suction creation to draw the sample into a disposable tip.
The piston does not come in contact with the
liquid.
b. Posittve Displacement Pipet
It operates by moving the piston in the pipet tip or barrel, much like a hypodermic syringe.
It does not require a diferent tip for each use.

c. Dispenser/Dilutor Pipet
t obtains liquid from a common reservoir and
dispensed it repeatedly.
It combines sampling and
dispensing
functions.

For Calibration of Pipettes:

Class A pipets do not require recalibration.
Distilled water is the calibrating medium for TD
pipettes while mercury is for TC pipettes.
Gravimetric and spectrometric methods used to verify
pipette volume accuracy and precision
0.1% phenol red solution in distilled
water used to compare the reproducibility of brands of pipet
tips

Volume Measurements:
1 lambda =1 microliter 0.001
( mL)
1 microliter 1.0milligram
Notes to Remember.
Plastic pipet tips are made primarily of polypropylene.
A s the fluid is allowed to drain into the receiving
with the
vessel, pipets should be held in avertical position
tips against the
side of the receptacle.
For volumetric TD pipette, it should not be shaken or hit
against the wall of the container during
draining because any disruption of the free-flowing liquid may result in an inaccurate deliver
of the
liquid.
Imperfect wetting or the presence of discreet droplets of water indicates that
the pipette is not
sufficiently clean.
Acid dichromate isa cleaning solution for glassware.

Calibration of Analytical Balance and Thermometer

Analytlcal Balance
Laboratory balances require calibration at regular intervals calibration intervals should coincide
with the requirements of the laboratory's licensing and accrediting organlzations.
The new mass standards and test weight accuracy classes appropriate for
include the American
laboratory balances
Society for Testing and Materials (ASTM) classes 1 and 2.
The operator must avoid direct contact with the weights by
using clean gloves or special lifting tools
e-g, forceps). Hand contact with the weights can cause corrosion.

34
Thermometer
2 types of thermometers: total immersion (freezers and refrigerators)
partialimmersion (water baths and heating blocks)
Noncertified thermometers can be calibrated by using an NIST SRM934
thermometer or an NIST
SRM 1968 gallium melting point celi.
Temperature-monitoring devices should be verified for accuracy at 6 or 12 month intervals.

Three Basic Approaches to Automation

Advantages
1. Increase the number of tests to be performed in a
2.
given period.
Minimizes variation of result from one laboratorian to another.
3. Eliminates the
potential error in manual analyses such as pipetting, calculation and transcription of
results.

1. Continuous Flow Analyzer

Liquids are pumped through a system of continuous tubing.
Samples flow through a common reaction vessel or pathway.
Air bubbles at regular intervals serve as
A
separating and cleaning media.
heating bath maintains the required temperature of the reaction to allow complete color
development (same with discrete analyzers) reaction rate is controlled by temperature.
-

Mixing of sample and reagents: by using a glass coil inserted into the flow path
Example: SImultaneous Multiple Analyzer (SMA), Technicon
Disadvantage: all tests are performed in parallei (measurement of every analyte configured on
the system for every sample)

2. Centrifugal Analyzer
It uses the force generated by
centrifugation to transfer specimen and reagents.
Liquids are placed in separate cuvets for measurement at the perimeter of a spinning rotor
(1000rpm)
I t uses acceleration and deceleration of the rotor to transfer the reagents and sample from one
chamber to another.
Mixing: centrifugal force (rotor) is utilized or bubbling of air
Examples: Cobas-Bio (Roche) and iL Monarch
Major advantage: Batch analysis (discrete-batch type system)

3. Discrete Analyzer
It is the most popular and versatile analyzer measures
only the tests requested on a sample.
t requires 2-6uL of the sample (minimum volume).
It employs a variety of syringe pipettes (positive liquid-displacement pipets) to aspirate and
dispense samples and reagents.
It is capable of running multiple-tests-one-sample-at-a-time.
Each sampie-reagent mixture handled separately in its own reaction vessel.
For dry slide technology (reflectance photometry), the spreading layer permits a rapid uniform
spreading layer over the reagent layer.
Miing: magnetic driven teflon stirring bar, forceful dispensing, magneticstirring bars, rotating
paddle, and ultrasonic energy

35
 Examples: Vitros, Dimension Dade, Beckman ASTRA System,
Integra and Analytics P Module
Hitachi, Bayer Advia, Roche Cobas
Major advantage: Random access capabilty- allows STAT samples to be easily tested
Reflectance photometry
I t is the
The
measurement of light reflected from solid surfaces.
Intensty of the reflected light from the reagent carrier is
light reflected from a reference surface. compared with the intensity of
A reflectometer is used to measure
analytes by measuring the quantity of light reflected by a
liquid sample that has been dispensed onto a grainy or fibrous solid
support.
Notes toFor
Remember
measurement, visible and UV light spectrophotometry are the common methods.
Abbott TDX uses fluorescent
polarization
for drug analysis.
Tablet form reagentsACA Star (Dade) and Paramax analyzers
Multilayered dry slide (thin film) reagents-Vitros analyzer
Corry over is the the transport of
quantity of analyte ar reagent from
into another, and
one specimen reaction
contaminating subsequent one.
a

Terminologies:
1. Batch testing all
samples are loaded at the same time, and a single test is conducted on each
2. Parallel testing more than one test is sample
analyzed
-

3. Random access
concurrently on a given clinical
specimen
testing- any test can be performed on any sample in any sequence
4. Sequential testing
mutiple tests analyzed one after another on a given specimen
5. Open reagent system -a system other than manufacturer's reagents can be utilized for measurement
6. Closedreagent system-a system where the operator can only used the manufacturers reagents
7. Pneumatic tube
delivery system- it provides point-to-point delivery of specimens to the
and offered several advantages over laboratory
specimen transport by humans

36
 PATIENT PREPARATION

Prior to blood collection, patients must be given correct instructions on how to prepare for each
laboratory test. Utmost care must be observed to minimize factors that may influence laboratory
results.

Pre-analytical
1. Exercise
Varlables-Factors Contributing to the Varlationof Results
Physical activity can have diferent effects on analyte concentrations-volume shifts between
the vascular and interstitial compartmets, volume loss by sweating and changes in hormone
concentrations.
Transient increased: lactate, fatty acid, ammonia
Long-tem increased (skeletal muscle enzymes): CPK, AST, LD and aldolase
Increased in hormones such as prolactin and growth hormone, while decreased plasma levels
are seen in follieule stimulating hormone, leutenizing hormone, estrogen and testosterone.
Vigorous hand exerclse (fist clenching) increases potassium, lactate and phosphate.
Elevated levels of proteins in urine (proteinuria) are observed.

2. Fasting8
Fasting requirement is between 8 to 12 hours.
Fasting specimen: FBS, GTT, lipids, ipoproteins, gastrin and insulin
Fasting for 48 hours may increase serum bilirubin.
Fasting for 72 hours may result to increase of plasma triglyceride in males while glucose
decreases in healthy women to 45 mg/dL.
Basal state collection is early morning blood collection, 12 hours after the last ingestion of food.
Basal state collection includes glucose, lipids, lipoproteins and electrolytes.
Basic metabolic panel: glucose, BUN, creatinine, sodium, potassium, chloride, CO2 and calcium

3. Diet
Metabolic products of food can increase in venousblood.
levels of urea and uric acid.
High protein diet can increase plasma
increased plasma urea and urine ketones.
Atkins dlet (high protein-low carbo diet) greatBy
show variation because of postabsorptive hormonal
Glucose, lipids and catecholamines may
effects. catecholamines from the
Caffeine increases concentration of glucose through the release of
adrenal medulla and braln tissue.
Increased in obese persons: glucose, cortisol, TAG and LD

4. Posture or postion
Preferred position during phlebotomy: upright position
or supine (lying) Oe
before
Recommendation: patlent should be seated/supine
for at least 15 minutes to 20 minutes
Mc
2007 cited by
or hemoconcentration (Young,
blood collection to prevent hemodilution
Pherson and Pincus, 2017). of the blood vessels
position causes constriction
Changing from supine to sitting or standing levels of albumin, enzymes and
calcium
and reduction of plasma volume: increased tissue causin8
into
Changing from sitting to supine causes shifting of water and electrolytes
calcium
hemoconcentration: increased levels of proteins, lipids, BUN, iron and

37
 Changing from standing to supine causes extravascular water to transferto the vascular system
and dilutes nondiffusable plasma constituents: decreased levels of cholesterol, triglycerides and
lipoproteins
Significant elevation of potassium after 30 minutes of standing is due to the release of
potassium from muscles.
Prolonged bedrest resuts to decreased plasma albumin due to fluid retention.
Renin plasma level is higher when standingthan supine.
Drugs bound to proteins are affected by postural changes
5. Touniquet application
One-minute application of tourniquet is recommended.
Effects of prolonged tourniquet application: hemoconcentration (venous stasis) and anaerobiosis
Increased levels due to prolonged tourniquet applicction: potasslum, proteins (albumin)
enzymes, lactate, cholesterol, and ammonia
The pressure from the tourniquet causes biological anaiytes to leak from the tissue celis into the
blood.
Prolonged use of a tourniquet with fist exercises can increase the serum potassium level byl
mmol/L
For accurate measurement of lactate, tourniquet should not be applied, and the patient should
not clench his fist at the time of the blood draw.
Tourniquet application and or muscular activity may decrease venous pO and pH.

6. Tobacco smoking (nicotine)

It can cause elevated hormone levels such as the catecholamines and cortisol. CH: ypre erunic ag i
Increased levels: glucose, growth hormone, cholesterol, triglyceride, ammonia, urea, lactate,
insulin and urinary 5-HIAA Ns t o 1u/L eigar
Decreased plasma levels of vitamin B12 is also observed.

7. Alcohol ingestion
It can cause increased plasma levels of urate, lactate, triglyceride and gamma glutamyl
transferase (GGT).
It causes hypoglycemia among patients with chronic aicoholism.

8. Stress
Increased: catecholamines, cortisol, ACTH, prolactin, albumin, glucose and lactate
Total cholesterol is increased with mild stress, while HDL cholesterol declines by almost 15%
(Dufour, 2003 cited by Mc Pherson and Pincus, 2017).

9. Drugs
Medications affecting plasma volume can affect protein, BUN, iron and calcium concentrations.
Therapeutic drug monitoring (TDM} specimen collection should be scheduled according to the
time of the last dose.
Hepatotoxic drugs can elevate liver function enzymes.
Diuretics can cause decreased plasma sodium and potassium.
Opiates cause increases in liver and pancreatic enzymes.
Analytic methods that are based on oxidation-reduction reactions may be influericed positively
or negatively by ingested substances such as ascorbic acid t(vitamin C).

38
Physlologic variation
t refers to changes that occur within the body such as cyclic changes (diurnal or circadian) or
those resulting from exercise, diet, stress, gender, age, drugs, posture or undertying medical
conditions.
Affected by age (increased levels): alibumin, ALP, cholesterol and phosphorus
Affected by gender (increased levelst Male- albumin, ALP, crestine, uric acid, cholesterol, BUN
Female- HDL, iron and cholesterol
Affected by recent food ingestion (increased levels): glucose, TAG, gastrin, free Ca
(decreased levels);: electrolytes (CT, K, P"), ALP and AMS

LABORATORY TESTS AFFECTED BY DIURNAL VARIATION

Peaks 4am-6am; lowest 8pm-12am;

Cortisol S0% lower at 8pm than at 8am; increased
with stress
Adrenocorticotropic hormone (ACTH) |Lower at night; increased wth stress
Plasma renin activity Lower at night; higher standingthan supine

Aldosterone Lower at night

Insulin Lower at night

Growth hormone Higher in afternoon and evening
Acid phosphatase (ACP) Higher in afternoon and evening
Increases with exercise
Thyroxine
Higher with stress; higher levels at 4am
Prolactin and 8am and at 8pm and 10pm
Peaks early to late morning; decreases up
Iron to 30% during the day

Calcium 49% decrease supine

23d ed., 2017
Source: Henry's Clinical Dingnosis and Management by Laboratory Methods,

39
 SPECIMEN cOLLECTION AND HANDLNG
"Proper patient identification is the first step in sample collection "- t h i s is the prime factor in order to

attainaccurate results in the dinical laboratory. Likewise, proper techniques in specimen collection must
be strictly followed including the observance on the confidentiality of results.

Patient Identdincation Procedures

1. Conscious Inpatients/Hospitalized patients
Verbally ask their full names including middle names.
Verify the name using the identification bracelet which includes first and last names,
hospltal/unit number, room/bed number and physician's name.
2. Sleeping patients
They are identifled in the same manner as conscious in-patients.
They must be awakened before blood collection.
3. Unconscious, Mentally Incompetent Patients
They are identified by asking the attending nurse or relative; 1D bracelet.
4. Infants and Children
A nurse or relatlve may identify the patient, or by means of an identification bracelet.
5. Outpatient/Ambulatory Patlent
Verbally ask their full names, address or birth date, and countercheck with driver's license, or ID
card with photo.
If the patient has idertfication card or bracelet, same manner as with
hospitalized patients.

3-WayID
To avoid misidentification, a phlebotomist may require what is referred to as 3-Way 1D, in which the
patient is identified by:
the patient's verbal ID statement
a check ofthe ID band
a visual comparison of the labeled specimen with the patient's 1D band before leaving the
bedside
Some facilities are also showing thelabeled specimen to the patient to ensure accurate labelling of the
tubes.

General Methods of Blood Collection:

An average human body contains approximately 5 quarts (4.73 L) of whole blood.
For adult males, they have approximately 5 to 6 liters of whole blood.
For adult females, they have approximately 4 to 5 liters of whole blood.
Whole blood is composed of approximately:
a. 60% ofplasma 3 quarts or 2.84 liters
b. 40% of cells -2 quarts or 1.89 liters

I.ARTERIAL PUNCTURE
Aprocess bywhich blood is obtained from a patient's artery.
Arterial blood is the oxygenated blood with a bright red color.
Use: for blood gas analysis and pH measurement
Sites: radialartery, brachial ortery, femoral artery, scalp artery and umbilical artery
Blood sample is collected without a tourniquet.

40
 Before biood is collected from the radial artery, modified Allen test should be done to
determine whether the ulnar artery can provide collateral circuBation to the hand after the radial
artery puncture.
The femoral artery is relatively large and easy to puncture, but extra care must be given to older
individuals because the femoral artery can bleed more than the radial or brachial.
Arterial bleeding is the hardest to control and usually requires special attention.
Mojor complications; thrombosis, hemorrhage, and possible linfection
Unacceptable sites: irritated, edematous, near a wound, or in an area of an arteriovenous (A
shunt or fistula

Arterlal Puncture Procedure

Prepare the artertal blood gas syringe according to established procedures. The needle (18-20
gauge for brachial artery) should pierce the skin at an angle of approximately 45-60 degrees (90
degrees for femoral artery) in a slow and deliberate manner. Some degree of dorsiflexion of the
wrist Is necessary with the radlal artery, for which a 23-25 gauge needle is used. The pulsations
of blood into the syringe confirm that it will fill by arterial pressure alone.

2 After the required blood is coilected, place dry gauze over the puncture site while quicktly
withdrawing the needle and the collection device.
3 Compress the puncture ste quickly, expel air from the syringe, and activate the needle safety
feature; discard into sharpscontainer.
Mix specimen thoroughly by gentty rotating or inverting the syringe to ensure anticoagulation.

Place in ice water (or other coolant that will maintain a temperature of 1-5° C) to minimize
leukocyte consumption of oxygen.
6 Continue compression with a sterile gauze pad for a minimum of 3 to 5 minutes (timed). Apply
an adhesive bandage.
Source: Heary's Clinical Diagnosis and Management by
Lab Methods, 22 ed, 2011

Modified Alen Test

both the ulnar (opposite the thumb side) and
1 Have the patient make a fist and occlude
compressing with two fingers over each
the radial arteries (closest to the thumb) by
artery.
observe if the patient's palm has become
2 Have the patient open his or her fist, and
bleached of blood.
from the thumb) only, and note if
3 Release the pressure on the ulnar artery (farthest
should become perfused with blood. Adequate
blood return ispresent.The palm
the radial
that arterial blood may be drawn from
perfusion is posltive test indicating
a
Serious consequences may
artery.Blood should not be taken if the test is negative. or tts
not followed, which may result
in loss of the hand
occur if this procedure is

function.

Lab Methods, 22d ed., 2011

Souroe: Henry's Clinical Dingnosis and Management by

41
. VENIPUNCTURE
A process by which blood is
obtained from a patient's vein.
Venous blood is the
For blood gases
deoxygenated blood with a dark red color.
measurement, venous blood is not the specimen of choice because it
reflects the acid-base status of an extremity not the usually
body as a whole.
Sites for Venipuncture:
1. Antecubital fossa
region
2. Veins on the wrist and dorsal
aspect of hands
3. Veins on the ankle
According to CLSI Standards, an attempt must have been made to locate the
on both arms before median cubital vein
considering an alternate vein.
Median cubital vein is the best site for
anchored vein.
venipuncture because it is the largest and the best
Cephalic vein is the second choice if the median cubitai vein is unsuitable; basilic vein is the
choice. third
Basilic vein should not be chosen unless no other vein is mare prominent due to its close
proximity to the brachial artery.
Veins on the dorsal part of hand or wrist be chosen
not acceptable.
area can
provided the antecubital veins are
Ankle vein should be used only if arm veins have been
determined to be unsuitable.
If petechiae appear after
venipuncture, it indicates that minute amounts of blood have escaped
into skin epithelium.

order of Draw (Rvacuated Tubeand Syringe)

1. Yellow top Blood culture tubes (Sterile)
2. Light blue top Trisodium citrate / (oaqulatyct,
3. Serum tube With or without clot activator or gel
separator
4.Greentop Heparin (Li, Na, NHa)
5. Lavender/purple top EDTA
6. Gray top (An 9tyoytic tube) - Ant t60quiot
Sodium fluoride and Potassium oxalate/lodoacetate and Heparin
A
y0ky to Agriiis ing
I0 hgNaf - Glit aguj ant

Notes to Remember:
Polymer barrier or gel separator has a specific gravity between the blood clot and the serum.
Polymer barrier moves upward to the serum-clot interface, creating a barrier separating serum
from fibrin serum maybe aspirated or measured directly frorm the collection tube
without
transferring to another container.
Blood samples for therapeutic drug monitoring should not be collected in tubes with
gel
separator or serum separator tube (SST) because some gels absorb drugs (phenytoin,
phenobarbital, lidocaine, quinidine, and carbamazepine) causing a falsely low resutt.
A gray-top tube containing fluoride and oxalate shoutd be used for lactate sarnple collection, as
it blocks glycolysis.
Sodum fiuorlde tubesare used to collect ethanol specimens to prevent either a decrease in
alcohol conocentration due to glycolysis or an increase due to fermentation by bacteria.
When blood is drawn from a winged set or intravenous catheter, a discard tube is used because
the air space in winged set leads to underfilling the first tube (Favaloro et al, 2012 cited by Mc
Pherson and Pincus, 2017).
Red top onda 55f piüta aqtr es ietoau
42
Procedurefor Venipuncture?
1. Greet the patient politely and wth gladness.
2. Ask the patient for his ful name including the middle name and birthdate. For hospital patients,
vertfy the the identification band with the requistlon fom. If a fasting specimen is required, confim
the complete fasting hour.
3. Decontaminate hands, put on gloves and prepare the materials preferably in the presence of the
patient.
4. Position the patiente's am in a downward and comfortable manner.
5. Apply the tourniquet 3 inches to 4 inches above the site, and instruct the patient to make a fist. Check
for potential sites by gently palpating the vein. Never leave the tourniquet longer than one minute.
6. Ifa suitable vein is not felt on the first arm, remove the tourniquet and try the other arm or other sites.
7. Ifa vein has already been chosen, release the tourniquet.
8. Decontaminate the patient's skin with an alcohol pad starting at the point where you expect to insert
he needle, and moving outward in even-widening concentric circes.
8. Allow the alcohol to evaporate to achieve maximum sterility, or remove excess alcohol with sterile
gauze pad. Do not blow on it. Do not touch the site after cleansing.
9. Re-appy the tourniquet and instruct the patient to make a fist. Avoid fist clenching or vigorous hand
exercise.
10. Pull the skin gentty with the thumb (if needed, hold the patient's arm below the site), and position
the needle paraliel or running in the same direction as the vein.
11. Insert the needle quickly, with the bevel side up at a 15 to 30 degree angle with the skin. A slight
"pop" should be felt as the needle enters the vein.
12. For evacuated tube, push the tube, and as the blood begins to flow, instruct the patient to open his
fist, and release the tourniquet. if it appears that the blood flow is slow, tourniquet may be left on
provided not longer than one minute until after the tubes have been filed. But always remove the
tourniquet first before withdrawing the needle.
For multidraw, carefully remove each tube from the holder with a gentle twist-and-pull motion.
Follow the order of draw.
For syringe, gently pull the plunger and as the blood begins to flow, instruct the patient to open
his fist, and release the tourniquet.
removed from the holder, withdraw
13. When blood collection is done or ali tubes have been filled and
over the
the needle with a quick motion and hold a dry sterile gauze pad (2- by 2-inch gauze pad)
site. Apphy pressure to the site for a minimum of 2 minutes.
time of collection, and the initials of the
14. Label the tubes with the patient's full name, date and
of the institution's protocol,
phiebotomist. Check the condition of the patient before leaving (as part accurate labelling of
the phlebotomists are showing the labeled specimen
to the patient to ensure

the tubes)
designated waste bins.
15. Discard all materíals in the

Precaution:
T h e sites adjacentto intravenous (V) therapy should be avoided.
However, in conditions were both arms are involved in therapy and the V cannot be
discontinued for a short time, a site below the IV line should be sought; the initial sample (5
mL) drawn should be discarded.
Collection of blood below the !V line must be written on the laboratory requisition form to
Inform the staff in the chemistry or other sections.

43
 VFluid Contaminatio: use ai's Cag bleog 5t,
p4s patAt cap itor tap
An increase of infused
ditaud Coess cap
tuwe
substances such as glucose, chloride,
decrease in urea and creatinine. Dextnt»e potassium and sodium, with a

As little as 10% contamination clectroy 1es (Iv Fluo )

with 5% dextrose will increase
mg/dL or more. in giucose a blood sample by 500

Notes to Remember.
1. Tourniquet Application
Tourniquet application should be 3 inches to 4 inches above the site and
minute. not longer than one
If blood pressure cuff is used
>Prior to blood collection, ask the tourniquet,
as a it is inflated 60 mmHg.

I f a patient is
patient for latex sensitivity to avoid allergic reactions.
allergic to latex, it is
removed and should not be brought into theimportant
very that all materials with such content be
room.
> If tourniquet is closer to the
site, the vein may collapse as blood is above the intended
venipuncture site.
>According to the CSiI, when a
tourniquet is used
during preliminary vein selection, it should be
released and reapplied after 2 minutes.
Tourniquet is applied to obstruct the returns of venous blood to the heart and distend
and it is discarded after each the veins,
>Studles have shown that reusable
phlebotomy.
tourniquets have the potential to transmlt bacteria, Including
methicillin-resistant Staphylococcus aureus (MRSA).
2. Disinfection of the site for puncture
>No traces of alcohol should remain on the skin because it may cause
contaminate glucose testing. hemolysis, may and

>For ethanol testing, benzalkonium chloride solution (Zephiran chloride, 1:750) should be used
for skin cleansing
> 70% alcohol followed by an iodophor is the most common
form of skincleansingbefore drawing
blood for culture.
According to the CLSI, chlorhexidine gluconate is the recommended skin disinfectant for blood
culture, for infants 2 months and older and patients with iodine sensitivity.
3. Needle specifications
>The color coding for needles indicates the gauge.
o For needles use in evacuated tubes/syringe,
yellow cap is 20 gauge, green cap is 21
gauge and black cap is 22 gauge.
The gauge of the needle is inversely related to the size of the
needle, the larger the gauge
number, the smaller the needle bore and length.
A 21-gauge needle is considered the standard for
venipuncture.
A 23-gauge is used for children; 23-or 25-gauge is for
winged infusion set (butterfly).
A 23-gauge butterfly Is most commonly used for small and
A 25-gauge needle is
difficut veins.
used
by speclally trained personnel to collect blood from scaip or other
tiny veins of premature Infants and other neonate.
Using a needle smaller than 23 gauge for arm veins may increase the chance of
specimen. hemolyzing the

44
 Needle length: 1 Inch or 1.5inches 21 to 23 gauge
%to % inch- butterfy needle
4. Tubes for Blood Collection
Blood collected in a syringe must be transferred into an evacuated tube using a syringe transfer
device attached to the tube, and not by "direct transfer" from a syringe into an evacuated tube.
Red top tubes without clot activators will take 60 minutes to completely coagulate blood.
Clot activators will cause the blood to clot within 30 minutes while tubes with thrombin particles
will clot blood in 5 minutes.
Never transfer blood collected in an additive tube into another additive tube, even if the
additives are the same. Different additives may interfere with each other or the testing process.
If the additives are the same, an excess of the additive is created, which can negatively affect
testing.

5. Other significant information

>Wear gloves before phlebotomy and change between patients.
f the needle orlancet touched any surface before blaod callection, replace it witha new one.
Geriatric, oncology or other hematologic patients can have fragile veins, so it is preferable to use
a winged blood collection set or syringe to minimize vessel wall injury and hemolysis.
Traumatic draw as a result of vessel wall injury can cause increase CK, myoglobin and potassium.
Renin blood level is collected after a 3-day diet, from a peripheral vein.
Ifa phlebotomist accidentally punctures an artery instead of a vein, he should immediately
apply pressure and report the case to the supervisor.

Sitesto be avoided (Venipunture)

1. Intravenous lines in both arms 5. Edematous arms
2. Burned or scarred areas 6. Partial/radical mastectomy on one or both arms
3. Areas with hematoma 7. Arms with arteriovenous (AV) shunt or fistula
4. Thrombosed veins 8. Casts) on arm(s)

Complicatons of Veniounctur
1. Immediate Local Complication
a. Hemoconcentration - is an increase in the number formed elements in blood resulting either from a
decrease or increase in plasma volume

b. Failure of blood to enter the syringe/vacutainer tube

Excessive pull of the plunger
Piercing the other pole of the vein
Transfixation of vein
Incorrect bevel position (bevel down)
Absence of vacuum
c. Syncope (fainting)
It is the transient loss of consciousness due to lack of oxygen in
the brain and results in an

inability to stay In an upright position.

If a seated patlent feels faint, the needle should be removed immediately,
the patient's head
should be owered between the legs and the patient should be instructed to breathdeeply.

45
2. Late Local Complication
a. Thrombosis isan abnormal vascular condition in which thrombus develops within a blood vessel
of the body
b. Thrombophlebitis isinfammation of a vein often accompanied by a clot which occursas a result
of trauma to the vessel wall

3. Late General Compiications -serum hepatitis and AlDS are acquired through
contaminated needles and needle stick

Causes of Hemolysis (hemolyzed specimen):

1. Using a needle that is too smal
2. Puling a syringe plunger back too fast
3. Expelling the blood strongly Into a tube
4. Forcing the blood from a syringe into an evacuated tube
5. Shaking or mixing the tubes vigorously
6. Performing blood collection before the alcohol has dried at the collection site

Causes of Hematom
1. The vein is fragile or too small for the needle size.
2. The needle penetrates all the way through the vein.
3. The needle is partly inserted into the vein.
4. The needle is removed while the tourniquet is still on.
5. Excessive probing.
6. Pressure is not adequately applied after
venipuncture.

l. SKIN PUNCTURE
A fingerstick to obtain blood for routine laboratory analysis is usualty preferred for children
older than one year old.
Length of the lancet: 1.75mm (preferred; to avoid penetrating the bone)
The depth of the incision should be < 2.0 mm for infants and children; and < 2.5 mm for adults,
to avoid contact with the bone.
The distance from the skin surface to bone or cartilage in the middie finger is 1.5-2.4mmn.
The cut should be oriented across the fingerprints to generate a large drop of blood
single deliberate motion.
using a

Preferred Sites:
1. Lateral plantar heel surface newborn
2.
Paimarsurfaces of the fingers (3 and 4th fingers)
3. Plantar surface of the big toe
4. Earlobes-least site

46
Skin Puncture Procedure
Select an appropriate puncture site.
For infants younger than 12 months old, this is most usually the lateral or medial plantar heel
a
surface.
For infants older than 12 months, children, and adults, the palmar surface of the last digit of the
b
second, third, or fourth finger may be used
The thumb and fifth finger must not be used, and the site of puncture must not be edematous or a

previous puncture ste because of accumulated tissue fluid.

Warm the puncture site with a wam, moist towel no hotter than 42' C; this increases the blood flow
2
through arterioles and capillaries and results in arterial-enriched blood.
Do not touch the
3 Cleanse the puncture site with 70% aqueous isopropanol solution. Allow the area to dry.
swabbed area with any nonsterile objec.
deliberate motion
Make the puncture witha sterile lancet or other skin-puncturing device, using a single
hold the heel with the forefinger at the arch
nearly perpendicuiar to the skin surface. For a heel puncture,
and the thumb proximal to the puncture site at the ankle. If using a lancet, the blade should not be longer
than 2 mm to avoid injury to the calcaneus (heel bonej.
Discard the first drop of blood by wiping it away with a sterile pad. Regulate further blood flow by gentle
excess tissue fluid.
thumb pressure. Do not milk the site, as this may cause hemolysis and introduce
Closed systems are available for collection
5 Collect the specimen in a suitable container by capillary action.
of nonanticoagulated blood and with additives for whole biood analysis. Open-ended, narrow-bore
volumes of 200 ul Both heparinized and
disposable glass micropipets are most often used up to
for the test ordered. Mix the
nonheparinized micropipets are available. Use the appropriate anticoagulant
specimer as necessary.
device.
Apply pressure and dispose of the puncture
of collection and patient demographics.
8 Label the specimen container with date and time
Indicate In the report that test results are
from skin puncture.
Lab Methods, 22od,2011
Source: Henry's Clinical Diagnoeis and Management by

Order offilling microcollectlonfubes:

1. EDTA
2.Other tubes with additves
3. Nonadditive tubes

Advantage of Skin Punture: venipunctures may cause

because large amount of blood required for repeated
1. For premature Infants
latrogenic anemla. reserved
therapy -accessible veins in sick infants must be
2. For sick infants which require parenteral
exclusively for parenteral therapy.
thinner and less elastic.
Skin puncture is often preferred in geriatric patients because the skin is
3.
tendencies
extreme obesity, severe burns, and thrombotic
Skinpunctureis useful inadutswith;
of a
central arch area of an infant's heel;fingers
Sites notaenerallyrecommendedfor skinpuncture:
infant less than one year old; thumb, Index and
fifth fingers and fingers
on the side of a

newborn or
mastectomy

47
Arterialized Capillary blood pHp(Q2
It is used for blood gas analysls (newborm and infants) for measuring pH and pCOz but not pOz
Earlobe is the preferred ste because ofvascularity, low metabolic requirements and ease with
which it can be arterialized
Lateral plantar heel surface is the most commonly used site.
tot trol ncatol ntel turfato mow Omn
Procedure:
1. Warm the earlobe or heel surface with paper towel saturated with wam water, 39-42" C for 3-5
minutes(heat dilates the capillaries and accelerates the rate of blood flow).
2Flick the earlobe with the index finger.
3. Cleansed the area with 70% alcohol.
4. Two heparinized tubes are placed in the center of the next drop of blood and filled to capacity
without air bubbles.
5. Both ends are sealed in clay after the insertion of flea.
6. Blood is stirred usinga magnet to draw the flea back ard forth across the length of the tube to mix
the specimen completely.

Notes to Remember.
Capillary "arterialization" should not be done if arterial blood pressure is below 95 mmHg or if
the area has a poor blood supply.
>Flea is a minute metal filling which may be inserted Into the capillary tube before collecting
blood to help mx the specimen while the blood is entering the tube.
The best method for blood gas collection in the newborn remains the indwelling umbilical artery
catheter.

Central Venous Access (CVA) Collection

Advantage: It eliminates multiple phlebotomies and useful in critical care and surgical
situations
Initial Procedure:5 ml of blood must be drawn and discarded to eliminatecontaminants
Disadvantage: not recommended for bacteriology (because organisms that grow on the walls of
the catheter can contaminate the blood specimen)

Order of Draw From Catheter Lines

1. Draw 3-5 mL in a syringe and discard
2. Blood for blood culture
3. Blood for anticoagulated tubes (lavender, green, light blue, etc.)
4. Blood for clot tubes (red, SST, etc.)

48
ReasonsIdealy,
for rapld separation of blood(aftercentrifugation
all measurements should be performed within 45 minutes to one hour after collection.
Serum or plasma should be separated from cells as soon as possible, preferably within one
hour.
"Adequate time for cdotting must be allowed to prevent latent fibrin formation, which may cause
undesirable clogging of automated chemistry analyzers.
Plasma should not remain in contact with the cells overnight. Even in the presence of the
anticoagulant, protein aggregation can occur in plasma that is stored in the refrigerator for a
few days or frozen for longer periods.
3000 relatve centrifugal force (RCF) for 10 minutes is the centrifugation requirement (required
RCF may vary depending on the manufacturer's recommendation).

1.To prevent ghycolysis

A pyuvat laut
2mg NaF/ml of blood prevents glycolytic enzyme (enolase-Mg-dependent
enzyme) or gtycolysis for up to 48-72 hour
The use of fluoride-containing tube is not necessary if plasma is separated from cells or glucose
is measured immediately.
Glucose concentration in unseparated serum and plasma decreases rapidly in the first 24 hours
and more slowBythereafter
Glycolysis occurs faster in newborns because their metabolism is increased, and in patientswith
leukemia because of high metabolic activity of WBCs.

2. Certain substances are very unstable

Failure to separate serum and plasma from red cells after 24 hours yield increases in total
bilirubin, electrolyes (sodium, caicium and magnesium) urea, albumin and total protein due to
movement of water into cells, resulting in hemoconcentration
Least stable in serumifnotremoved from the clot within 30 minutes: K', P and glucose
Unstable after 6 hours when the serum was not separated from the clot: albumin, bicarbonate,
chloride, C-peptide, HDL-cholesterol, iron, LDL-cholesterol, and total protein

3. To preveent shift of electrolytes.

This will result to false increase of potassium and subsequent decrease of sodlum in
Na" K). spstucio Katenia f
serum plasma.
and
4. To prevent hemolysis.
Effects of hemolsis: ElyAhocyhiC en)yme
a. Increased levels of the following analytes: enzymes (LD, ACP, ALT, AST); electrolytes (magnesium,
phosphorus and potassium); total protein; albumin; cholesterol and iron.
b. It interferes with the color reactions.
c. It increases bilirubin levels.
When serum blirubin approaches 430mmol/L (25 mg/1), interference may be observed in
assays for albumin (4-hydroxyazobenzene-2-carboxylic acid [HABA) procedure), cholesterol
(using ferric dhloride reagets), and total protein (Biuret procedure).
d. It inhibits lipase enzyme.

49
Interfering Conditions in the Measurement of Analytes
Most common interfering conditions: hemolysis, icterus and lipemia
Hemolysis and icterus strongly absorb specific wavelengths of light.

1. Hemolysis
Severe hemolysis causes a slight dilutional effect on the analytes preset in serum or plasma.

2. Icteric sample
Serum biliubin reaching 25.2 mg/L (430 mmol/L} which means icteric specimen, interferes with
the measurement of total protein, albumin, cholesterol and
glucose.
Bilirubin in a specimen is not readily removed and so
may cause spectral interference through
its high absorbance at wavelengths between 340 and 500 nm.

3. Lipemia
It occurs when serum triglyceride exceeds 4.6 mmol/L (40C mg/dl).
Itscatters light and eventually blocks transmission of light.
t can potentially be cleared from a serum or plasma specimen by ultracentrifugation.
Lipemia inhibits amylase, urate, urea, CK, bilirubin, and total protein. fake
Corrective measures for artifactual absorbance (lipemia): blanking technique and dual-
wavelength reading
03proipid- most a bundoi* npic
Storage and Transportof Specimens:
During storage (ambient temperature, refrigeration or freezing), the concentration of a blood
constituent in the specimen may change as a result cf various processes, inc!uding adsorption
to glass plastic tubes, protein denaturation, evaporation of volatile compounds, water
or
movement into cells resulting in hemoconcentration of serum and
plasma, and continuing
metabolic activities of leukocytes and erythrocytes.
The ice crystals formed during storage cause
disruptive effects to molecular structure
particularly to large protein molecules.
Serum or plasma must be stored at 4° C to 6° C if analysis is to be
delayed for ionger than 4
hours.
LDH 4 and 5 isoenzymes (decrease) and alkaline phosphatase
(increase) are affected by low
temperature storage prior to testing.

Specimen Conslderations:
Specimens that require chilling (4'C) during transport and storage of specimens:
blood gases, cathecholamines, gastrin, lactic acid, renin, PTH and ammonia,
pyruvate . i
Photosensitive analytes: biirubin, beta-carotene, folate, porphyrins and vitamin A
and 86
ICt abe cOat¢d

Notes to Remember
Plasma may be used in medical emergencies because samples do not have to clot before
centrifugation.
Increased of substances (proteins and urea) in unseparated serum or
movement of water into cells resulting to hemoconcentration.
plasma is also due to
Rimming the tube should be avoided because it may cause hemolysis and aerosol infection.
Normally platelets release potassium during clotting, so serum has a slightly higher value of
potassium than plasma from the same individual; this difference is accentuated when
platelet count is extremely elevated. the

50
 Excessive centrifugation (>3000 RCF for tubes without gel separator) may cause cell
slight elevation in LD and potassium, however, insufficient centrifugation (« 1000 RCFlysis
and
or < 10
minutes) may cause incomplete barrier formation in gel tubes or cell contamination of the
specimen.
Whole blood or plasma transfusion may cause increase plasma proteins, bilirubin, LD and
potassium but decrease sodium and chloride.
>Electrolyes are affected by evaporatlon ofspecimen prior to testing.
Collection of Other Body Fluids:
1. Cerebrospinal Fluid (CSF)
Most common method of collection: umbar puncture (between the third and fourth lumbar
vertebrae, or between the fourth and fifth lumbar vertebrae)
Other methods of collection: cisternal puncture and lateral cervical
puncture
Purpose of collectionto establish a diagnosis of infection (bacterial, fungal, mycobacterial, or
amebic meningitis), malignancy, subarachnoid hemorrhage, multiple sclerosis, or demyelinating
disorders
Required pressure before collection:between 90 and 180 mm Hg
Volume that can be collected: 20 mi of CSF (not more than 2 mLcan be removed when the
pressure is greater than 200 mm Hg)
Purpose of the CSF Vals/Tubes:Tube #1 goes to chemistry for glucose and protein analysis, or to
immunology/serology; Tube #2 goes to microbiology for culture and Gram stain; Tube #3 goes
to hematology for cell counts (tube #3 is the least likely to be contaminated by a bloody tap at
collection)
Complications of Lumbar Tap: cerebellar tonsillar herniation in patients with elevated
intracranial pressure; asphydiation in infants; paresthesia; headache; and, rarely, hematomas

2. Urine
Random specimens may be collected at any time, but a first-morning-voided aliquot is optimal
for constituent concentration, as it is usually the nost concentrated and has a lower pH caused
by decreased respiration during sieep.
For 24-hour urine collection, the first morning specimen should be discarded, record the time,
and collect the succeeding voiding for the next 24 hours overcollection occurs if the first
morning specimen is included in this routine.
24-hour urine creatinine-to assessthe completeness ofthe specimen
Sodium fluoride can be added to 24-hour urine for glucose determinations to inhibit bacterial
growth and cell gycolysis.
Pediatric collections require special attention to avoid stool contamination.

3. Synovial fuld, Pleural Fluid, Pericardial Fluid, and Peritoneal Fluld

Synovial fuld is collected by arthrocentesis,an aspiration of the Joint using a syringe, moistened
with an anticoagulant,usualy 25 units of sodium heparin per ml of synovial fluid.
Synovial fluid differs from the other serous fluids in that it contains hyaluronic acid (mucin) and
may contain crystals.
Sodium heparin is the preferred anticoagulant for synovial fluid.
Some hospitalstransfer synovial fluid to aerobic and anaerobic blood culture bottles
formicrobiologic culture.

51
 Thoracentesis is a surgical procedure to drain fluid (effusions) from the thoracic cavity and is
helpful in dlagnosing infammation or neoplastic disease in the lung or pleura.
Perlcardiocentesis and peritoneocentesis refer to the collection of fuid from the pericardium
(errusion) and the pertoneal cavities (ascites), respectively. These cavities normaly contain less
than 50 ml of fluid.
Pleural fuid is an utrafiltrate of the blood plasma. It is formed continuousity in the pleural cavity.

Notes toremember
Commonhy encountered pre-analytical errors: improper fling of sample tubes, wrong choice of
tubes or containers and selecting the incorrect test.

Ten Common Errors in Specimen Collection

1 Misidentification of patient
2 Mislabeling of specimen
3 Short draws/wrong
anticoagulant/blood ratio
Mixing problems/clots
5 Wrong tubes/wrong anticoagulant
6 Hemolysis/lipemia
7 Hemoconcentration from prolonged
tourniquet time
8 Exposure to light/extreme
temperatures
9Improperty timed
specimens/delayed delivery to
laboratory
10 Processing errors: Incomplete
centrifugation, incorrect log-in,
improper storage
Source: Henry's Cinical Dingnosis and Management by Lab Methods,22 ed, 2011

Reasons for Specimen Reecton:

1. Hemolysis/Lipemla
2. Clots in an anticoagulated tube
3. Nonfasting specimen (if required)
4. Wrong blood collection tube
5. Short draws
6. Improper transport (temperature)
7. Discrepancies between requisition and
specimen label
8. Unlabeled or mislabeled specimen
9. Contaminated
specimen/leaking container

52
ANTICOAGULANTS
1. Oxalate
It combines wth calcium to form an insoluble salt.
t interferes with Na', k, and most BUN (urease) measurements.
Concentration: 1-2 mg/mL of biood.
2. Cttrate
It combines wth caldum in a nonHonized form.
Concentration: 3.2% or 3.8% (0.105 M or 0.129 M) in a ratio of 1 part to 9 parts of blood
An insufficlent blood volume (short draw) leads to falsely increase clotting time.

3. Ethylenedamine tetraacetic acdd (EDTA)

It combines with caicium in a process called cheiation.
Preporation: di-potassium (KEDTA) in plastic tubes and tri-potassium (KEDTA) in glass tubes
Concentrotion: 1-2 mg/mL of blood
Use: Carcinoembryonic antigen (CEA), TDM and lead
KaEDTA is the spray-dried form while the KaEDTA is the lliquid form.
Excess EDTA, which resuts when tubes are underfiled, can cause red blood cells to shrink and
thus, change blood count results.
Blood specimens for nuceic acid testing used EDTA to inhibit enzymes that might lyze it.

4. Flouride
It forms weakly dissociated calcium components.
It interferes with the measurements of Na', K', and BUN (urease method).
Concentration: 10mg/ml of blood

5. Heparin (mucoftin polysutfuric acid)

I t acts as antthrombin and antithromboplastin, anti-Factor X; ideal universal anticoagulant.
t accelerates the action of antithrombin (l, neutralizing thrombin and preventing the formation
of fibrin.
Preparation: Sodium, itthium, potassium and ammonium salts
Concentrotion: 0.2 mg/ml. of blood
Use: Tests for K", NH, carboxy/methemoglobin, pH and blood gas and cytogenetic studies
Lithium heparin: measurements of glucose, BUN, ionized calcium, electrolyte and creatinine
Lithium heparin may be used for most chemistry tests.
Sodium heparin is the injectable form used for anticoagulant therapy.
ithium and ammonium heparin are the preferred anticoagulants for microcollection tubes.
Heparinined plasma is preferred over serum for potassium tests because when blood clots,
potassium is released from platelets into the serum and can false increase results.
Heparin is not the choice for nucleic acid testing because it can be coextracted with DNA and
inhibits DNA polymerase in polymerase chain reaction.
Hospitalized patients are likelyto be receiving heparin (especially undercritical care), which can
delay clotting in blood collection tubes even with activators and lead to fibrin strands that can
clog up aspiration probes on instrumentation.

Underfilling bloodcolectlontubescan affect:

RBC morphology and lipids in EDTAtubes
Binding of electrolytes and troponin to heparin in some plasma tubes

53
 BLOOD COLLECTION TUBES

Collection Tubes Additive Specimen Clinical Use

(Cap Coior Code)
Red None Serum General Chemistry
Gold With gel separator Serum General Chenuistry
Red/Gray
Orange PasiL/Gias : M NKN Thrombin ICI At: Serum General Chemistry
Yellow/GrayS KÁSPniKite
Royal blue None Serum Trace elements, toxicolog
trace element-free tube) nutritional studies and TDM

Sodium heparin, K, EDTA Whole blood/Plasma Toxicology and nutritionalstudies

Tan BroFap Sodlum heparin (glass tube)
K EDTA (plastc tube)
Green and Gray/Light green LIthium heparin
Pink Ka EDTA (with crossmatth
label required by AABB)
Black
Buffered sodium citrate
(4:1 ratio of blood to
anticoagulant)
Whoe blood/Plasma Lead test
Whole blood/Plasma General Chemistry
Whole blood/Plasma ABO and Rh typing.
Antibody screening and
Molecular diagnostics
Whole blood/Plasma ESR-Westergren Method

Yellow
LUithilum heparin with gel General Chemistry
Sodium polyanethol Whole blood/Plasma Blood culture
Sulfonate (SPS)

Acid citrate dextrose (ACD) HLA phenotyping and Paternity test

White EDTA and gel Whole blood/Plasma
Light blue Trisodium citrate Whole blood/Plasma
Molecular diagriostic
Coagulation test and
Heparin monitoring
Citrate, theophylline,
adenosine and
dipyridamole (CTAD)

Thrombin and soybean

trypsin inhibitor
AABB-American Association of Bilood Banks
HPR RP
Notes to Remember.
Polypropylene is used in several tube designs, including specimen tubes and test
Polystyrene or other high-impact plastic-type containers
tubes.
urne specimen.
are
commonly used for collection of
Tubes containing gels are not used in blood bank
interfere with the immunologic reactions. or for immunologic testing, as the gel may
Respinning gel tubes can cause increase potassium.
etende

54
 CARBOHYDRATESs

These are hydrates ofaldehyde or ketone derivates based on the location of the CO functional

group.
Corbohydrates: monosaccharides, disaccharides, oligosaccharides,polysaccharides
.cietAC
Glycol aldehyde is the simpliest carbohydrate (CH0).
Gucose is the only carbohydrate to be directly used for energy or stored as glycogen.
Glucose does not accumulate in the muscle. It does not enter the muscle cell freely, and when it
enters the cell with the help of insulin,it is quickty metabolized.
The brain is completely dependent on blood glucose for energy production- 2/3 of glucose
utilzatlon in resting adults accours in the central nervous system (CNS).
Glucose metabolism generates pyruvic acid, lactic acid and acetylcoenzyme A as intermediate

products.
The complete oxidation of glucose yields carbon dioxide, water and adenosine triphosphate. ti

Reducing and Nonreducing Sugars;

Reducing substances/sugars: giucose, maltose, fructose, lactose and galactose
The presence of a double bond and a negative charge in the enol anion makes glucose an active
reducing substance.
Sucrose is the most common nonreducingsugar
Nonreducing sugar do not contain an active ketone or aldehyde group.

PANCREAS
I t is both an endocrine and exocrine organ in the control of carbohydrate metabolism.
oIt is an endocrine gland, it secretes the hormones' insulin, glucagon and
somatostatin from differet cells residing in the islets of Langerhans in the pancreas.
o It is an exocrine gland, it produces and secretes an amylase responsible for the
breakdown of ingested complex carbohydrates.

Insulin
I t is the primary hormone responsible for the entry of glucose into the cell.
t is synthesized by the B-cells of the islets of Langerhans in the pancreas.
Itis normaly released when glucose levels are high.
It is the only hormone that decreases glucose levels hypoglycemic agent.
It is stored from sources such as liver, fat and muscle.
It has a reciprocal relationship with glucagon.
It promotes glycogenesis, fipogenesis and glycolysls; decreases glycogenolysis.
I t enhances membrane permeability to cells in the liver, muscle, and adipose tissue.
Serum insulin measurements may be falsely low in the presence of hemolysis. An insulin-
this
degrading enzyme found red blood cells as well as in other tissues is responsible for
In
problem.

55
 Glucagon
-ct t is the primary hormone responsible for increasing glucose-hyperglycemic agent.
tis synthesized bythe a-celsbf the islets of Langerhans in the pancreas.
I t is released during stress and fasting states.
I t enhancescatabolic functions during fasting periods;, promotes gycogenolysis.
Fastingplasmaglucagon concentrations is normaly 25-50 pg/mL
Natip0san ng qurt,
Qther hermonea that tend to increae atucoae concentration
1.Cortisol and corticosteroids (Glucocorticoids)
These are secreted by the cells of thé zona fasciculata and zona reticularis of the adrenal cortex.
They decreased intestinal entry of glucose into the cell.
They promote gluconeogenesis and lipolysis.
2. Catecholamines
These are released from the chromaffin cells of the adrenal medulla
They Inhibit insulin secretion and promotes glycogenolysis and lipolysis.
3. Growth hormone
(Somatotrophic)
It is secreted by the
anterior pituitary gland.
t decreases entry of glucose into
the cell.
I t promotes glycogenolysis and
glycoiysis.
4. Thyroid hormones
t promotes glycogenolysis,
gluconeogenesis and intestinal absorption of glucose.
5.
Adrenocorticotropic hormone (ACTH)
It stimulates release of cortisol from the adrenal cortex.
t
promotes glycogenolysis
and gluconeogenesis.
6. Somatostatin
tis produced by the delta cells of the islets of
Langerhans of the pancreas.
It is also
synthesized in the paraventricular
and arcuate nucleiof the hypothalamus (a
neuroendocrine hormone).
It primarily inhibits the action of insulin,
growth hormone and glucagon.
Clinlcal Conditions of Carbohydrate Metabollsm:
1. Hyperglycemia
It is an increase in blood glucose concentration.
it is toxic to beta cell function and
impairs insulin secretion.
Causes: stress, severe infection, dehydration or pregnancy,
pancreatectomy,
hemochromatosis, in_ulin deficiency or abnormal insulin receptor.
FBS level: 2126 mg/dL

Laboratory Findings in Hyperalycemia:

1. Increase glucose in plasma and urine
2. Increase urine speciflic gravty
3. Ketones in serum and urine
Decrease blood and urine pH (acidosis)
Electrolyte imbalance (Na, TK,LHCO
In the presence of normal renal function, plasma))glucose reaches a
"period of plateau" around
300mg/dL to 500mg/dL, that is, glucose urinary excretion will match the
the plateau overproduction, causing

56
 Serum osmolality is high as a resut of hyperglvcemia; sodium concentrations tend to be lowerdue
in part to losses (polyuria) and in part to ashift of water from cells because ofthehypergycemia.
Type 1DM patients are more likely to produce ketones as opposedto type 2 DM.
Ketoacidoscis resuting to pH Imbalanceresults from dehrydration, electrolyte imbalance and acidosis
Hyperkalemia is almost always present as a resut of the displacement of potassium from cellsin
acidosis.
Bicarbonate and total carbon dioxide are usually decreased due to Kussmau-Kien respiration (deep
respirations).
2. Hypogtycemta
it results from an Imbalance between gucose utilization and production.
It involves decreased glucose levels and can have many causes.
The warning signs and symptoms of hypoglycemia are related to central nervous system.
A diagnosis of hypogtycemia should not be made unless a patient meets the criteria of Whipple's
triad low blood glucose concentration, typical symptoms and symptoms alleviated by glucose
administration.
Glucose tolerance tests should be performed with great caution in patients with suspected
hypoglycemia because the procedure can induce severe reactive hypoglycemia, causing loss of
consciousness and even shock
Diagnostic test: 5-hour glucose tolerance test (hypogiycemic "dip" often is not seen until after 3 hours)

Hypoglycemnic values
65mg/dL to 70mg/dL glucagon and other gycemic hormones are released into the circulation
60 mg/dl-strongty suggest hypoglycemia (series of random fasting serum specimens)
50mg/dl to 55 mg/d! -observable symptoms of hypoglycemia appear
Hypogtycemic symptoms with a plasma glucose level s55 mg/dL (3.0 mmol/L) in an individual
who is not recelving medications for diabetes require further evaluation.
A blood glucose level s50 mg/dL (2.8 mmol/) in infants is considered abnormal and requires
diagnostic assessment
Healthy males will maintain plasma glucose of SSmg/dl to 60 mg/dl for several days, while
healthy females will produce ketones faster and allow plasma glucose to decrease to 40mg/dL

Svmptoms ofHypcalvcemia:
1. Neurogenic tremors, palpitatlons, anxiety, diaphoresis
2. Neuroglycopenic diziness, tingling, blurred vision, confusion, behavioral changes

Cassification of Hyooalycema:
1.Drug administration Insulin, alcohol, salicylates, sulfonamides, pentamidine
2. Critical illnesses hepatic fallure, sepsis, renal failure, cardiac failure, malnutrition
3. Hormonal deficiency epinephrine, glucagons, cortisol, growth hormone
-

4. Endogenous hyperinsulinism -pancreaticbeta cell disorders

5. Autoimmune hypoglycemia insulin antibodies
6. Non-beta cell tumors- leukemia, hepatoma, pheochromocytoma, lymphoma
7. Hypogtycemia of infancy and childhood galactosemia, GSD, Reye's syndrome
8. Alimentary (reactive) hypoglycemia post-gastricsurgery
9.Idiopathic (functionai) postprandial hypogtycemia

51
Notes to Remember.
>Alcohol consumption can inhibit hepatic gluconeogenesis and increaseghya»gen phosphorylase
activity, depleting hepatic gycogen stores and resuiting in hypoglycemia
AlcohoHnduced hypogtycemia is usually seen in the settingof a history of alcohol intake of 50
300g without food intake for the preceding 6 to greater than 36 hours.
A decrease in gycogen reserves coupled with failure of gluconeogenesis and enhanced glucose
utilization may be the cause of hypoghycemia in patients with severe sepsis.
Hypoglycemia in end-stage renal disease (ESRD) can be related to defectivegluconeogenesis,as
well as impaired hepatic gtycogenolysis due to poor nutritional status.
Infants and children with a deficiency of cortisol and growth hormone are prone to deveiop
hypoglycemia, especialy during an acute illness.
Alimentary (reactve) hypoglycemia occurs usualy within 4 hours after eating a meal.

DIABETES MELLITUS
t is a group of metabolic disorders characterized by hyperglycemia resulting from defects in
insulin secretion, insulin receptors or both.
Fasting plasma glucose concentrations 126 mg/dl on more than one testing are diagnostic of
DM.
Glucosurla occurs when the plasma glucose level exceeds 180 mg/dl (9.99 mmol/1) with normat
renal function.
Ketosis develops in DM from excessive synthesis of acetyl-CoA, as the body atternpts to obtain
required energy from stored fat in the absence of an adequate supply of carbohydrate
metabolites the presence of ketone bodies is a frequent finding in Individuals with severe,
uncontrolled diabetes.
In severe DM, the ratio of B-hydroxybutyrate to acetoacetate is 6:1.
The entire process of ketosis can be reversed by insulin administration.

Classification of Diabetes Mellitus

A. TYPE 1 DIABETES MELI US
Formerly knownas: Insulin Dependent Diabetes Mellitus (1DDM)
Juvenile Onset Diabetes Mellitus
Brittle Diabetes
Ketosis-Prone Diabetes
It is a resut of cellular-mediated autoimmune destruction of the B-cells of the pancreas.
Dlabetic individuals have insulinopenia (absolute insulin deficlency) due to loss of pancreatic B-
cells, and depend on insulfin to sustain life and prevent ketosis.
80-90% reduction in the volume ofthe Pcell is required to induce symptomatic type 1 DM- t is
only after most of the beta cells are destroyed that hyperglycemia develops.
There is genetic association between type 1 DM and HLA DR3 and DR4 the major locus is the
major histocompatibility complex on chromosome number 6.
Individuals at greater risk of developing this type of diabetes have high titers of multiple
autoantibodies -glutamic acid decarboxylase (GAD65) & insulin autoantibodies (IAA).
IAA are more commonin young children who develop type 1 diabetes, whereas GAD65 is more
common in adults.
Signs and symptoms: polyuria, polydipsia, polyphagla, rapid welght los, hyperventilation,
mental confusion and possible loss of consciousness.
Complications: microvascular disorders - nephropathy, nevropathy and retinopathy

58
 Idiopathic Type 1 DM
itis a fom oftype 1 diabetes that has no known etiology; it is strongty inherited; it does not
have -cell autoantibodies and have episodic requirements for insulin replacement.

B. TYPE 2 DIABETES MELLITUS

Formerly known a_: Non-Insulin Dependent Diabetes Mellitus
Adult Type/Maturtty Onset Diabetes Mellitus
Stable Diabetes
Ketosis-Resistant Diabetes
Receptor-Deficient Diabetes Mellitus
tis characterized by hyperglycemia due to an individua's resistance to insulin; there is relative
insulin deficiency.
t is associated with strong genetic predisposition and not related to an autolmmune disease.
It has been described as a geneticise's nightmare.
The individuals are at risk of developing macrovascular and microvascular complications.
t has milder symptoms as compared to type 1 DM, however, untreated type 2 DM will result
to nonketotic hyperosmolar coma due to overproduction of glucose (>300 mg/dL) accompanied
by severe dehydration, electrolyte imbalance (Na +TK) and increased BUN and creatinine.
Risk foctors: obesity, family history, advanced age, hypertension, lack of exercise, GDM,
impaired glucose metabolism

Recommendation:
t is recommended that adults ages 45 and older be screened for diabetes every 3 years, but
screening should be performed earlier and more frequently if the individual is at high risk.

COMPARISON BETWEEN TYPE 1 DM AND TYPE 2 DM_CANGENITAUY A'D G0-907.

TYPE 1 DM TYPE 2 DM

Pathogenesis B-cells destruction Insulin resistance ( RC

Incidence rate 5-10% 90-95%
Onset Any; tnost common to Any; most common with advancing
childhood/teens age, race/ ethnicity, hypertension,
dyslipidemia, polycystic ovarian
syndrome
Risk factors Genetic, autoimmune Genetic, obesity, sedentary
lifestyle, polycystic ovarian
syndrome, dyslipidemia and
hypertension
Cpeptide leve!s Decreased or undetectable 0 Detectable ) nomoi exe
Pre-diabetes Autoantibodies (+) Autoantibodies (-)
Symptomatology Symptoms develop abruptly Symptoms develop gradually
(some patients asymptomatic)
Ketosis Common; poorly controlled Rare
Medication insulin absolute (ParCntcol)Oral agents i Mctrormin / NSUUN
FORYIERLY KNOWN 10OM NIDOM CFLUNE TO DET
JuVENALE CNSET M HOULT ONSET DM

VP-DIADETES:GAO 5
1A

pt inonmCe: DM x
DKG Diobcn Ken kideMS
Ketoe Bodies 59
C.GESTATIONAL DIABETES MELLITUs (GDM)
t is a disorder characterized by impaired ability to metabolize carbohydrate usually caused bya
deficiency of insulin, metabolic or hormonal changes.
t occurs during pregnancy and disappears after delivery but, in some cases, returned years
later.
t is a type of glucose intolerance with onset or first recognition during pregnancy (diabetic
women who become pregnant are not included in this category).
Screening should be performed between 24 and 28 weeks ofgestation. Tnnts
The screening and dlagnosis of GDM is by the performance of a 2-hour 0GTT using 758 glucose
load.

Revised
1. FBS
Diagnostic Criteriafor GDM:
292 mg/dL
2.1-hour GCT 2180 mg/dL
3.2-hour OGTT= 2153 mg/dL
Interpretationof the Revised Criteria:
GDM is diagnosed if one of the three criteria is met.
Fetal inslin secretion is stimulated in the neonate of a mother with diabetes, but upon delivey
and the umbilical cord is severed, the infant's oversupply of glucose is immediately terminated.
Infants borm to dlabetic mothers are at increased risk for respiratory distress syndrome,
hypocalcemia and hyperbilirubinemia.
After giving birth, women with GDM should be evaluated 6 to 12 weeks postpartum.
GDM converts to DM within 10 years in 30%-40% of cases.

D. OTHER SPECIFIC TYPES OF DIABETESs

1. Pancreatic disorders/Pancreatectomy tCatmecokomines
TUMOR O AMFOULLA
2. Endocrine disorders- Cushing's 3ynarbme, pheochromocytoma,
acromegaly and hyperthyroidism
3. Drugs or chemical inducers of 6-cell dysfunction (dilantin and pentamidine) and impair insulin action
(thiazides, glucocorticoids)
4. Geneticsyndromes-Down syndrome, Klinefelter's syndrome, Rabson-Mendengall syndrome,
Leprechaunism, Huntington's chorea and Turner syndrome
S. Exocrine disorders cystic fibrosis, neoplasia and hemochromatosis

SlucoseMethodologies
The standard clinical specimen is venous plasma glucose.
Fasting glucose in whole blood is 15% lower than in serum or plasma.
A serum specimen is appropriate for glucose analysis if serum is
separated from the cells withir
30 minutes, but if serum is in contact with cells for longer than 30
minutes, a preservative such
as sodium fluoride that inbibits glycolysis should be added.
Venous blood glucose is 7 mg/dL lower than capillary blood due to tissue
blood glucose is same with arterial blood glucose.
metabolism; capillary
CSF glucose concentrations should be approximately 60% of the plasma concentrations.
Peritoneal fluid glucose is same with plasma glucose.
Lower plasma glucose compared to adults is observed in infants due to
decrease glycogen
reserve.

60
 Plasma glucose levels increase with age fasting, 2 mg/d/decade; postprandial,
Amg/dL/decade; glucose challenge, 8-13 mg/dl/decade.

Soecimen Handling and Storage

At room temperature (20-25°C), gycohysis decreases glucose by_ mg/di/hour in normal
uncentrifuged coagulated blood.
At refrigerated temperature (4°C), glucose is metabolized at the rate of about 2 mg/d/hour.
With long-term specimen storage, even at -20" C, glucose values decrease significantly and
progresst/ely.
WBC and RBC metabolize gucose resuting to decrease value in clotted, uncentrifuged blood.
Leukocytosis can lead to excessive dycolysis.
In serum specimens without bacterlal contamination or leukocytosis, resuts remain clinicaly
acceptable even after a delay of up to 90 minutes before separ tion of serum and cells.

LCHEMICAL METHODS
A. Oxidation Reduction Method
1. Alkaline Copper Reduction Method
Principle: Reduction of cupric ions to cuprous ions forming cuprous oxide in hot alkaline
solution by glucose.

Alkaline Copper Tartrate Glucose Cuprous lons

Heat

a. Folin Wu Method
Cuprous lons Phosphomolybdate

Phosphomolybdic Acid or
Phosphomolybdenum Blue

b. Nelson Somogyi Method

Cuprouslons+ Arsenomolybdate

Arsencmolybdic Acid or
Arsenomoybdenum Blue

c. Neocuproine Methodf 2,9 Dimethyl 1,10 Phenatroline Hydrachioride)

Cuprous lons Neocuproine

Cuprous-Neocuproine Complex
(Yellow or Yellow Orange)

d. Benedict's Method (Modification of Folin-Wu)

Itis used for the detection and quantitation of reducing substances in body fluids like blood and

urine.
Stabilizing agent: Citrate or tartrate

61
2. Alkaline Ferric Reduction Method (Hagedorn Jensen)
to a colorless ferrocyanide by glucose (Inverse
I t invoves reduction of a yellow ferricyanide
Colorimetry).

B. Condensation Method
Ortho-toluldine (Dubowski Method)
Glacial HAC (vtne gor)
Glucose+ Aromatic amines heatglycosylamine+ schiff's base
CGREEN CO0R)

I1. ENZYMATIC METHODS

Acts on glucose but not on other sugars and not on other reducing substances.

1. Glucose Oxidase Method

I t measuresthe B-D glucose. utarotoyc 'A other gu,e t) 0 gBuos
I t also measures CSF and urine glucose.

a. Colorimetric Glucose Oxddase Method (saifer Gernstemfield Method)

Glucose oxidase
Glucose + O Gluconic Acid + H202

Peroxidase
H20+Chromogenic Substances Oxidized Chromogenic Substance. + H0

b. Polarographic Glucose Oxldase

I t measures rate of oxygen consumption which is proportional to glucose concentration.
Glucose oxidase in the reagent catalyzes the oxidation of glucose by oxygen under firstorder
conditions, forming hydrogen peroxide.
The enzymatic conversion of glucose is quantitated by the consumption of oxygen on an oxygen-
sensing electrode.
The hydrogen peroxide is prevented from re-forming oxygen by adding molybdate, iodide,
catalase and ethanol.

Glucose oxidase

Glucose + O Gluconic Acid+ H20

H20+CHsOH Catalase CH,CHO+ 2H20

H02+2H +21 Mobybdate lh+2H20

BSCORBIC o t t t NTEKERNCE
2. Hexokinase Method
tis the most specific glucose method; reference method.
Plasma colfected using heparin, EDTA, fluoride, oxalate or citrate may be used for
this test.
Other samples: urine, CSF and serous fluids
REPE RENCE MD : MO sPECHC MT0
62
 Hexokinase
Glucose ATP Glucose-6-Phosphate + ADP

Glucose-6-Phosphate + NADP
6phosphate dehydrogenase (G-6Pt) nOST SPrcRt RAT

6 Phosphogluconolactone + NADPH
NAOPH RL0ucd NAOP . Hob rol*
Notes to Remember,
Fase decreased values of glucose (glucose oxidase method): ascorbate, blirubin, uric acid,
giutathione, creatinine, -cysteine, -dopa, dopamine, methyldopa, and citric acid.
The presence of bleach in the glucose oxidase method can cause false increased of glucose.
Hemolysis and icterisia affect hexokinase method causing false decreased ofplasma glucose.
Hemotyzed samples contalning> 0.5g hemoglobin/dL are unsatisfactory for hexokinase because
phosphate esters and enzymes released from RBCs interfere with the assay generating NADH.
Hexokinase method is not affected by the presence of ascorbic acld or uric acid.
The enzymatic converslon of glucose to product is quantitated by a color change reaction at the
last of a series of coupled chemical reactions (kinetic analyis).

3. Glucose Dehydrogenase Method

I n this method, glucose is reduced to produce a chromophore that is measured
spectrophotometricaly or an electrical current.
The amount of NADH generated is proportional to the glucose concentration.
t provides results in close agreement with hexokinase procedures.
Mutarotase is also added to shorten the time necessary to reach equilibrium.

a-D glucose +mutarotase B-D glucose

Glucose dehydrogenase

B-Dglucose + NAD D-gluconolactone+ NADH

Oiaphorase
MTT+ NADH MTTH (blue color)+ NAD

4. Dextrostics (Cellular Strip)

it is important in establishing correct insulin amount for next dose.
tis effective in reducing the rate of development of diabetlc complications.
An individual with a capillary glucose of 2140 mg/dL (7.8 mmol/), should be rescreened with a
fasting plasma glucose, HbAlc, or OGTT using venous samples.
Whole blood capilary glucose values obtained with point-of-care devices are useful for the
detection of Iyperglycemia and hypoglycemia in individuals with diabetes, and help to monitor
and direct therapy, but should not be used to diagnose diabetes or hypoglycemic disorders.
ecawde testing / deuetroi *d /toa Mext Tet: F89. MBho
cynatio ng inod guO oddax

Estpowishing torcut huin ont x dok

PoCT TNg

63
5. Interstitial Glucose Measurtng Device
I t is used for continuous monitoring of glucose levels in people with diabetes
It utilizes electrochemical methods to automatically and frequently measure glucose levels in
the interstitial fluid of dermis or subcutaneous fat tissue, and require repeated calibration to
plasma or whole blood glucose levels.
The resuts of this test provide information about glucose patterns over hours to days.
This glucose "trend analysis" can reveal useful findings for modifying treatment, such as
unsuspected nocturnal hypoglycemia or postprandial hyperglycemia.
This devise used for glucose measurement is only supplemental may supplement but cannot
replace coventional home blood giucose monitoring.
Interstitlal glucose is in slow (5-30 minute) equilibrium with capillary blocd glucose and
therefore is not equal to blood glucose, except in stable systems.

Samples for GlucoseMeasurement

1. RBS Random
( Blood Sugar), sudgen oss o unmioustess
I t is requested during insulin shock and hyperglycemic ketonic coma.
2. FBS (Fasting Blood Sugar)
It is a measure of overall glucose homeostasis.
Requirement: NPO (Non-Per Orem) at least 8 hours before the test
3. 2-Hour PPBS (2-Hour Post Prandial Blood Sugar)
It measures how well the body metabolizes glucose. HYPERL CEVIA 4o ngkll
4. GTT (Glucose Tolerance Test) wYPOGLy CEMIA 0mgld
It is a multiple blood sugar test.
t is used to determine how well the body metabolizes glucose over a required period of time,
same with 2-HPPBS.
It should be performed to diagnose gestational diabetes.
t is not generally recommended for routine clinical use in the diagnosis of diabetes.

Knds of Glucose Tolerance Tests

a. Oral Glucose Tolerance Test (0GTT)
a. Janney-1saacson Method (Single Dose Method)- most common
b. Exton Rose Method (Divided Oral Dose or Double Dose method)
Added Plasma Glucose After Intake of GlucoseLoad
3 0 mins = 30-60 mg/dLabove fasting
1-hour 20-50 mg/dL above fasting
2-hour = 5-15 mg/dL above fasting
3-hour = fasting level or below

b. Intravenous Glucose Tolerance Test (IVGTT)

It is used for DM patients with gastrointestinal disorders.
Fasting blood sample is also required.
Glucose load:0.5 g of glucose/kg bodyweight ( given within 3 minutes) administered
intravenously
The second blood collection is after 5 minutes of IV glucose.

64
Indications
Those
(1VGTTE
.
who are unable to
b. Those with altered tolerate a large carbohydrate load.
gastric
c. Those who
had previous physiology.
d. Those with chronic operation or surgery in the intestine.
malabsorption syndrome.
Requirements
L.
for OGTI
Patient should be ambulatory. P SHOuO NO1 oe eep kiDDEN
depletion and inactivity or dapot
CHO naKaKalakad!
bed rest impair
150g carbohydrate/day for 3 daysglucose
2. Unrestricted diet of tolerance.
prior
synthesis of inducible glycolytic enzymes. testing. 09
to stabilize the to /mor
3. The patient should not smoke and drink alcohol
4. prior to testing. : CONVALEICENT PO
Fasting for 8 to 14 hours. RECOVERY
5. Glucose Load
75 gms WHO standard glucose load
100gms Prcgnont Suspccted 60M
1.75g of glucose/kg body weight childrenlto a maximum
-

of 758)
Procedure
1.
for OGTI: more tman pmins- SI0P/KEJEC n091 IMPOR THNT/ 0 R AMPLE
Instruct the patient to drink the (ERO
2. Collect blood
gBucose load (within 5 minutes). CONFIKA
sample after 1-hour, 2-hour, 3-hour, respectively. OPToAIAL
3. Collect the fasting blood
sample. (urine nmay also be collected)
The patient should avoid exercise, eating,
drinking (exeepeWater) and smoking during the test.
For nonpregnant women and adults, only the fasting and the 2-hour sample may be measured
(or according to the physician's request).

Criteria for Fasting Plasma Glucose (FPG):

1. Non-Diabetic (Prediabetes) <100mg/d
2. Impaired Plas1na Glucose (PG) Ponial = 100-125 /mg/di
DM
3. Diabetes Mellitus 2126 mg/d

Categories of QralGlucose Tolerange Test:

1. Normal/Non-diabetic 2-hr plasma glucose (PG) <140mg/d
2. Impaired GTT 2-hr PG 140-199 mg/d
3. Diabetes Mellitus 2-hr PG 2200mg/dl

Diagnostic Citeria for Diabetes Mellius: 2 200mg/di (with symptoms of DM)

1. R8S
2. FBS 2126 mg/dl
3.2-hr Post Giucose Load 200mg/dl
26.5% (using NGSP-certified method)
4. HbAie

Impaired Fasting Glucose

is characterized by fasting blood glucose concentration between normal and
diabetic values (FBS = 100-125 mg/dL).

DM PATIEN
NORMIAL NON OM
70 100ng ld F89:1 moidL
65
a thr 617: t00 ng dt
Lr-OSTT:140mg
Impalred Glucose Tolerance
t i s characterized by fasting blood glucose concentration less than those required for
the diagnosis of diabetes, but the OGTT Is between normal and diabetic values
Hour 0GTT 140-199mg/dl).
(2

Notes toRermember, M oir a guove tod, dlena quoo should rcaum to rash ng tcve
Normally, serum glucose levels rise and then fall within about a 2-hour period.
If the glucose levels remain elevated, however, the diagnosís of diabétes mellitus may again be
made.
glucose is detected in the urine at any point, evidence for this condition is also obtained,
although absence of urinary glucose does not in any way rule out diabetes mellitus.
Lob Keut: t) gluuoauria hut nt 0ormoghycmla
ob eNal ublor
5. GLYCOSYLATED HEMOGLOBIN (HbAte)
t is known as
glycated hemoglobin.
t is the largest subfraction
of normal hemoglobin A in both diabetic and non-diabetic
individuals.
t is the hemoglobin A that is irreversibly glycosylated at one or both N-terminal valines of the B
chains of the tetrameric hemoglobin molecule, including hemogtobin that may aiso (but not
solely) be glycosylated on lysine residues.
It represents a "weighted" average of glucose levels, with youngest
erythrocytes contributing to
greater extent than older ones.
a
opli Once Tot. ng icrm nonitoringter
Diagnostic Sianificance
t is a reliable method in the
monitoring of long-term glucose control.
It reflects the average blood glucose level over the
previous 2-4 months.
HbAlc levels should be performed every 3 to6 months in
individuals with diabetes to monitor
glycemic control using a certified method, traceable to the Diabetes Control and
Trial (DCCT) reference method. Complications

Measurement:
Dietary status on the day of the test has no effect on HbAe
tis recommended that HbAe be measured twice a year for patients who are meeting treatment
goals, and quarterly for non-compliant patients.
Specimen: EDTA whole blood
Methods: Electrophoresis, Immunoassay, HPLC, Affinity Chromatography
DIRECT LY9IS RED tEL ® gb
Interpretationof Resuits:
5.7%-6.4%- indicates increased risk for diabetes (pre-diabetes)
26.59% on at least two ocasions indicative of diabetes mellitus; can be used
diabetes using a method that is National Glycohemoglobin Standardization
todiagnose
and standardized to the DCCT assay. Program certifed
nio Older red blood cells and iron deficiency anemia have high HbA levels.
HbA is not suitable for patients with shortened RBC lifespan disorder since
value.
they have low HbAe
For every 1% change in the HbA Value, 35 mg/dl is added to plasma glucose.
High levels of serum glucose also result in the glycosylation of Hb.

66
 Hyperglycemia has been associated with a decrease in erythrocyte survival, suggesting that
HbAie leves in poorly controlled patients may underestimate thelr mean plasma glucose
concentration.

Factors Affecting HbAic Results:

Shortened red blood cell survival, lower mean blood cell age, need for transfusions (with certain
hemoglobinopathies) hemolytic conditions, presence of carbamylated hemoglobin (uremia,
hypertrigtyoceridemia and hyperbilirubinemia), opiate and alcohol use, lead poisoning and
salicylates.

CHROW& HLANEANIk
6. FRUCTOSAMINE HComanive ot, Montor A wH66 VARuaNTS (4KBO LPePAN)
t is also known as glycosylated or glycated
albumin/plasma protein ketoamine.
I t i s a reflection of short
term glucose control (3-6 weeks).
May be useful for monitoring diabetic individuals with chronic hemolytic anemias and
hemoglobin varlants (Hb Sor Hb C)-decreased RBC lifespan.
t should not be measured cases of low plasma albumin (<30
in g/L)-low fructosamine.
Methods: Affinity chromatography, HPLC and Photometric
Reference value: 205-285 umol/L trUM
Interference: high uric acid, triglyceride and bilirubin levels; and the presence ofheparin or
hemolysis

Inbom EmorsotCarbehvdrate Metabelism

1.Galactosemla
I t is a congenital deficiency of one ofthree enzymes involved in galactose metabolism.
I t is caused by falilure tothrive syndrome in infants.
3 enzymes: galactose-1-phosphate uridyl transferase (most common deficiency),
galactokinase (GALK} and uridine diphosphate galactose-4-epimerase (GALE)
Galactose-1-phosphate uridyl transferase converts galactose-1-phosphate to glucose.
Laboratory features: elevated blood and urine galactose
Clinical features: jauncide, hepatomegally, easy bruisability, galactosuria, E. coli sepsis, cataract,
hypotonia and sensory neural deafness
Diagnastic test: Erythrocyte galactose-1-P04 uridyl transferase activity

2. Essential fructosuria
t is an autosomal recessive disorder characterized by fructokinase deficiency.
.Fruktokinase catalyzes the conversion offructose to fructose-1-phosphate.
Diognostic indicator: the presence offructose in urine (fructosuria)

3. Hereditary fructose Intolerance

.tis a defect offructose-1-6-biphosphate aldolase Bactivity in the liver, kidney and intestine.
I t is also characterized by the inability to convert fructose-1-phosphate and fructose-1,6-
biphosphate into dlihydroxyacetone phosphate, glyceraldehydes-3-phosphate and
gyceraldehydes.
Cinicalfeatures: Irritability, 1ethargy, seizures and hepatomegaly

67
4. Fructose-1,6-biphosphate deficiency
t is a defect in
fructose-1,6-biphosphate results in failure of hepatic glucose generation by
gluconeogenic precursors such as lactate and ghycerol.
Clinical features: hypoglycemia, lactic acidosis, convulsions and coma

5.Glycogen Storage Disease (GSD)

I t is an inherited autosomal recessive trait.
This disease is a
breakdown of
consequence of inherited deficiencies of enzymes that control the synthesis or
glycogen-abnormalquality
Cause liver damage: types I, I, V, Vi, 0
or quantity of glycogen is found in these disorders.
IX,
Cause muscular defect: types V, VMI
Signs of liver glycogenoses: hypoglycemia
and
Symptoms of muscle ghycogenoses:
hepatomegaly
muscle cramps, exercise intolerance, fatigue, and weakness
Von Gierke disease is the most
common GSD; it is associated with hyperlipidemia.
Other GSDs: deficiencies of LDH,
PK, phosphoglycerate kinase and mutase
Intravenous Galactose Tolerance Test
Is for type 1 6SD( glucose levels).
Serial blood specimens are collected for 2 hours
at 15 minutes interval for testing

GLYCOGEN STORAGE DISEASE

Type of Synonym Enzyme Deficient Clinical Features
GSD (Tissue Affected)
Von Glerke
1b Glucose-6-Phesphatase Hepatomegaly, retarded growth, seizures
Glucose-6-phosphatase
translocase Sameas Ib; recurrent bacterial infections
Pompe 1.4-Glucosidase
lla Cori Forbes De brancher Cardiomegaly, infantile death
Hepatomegaly, muscle weakness,
(liver arvd muscle) retarded growth, cardiomyopathy
Ib De brancher (liver) Same as lla except muscle weakness
IV (Andersen AC (Branche Cirrhóss, esophageal varices, ascites
V
c Ardle lcle Phosphorylase. Myoglobinuria, muscle cramps
Hers Lver Phosphoryiase Hepatomegaly, hypoglycemia
VII Tarui Phosphofructokinase
VIll
Painand stirfness on exertion
Adenyl kinase Urinary excretion of cathecolarmines
IXa Phosphorylase kinase (liver)
Hepatomegaly, hypoglycemia, delay in
IXb motor development
Phosphorylase Hepatomegaly, retarded growth, muscle
(liver and muscle) hypotonia
Cyclic AMP-dependent kinase Hepatomegaly
X Fanconi-Bickel Glucose transporter-2
Gycogen synthase Hepatomegaly, rickets
No hepatomegaly; hypoglycemic
symptoms in mornin8: growth delay

68
Notes toRemember.
1. CSF Glucose
t is about 40 to 60% of the blood plasma glucose level.
Any changes in blood sugar are reflected in the CSF approximately one hour later because of the
lag in CSF gucose equilibrium time.
For comparison, a blood glucose specimen should be collected at least 60 minutes before the
lumbar puncture.
Amarkedly decreased CSF glucose (e 40nmg/dl) &increased WBC count (neutrophils) is seen in
bacterlal meningitis.
Increased: diabetes mellitus
Decreased: bacterlal meningitis, tuberculosis, fungal and amebic meningitissubarachnoid
hemorhage; systemic hypoglycemia
Reference value: Child 60-80 mg/dL
Adult 40-70 mg/dL
Normal CSF-to-glucose ratio: <0.5
2.C-Peptide Test
tis formed during the conversion of pro-insulin to insulin.
The amount of circulating C-peptide provides reliable indicators for pancreatic and insulin
secretions (B-celi function).
t can be used to monitor individual responses to pancreatic surgery.
This test mainly evaluates hypoglycemia and continuous assessmentof B-cellfunction.
Specimen: fasting serum
Method for testing: Immunometric assay
Increased: insulinoma, type 2 DM, ingestion of hypoglycemic drugs
Decreased: Type 1 DM
Reference value: 0.90-4.3 ng/mL (CF: 0.333)
Normal ratio ofC-peptide:insulin 5:1 to 15:1

3. Ketone Test
Normal ratio of B-hydroxybutyrate and acetoacetic acid is 1:1 (0.5-1.0 mmol/leach).
The ratio of p-hydroxybutyrate to acetoacetate is greatly increased in diabetic ketoacidosis
(DKA) due to the altered redox state and elevated levels of NADH in the hepatic mitochondria.
An increased in serum acetone is indicative of a defect in the metabolism of carbohydrate.
B-hydroxybutyrate levels are high in diabetic ketoacidosis (DKA) and fal with treatrment,
whereas acetoacetic.acid and acetone levels rise with treatment.
A ketone test Is recommended when plasma glucose reached 300 mg/dL
The entire process of ketosis can be reversed through restoration of an adequate level of
carbohydrate metabolism.
Calculation ofthe anion gap is commonly used to monitor recovery from DKA.
Specimen: fresh urine or serum (important for individuals with type 1 DM to detect ketosis)

Ae 1

69
 Methods
Gerhardt's ferric chloride test
a. reacts only with acetoacetate
-

b. Nitroprusside test 10x more sensitive to acetoacetate than

to acetone
c. Acetest tablets detects acetoacetate and acetone (lesser degree)
d. Ketostx-detects acetoacetate better than acetone
f.KetoSite Assay detects B-hydroxybutyrate; not widely used
Strips and tablets for ketone testing (using sodium ntroprusside); enzyme test for B-
hydroxybutyrate

False result
a. False-positive presence of angiotensin-comverting enzyme inhibitors and other
medications
sutfhydry!
b. False-negative old strips; strips with excessive contact with
air; after ingestion of large amounts of
vitamin C

icmenbcr

nnoninMa

70
 LIPIDS AND LIPOPROTEINSs

LUPIDS
These are commonly referred to as fats, composed mostly of carbon-hydrogen bonds.
These are primary sources of fuel, they provide stability to cell membrane and allow for
transmembrane transport.
These are insoluble in blood and water, but soluble in organic solvents (chlorofom and ether).
They require special transport mechanisms (lipoproteins) for circulation in the blood.
Major lipids: PhosphoHipids, Cholesterol, Triglycerides, Fatty acid and
Fat soluble vitamins (ADEK)

PHOSPHOLIPID (Conjugated Lipld)

I tis the most abundant liplds derived from phosphatidic acid.
I t originates in the liver and intestine.
t i s produced from the conjugation oftwo fatty acids and a phosphorylated glycerol.
t is an amphipathic lipid, which means it contains polar hydrophilic (water-loving) head groups
and non polar hydrophobic (water-fearing) fatty acid side chains the saturated fatty acid
content of plasma phospholipids is reported to be an independent risk factor for atherosclerosis.
It is similar in structure to triglycerides, except that they contain two fatty acids.
In the lungs, it is produced by type l pneumocytes in the form of lamellar bodies.
Reference value: 150-380 mg/dL (serum) o-g/ tMNIO1IC uio
membrone Stobiliacr Reduco surrace Tcnsions, Lung 9urrautont Gestano0gl Markcr
ongc1a di sorder
Functions:
Phospholipids alter fluld surface tension (surfactant) it decreases surface tension within the
alveolar space, thus allowing effective gas exchange, and prevents alveolar collapseduring
expiration.
It participates in cellular metabolism and blood coagulation.
They are also important substrates for a number of lipoprotein metabolizing enzymes (e.g.,
LCAT, LPL, HL}; therefore changes in the composition could adversely affect the function of
these enzymes.
Deficiency of surfactant leads to neonatal respiratory distress syndrome (RDS).

Forms ofPhospholipids: ays Rand:NG SURFOTANTS

1. Lecithin/Phosphatidyl choline 70% LmgldL Man
20% Fctal ung Maturity
2. Spingomyelin
3. Cephalin 10%
Phosphatidyl ethanolamine
Phosphatidyl serine
Lysolecthin + Inositol Phosphatide

Spingomyein
it is the only phospholipid in membranes that is not derived from glycerol but from an amino
alcohol called sphingosine.
t is an essential component of cell membranes (RBC and nerve sheath).
t accumulates in the liver and spleen of patients suffering from Niemann-Pickdiseaseflipid storage
disorder).

71
Notes to Remember
The only unique structural feature common to all phospholipids is the presence of lipid-bound
phosphate.
The lipid concentration in amniotic fluid refiects thelipidspresent in intrapulmonary secretion.
Maturelungfunction correlates strongty with 1/5 ratio2
Fetal lung maturity testing should be at less than 39 weeks and uncertain gestational age.
Spingomyelin serves as a reference material during 3 trimester of pregnancy because its
concentration is constant as oppose to lecithin.

Methods
Quantitative analysis of phospholipids is rare in laboratory medicine - it provides little added
information in cases of dysbetalipoproteinemia. /
Phospholipids can be measured in disorders characterized by altered phospholipids composition
and lipoprotein distribution.

1. Estimaton of serum lHpld

Each mole of
phosphorus
phosphorus contributes about 49% to the total phospholipid mass; thus
phospholipid mass can be determined by multiplying the
phospholipid phosphorus
concentration (expressed in mg/dL) by 25.

2. Status of fetal lung maturation

The status of
Lecthin/SpingomyeBin (LS) ration
-

fetal
lung maturation is estimated from the evaluation of puimonary surfactant in
amniotic fluid the lecithin/sphingomyelin ratio (L/S) and
phosphatidyl glycerol (PG) by
chromatography or the microviscosity by fluorescence polarization are used.
TLC followed by densitometric
quantitation is the method for L/S ratio.
Microviscocity of amniotic fluid can be measured fluorescence
by polarizaticn.

CHOLESTEROL/3-hydroxy-5,6-cholestene
It is an unsaturated steroid alcohol containing four rings, and it has
similar to fatty acid.
a ingie C-H side chain taii

u t is amphipathic hydroxyl group in the A-ring is the hydrophilic part of cholesteroi.

I t is found on the surface of lipid layers;
synthesized in the liver.
t isalmost exclusively synthesized by animals; not catabolized by most cells does not serve as
a source of fuel.
i t s transport and excretion is promoted by
estrogen.
t should be measured in all adults 20
years of age and older at least once every 5
individuals). years ( healthy
Reference value: Desirable = <200 mg/dL
Interpretotion of Result: Borderline 200-239 mg/dt
High Cholesterol =
2240 mg/dL
Functions:
Precursorof five major classesof steroids: progestins, glucocorticolds,
androgens and estrogens. mineraiocorticoids,
tis an important constituent in
the assembly of cell membranes and bile
acids.

72
 A small amount of cholesterol, after first being converted to 7-dehydrocholesterol, can also be
transformed to vitamin Da in the skin by irradiation from sunlight.

Dlagnostic sianBficance:
t evaluates the risk for atherosclerosis, myocardial and coronary arterial occlusions.
There is a direct relationship between elevated serum cholesterol and myocardial infarction.
It is used as thyroid, liver and renal function tests; and for DM studies.
t is essenträl in the diagnosis and management of lipoprotein disorders.
tis also used to monitor effectiveness of lifestyle changes and stress management.
ChoAE T.: THYRONO TEST T44 mORMONES )
Formms of Cholesterol
1. Cholesterol Ester (CE) - 70%
It is found in plasma and serum.
t is the cholesterol bound to fatty acid (hydrophobic form).
Because it is not charged, it is classified as a neutral lipid arnd are not found on the surface of
lipid layers but Instead are located in the center of lipid drops. and lipoproteins, along with
triglycerides
It undergoes esterification by LCAT.
Excess cholesterol is re-esterified by the microsomal enzyme acyl: cholesterol acyl transferase
(ACAT) and is stored until it is needed.

Lecithin-Cholesterol Acyl Transferase (LCAT)

It is normaly present in human plasma, it catalyzes the esterification of cholesterol (HD) by
promoting the transfer of fatty acids from lecithin to cholesterol which results in the formation
of lysolecithin and cholesterol ester.
It is synthesized in the liver.
It enables HDL to accumulate cholesterol as cholesterol ester.
Activator of LCAT: Apo-1

2. Free Cholesterol (Fc) - 30%

It is found in plasma, serum and RBCs.

It is a polar nonesterified alcohol.
It is produced via lysosomal hydrolysis and becomes available for membrane, hormone, and bile
acid synthesis.
Free cholesterol and phospholipids (with moderate hydrophobicity) are found on the surface of
lipoproteins.

Methods:
Total cholesterol (TC) concentration is measured rather than its forms.
Cholesterol may be assayed from nonfasting blood samples-fasting has litle effect on TC.
Cholesterol increases with age, with women having lower values than men before age 45.
Serum total cholesterol increases at 2 mg/dL/year between 45 to 65 years old.
Decrease level of estrogen anmong postmenopausal women contributed to the increase of serum

cholesterol.
Either piasma or serum can be used for measurements.

73
Patient Preparation;
Two weeks
prior to testing, the person should be in its usual diet -before individuals'
cholesterol levels are measured,it is important that they be
on their usual diet for 2 weeks and
are neither gaining nor
losing weight.
L. Chemical Methodss
Principle: Dehydration and oxidation of cholesterol to form a colored
The presence of double compound.
bonds and hydroxyl group in the sterol's structure makes it possible for
cholesterol to carry out a colorimetric assay.

1. Liebermann Burchardt Reaction

End product Cholestadieny! Monosulfonic Acid (green end color)
2. Salkowski Reaction
End product Cholestadienyl Disulfonic Acid (red end color)
PrecautionS:
1. Avoid hemolyzed blood -falsely increases cholesterol levels
2. Avoid icteric specimens 5mg% to 6mg% increase in
chole/mg bilirubin above normal; bilirubin can nm
interfere with TC measurements because of its own spectral properties; bilirubin absorbs light at 500
3. Avoid water contamination.
4. Precise and accurate
timing for coBor development must be observed
Color Developer Mixture (Liebermann Burchardt Reagent):
a. Glacial Acetic Acid
b. Acetic anhydride
c. Concentrated H;S04

General Methods:
1.One-Step Method
2. Two Step Method
3.Three-Step Method-
4. Four-Step Method
Colorimetry (Pearson, Stern and Mac Gavack)
Extraction+ Colorimetry (Bloors)
nnon Saponification + Exttatön * Colorimetry (Abell-Kendai)
Saponification Extracton + Colorimetry +Precipitation
(Schoenheimer, Sperry, Parekh and lung)

I. Enzymatic Methods
Advantages: rapid; use microliter quantities of sample; do not require a preliminary extraction step
I t is thee most common method of
quantifying
the cholesterol oxidase reaction is
to measure the amount of hydrogen peroxide produced.
f the cholesteryl ester hydrolase step is omitted, they can be used to
cholesterol.
measure unesterified
Interference: elevated levels of ascorbic acid (low TC); hemoglobin and bilirubin
Hemoglobin has a pseudo-peroxidase activity that can consume the hydrogen peroxide
produced in reaction.
Hemolyzed products (catalase) do not significantly affect cholesterol measurements even at
abnormally high plasma concentrations.
The color of the hemoglobin falsely increased total cholesterol.
Bilirubin is oxidized by H20, which results to losing its absorbance at 500 nm.

74
 Bilirubin exceeding 5 mg/dlL decreases cholesterol by 5% to15%

Cholesterol Oxidase Reaction: ENZ7 MATO MrD CRE

he art yoROGEN PEKDO4E
Cholesterol esterase
Cholesterol Ester + H20 Cholesterol+ Fatty acid
Cholesterol oxidase
Cholesterol+02 Cholest-4-en-3-one + H20
Peroxidase
H202+ phenol +4-aminoantipyrine Quinoneimine dye

en Levy and Brodie Method) CHEMMCH ,1-STP MmO

it uses hexane extraction after hydroiysis with alcoholic, KOH followed by reaction with
Liebermann-Burchardt color reagent. Colori mctry aponni fication
exTaution
Decreased Cholesterol: 5
increased Cholesterol:
1. Hyperlipoproteinemia types 11, 1, V 1. Severe hepatocellular disease (AKRHOIE)
2. Biliary cirrhosis 2. Malnutrition
3. Nephrotic syndrome 3. Severe burns
4. Poorly controlled diabetes mellitus 4. Hyperthyroidism
5. Alcoholism 5. Malabsorption syndrome
6. Primary hypothyroidism
1. Diobctes Melitus CHOLEL THYROIDISM
chok Thyradi sm
TRIGLYCERIDE/TRIACYLGLYCEROL (Neutral Fat) ' MARKER O hiHEo sOE RONS
It contains 3 molecules of fatty acid and one molecule of glycerol by ester bonds.
and water insoluble.
-Iukar lt does not contain charged or hydrophilic groups very hydrophobic
t is the main storage lipid in man (adipose tissue) constitutes 95% of stored fat and the
hea
predominat form of glyceryl ester found in plasma.
40-0 It allows the body to compactly store long carbon chains (fatty acids) for energy that can be
used during fosting states between meals.
their fatty acids are released to the cells
Eunction: When triglycerides (TAGs) are metabolized,
and converted into energy provides excellent insulation.
The breakdown of TAG is facilitated by lipoprotein lipase (LPL), epinephrine and cortisol.
ingests, absorbs, resynthesizes, and transports about 60g-130g of fat daily
in
An average person
the body, mostly in the form of trigtycerides.
People with low caloric intake have relativeiy low triglyceride levels.
Fasting requirement: 10 hours to 12 hours
Reference value: <150 mg/dL normal
150-199 mg/dL borderline high
200-499 mg/dL high TAG
>500 very high TAG (acute and recurrent pancreatitis)
uPEMI Ouuns e t g y +oomg Id (eCTCy hgn) WpO rom
Diagnostic siantficance
It evaluates suspected atherosclerosis and measures the body's ability to metabolize
fat.
VLDL
Fasting TAG2 200 mg/di are at risk for coronary artery disease because of atherogenic
remnants.

75
 TAG and cholesterol are the most important lipids in the management of coronary artery
disease (CHD).

Methods
Prior to venipuncture,
idealy patients should undergo fasting for 10 hours to 12 hours.
Postural changes decrease TAG levels by almost 50%
if processing of samples will be
(upright to supine position).
delayed and lipemic serum will be frozen at -20°C, the specimen
should be warmed thoroughly to 37C and mixed before
the test.
Interference: ascorbic acid, bilirubin and hemolysis
Bilirubin interferes both spectrally and
cause the dilution
chemically; hemolysis can spectrally interfere and may
of lipid constituents.
TAG level increases at 2
The strategy for
mg/dl/year between 45 to 65 years old.
determining trighyceride concentration is to hydrolyze all of the fatty acid esters
of triglycerides to
produce glycerol.
Either plasma or serum can be
used for measurements.
L. Chemical Methods
1.Colorimetric Method (Van Handel &
Zilversmith} [REr MTO]
Alcoholic KOH
Triglycerides Glycerol+Fatty Acid (FA)
Glycerol Oxidized by Periodic Acid Formaldehyde (HCHO)
HCHO+ Chromotropic Acid ( + ) Blue color compound
2. Fluorometric Method (Hantzsch Condensation)
Alcoholic KOH
Triglycerides Glycerol FA
Glycerol Oxidized by Periodic Acid Form ldehyde (HCHO)
HCHO+Diacetyl Acetone + NH3 Diacetyl Lutidine Compound
I. Enzymatic Methods
There methods are relatively specific, rapid, and easy to.
Major interference: Glycerol is normally present in plasma in
concentrations below 0.163
mmol/L (1.5 mg/dL), equivalent toa triglyceride concentration of about
A blank assay,
14 mg/dL
without the addition of lipase, provides a measure of preexisting
increased readings in the blank indicate the presence of glycerol
be corrected accordinghy. glycerol, and measured TG values can
The analyses are performed directly in plasma or serum, and are not
phospholipids or glucose. subject to interference by

Glycerol Kinase Method R]

t involves hydrolysis of triglycerides to free fatty acids and glycerol, followed by
phosphorylation of glycerol to glycerophosphate. the

INF endogenous gycroi

76
1. Reaction (A)
In this reaction, the disappearance of NADH is measured at 340 nm.

Lipase
Triglycerides Glycerol+FA

Glycerol kinase
Glycerol+ ATP Glycerol PO +ADP

Pyruvate kinase

ADP+Phosphoenol Pyruvate ATP Pyruvate

LDH
Pyruvate+ NADH+H* Lactate + NAD

2. Reaction (B)
Upase
Triglycerides Glycerol+ FA
Glycerol kinase
Glycerol+ATP Glycerol POs+ADP
Gtycerol PO4 dehydrogenase
Glycerol PO +NAD Dihydroxyacetone POa + NADH

Diaphorase
NADH+tetrazolium dye Formasan+ NAD

CDC reference method (Modifled Van Handel and Zilversmith) T

is a time-consuming manual method which cannot be automated.
It involves alkalline hydrolysis (saponification) using alcoholic KOH, soient extraction with
chioroform and the extract ls treated with silicle actd (chromatography) to Isolate TAG, and
a color reaction with chromotropic acid, giving rise to a pink end color.
Interfering substances are removed during the extraction (with chloroform) and adsorption
(silicic acid chromatography) steps.
The purpose of the sllicic acid is to remove phospholipids from the chioroform extract.
aasoret

Glycerol+Sodium periodate Formaldehyde +Formicacid

Formaldehyde +Chromotropic acid Colored end product PMK

IncreasedTAG Decreased TAG:
1. Hyperlipoproteinemia types I, Ib, I,
IV,V 1. Malabsorption syndrome
2. Alcoholism
2. Hyperthyroidism
3. Nephrotic syndrome
3. Mainiutrition
4. Hypothyroidism
4. Brain infarction
5. Pancreatitis

FATTY ACIDS
These are Iinear chains of
Theycan be classifiedas
carbon-hydrogen bonds that terminate with a carboxyl group.
These are mostly found as constituents of
These are mainly dertved from phopholipids or triglycerides.
hydrolysis of triglycerides in adipose tissues.
Only small amount is present in plasma (free unsterified form), most is bound to
The polyunsaturated and cis-monosaturated fatty acids are not associated with albumin.
elevated
LDL cholesterol. serum
Example: palmitic acid, stearic acid, oleic acid, inoleic acid and
arachidonic acid
Reference value: 9-15 mg/dL

Classificaton:
1. As to chain
Short-chain (4-6 carbon atoms), medium-chain
(8-12 carbon atoms) or long chain (>12 carbon
atoms)
2. As to the number of C=C bonds
Saturated (without double bonds) fatty acids or
unsaturated (with double bonds) fatty acids
Functions:
They are very important sources of energy.
They provide the substance for comversion to glucose
(gluconeogenesis).
Lipld and Upoprobeln Transport and Metabolism
Lipids in the circulation are organized into large
characteristics of different classes. lipoprotein particles with apolipopoteins
The most hydrophobic lipids, such as
cholesterol esters and triglycerldes, are
of the lipoprotein particle. located in the core
Lipids with some hydrophilicity, such as free cholesterol and
surface with polar groups pointing outward. phospholiplds, are artanged on the
Lipid transport and dearance depend on
lipid in the diet. apolipoprotein concentrations and on the amount of

LipidAbsorotion:
The dietary or exogenous pathyway of lipid transport involves
and cholesterol (Ch)
through the intestine, with formation andabsorptlon
release
of triglycerides
of
(TAG)
Into the lymph and into the blood chylomicrons (CM)
The CMs released TAG
by way of the thoracic duct.
to adipose tissue as they cireulate. The ipoproteln lipase (LPL) Hberate
fatty acids (FA) from TAG, thereby reducing the size of
taken up by the lver. The free FAtiberated from TAG CM to become remnants which is in turn
Is taken up by muscle
and adipose tissue.

18
 In the endogenous pathway, production of TAG from FA by the liver take place, with synthesis of
vLDL partickes containing apo B100 and apo E. These VLDL particles are then converted by
lipoprotein lipase (LPL) to IDL that can either be removed by the liver through ApoE or be
converted to LDL The Ch-rich LDL particles can be taken up by the liver or into other tisssues for
steroid synthesis or part of cell membranes.
The HDL particles mobilize Ch from tissues and reintroduce it for continued metabolism or
excretion. LCAT catalyzes the esterification of Ch in HDLa, converting HDL to HD This fraction
of Ch can be transfered to VLDL to participate in the metabolism of membrane and steroid
synthesis, or be taken up by the liver and then excreted into bile.

Role of CholesterolEster TransferProtein (CETP):

CETP isproducedin the liver, and it is part of HDL
The CETP pathway is considered to be an alternative pathway for HDL metabolism.
CETP transfers triglycerides from apoB lipoproteins in exchange for cholesterol esters in HDL.
CETP catalyzes the transfer of cholesterol esters to apoB-100- containing particles in exchange
for triglycerides.
CETP connects the forward and reverse cholesterol transport pathways.
Deficiency of CETP leads to production of large cholesterol-laden HDL.

Enames thatparticipate:inlipoproteinmetabolism (lipolytic enzymes):

1. poprotein lipase (LPL hydrolyzes TAG and cholesterol esters in lipoproteins;hydrolyzes TAG
releasing fatty acld and gycerol; it is present on the surface of capillary endothelial cells in adipose
tissues, cardiac and skeletal muscles
2. Hepaticlipose-hydrolyzes TAG and phospholipids from HDIL; hydrolyzes lipids on VLDL and iDL
3. Lecithin Cholesterol Aryl Transferase (LCAT) catalyzes the esterification of cholesterol from HDL;
enables HDL to accumulate cholesterol as cholesterol ester.
4. Endothelial lipase hydrolyzes phospholipids and TAG in HDL
5. ATP-binding cassette protein A1 (ABCA1) -for efflux of cholesterol from peripheral cels into HDL

LIPOPROTEINS
These are large macromolecular complexes of lipids with speciallzed proteins known as
apolipoproteins.
Main purpose: To transport TAG and cholesterol to sites of energy storage and utilization
Cholesterol and trglycerides travel in plasma not as free-floating molecules, but as part of
water-soluble complexes called lipoproteins.
Vitamin é (fat-soluble vitamin) depends upon chylomicrons for absorption and relies upon VLDL
and LDL for dellvery to tissues.

Apolipoprotein
t helps to keep the ipids in solution (solubility) durlng irculatlon through the blood stream.
t aids in the solublization of the lipids and also in thelr transfer from the gastrointestinal tract
to the liver, which contains specific receptors for
apolipoproteins.
tinteractswith specificcel-surface receptors and direct the lipids to the correct target organs
andtissues in the body-present on the surface of lipoprotein particles.
t containsastructural motif called an "amphipatic helia-abity of proteins to bind to lipids.
it malntalns the structural integrity of the
lipoprotein (LPP) complex.
79
 Maior Lipoproteins
1. Chylomicron (CM)
It is the largest and the least dense of
the lipoprotein particles.
It is produced in the intestine from
dietary fat; con1pletely cleared within 6 to 9 hours post
prandial.
It
transports exogenous/dietary TAG to liver, muscles and fat
depot.
Major composition: 90% TAG ( non-fasting plasma) +1-2% protein
Apolipoproteins: Apo B-48, Apo A-1, Apo Cand Apo E
Density:< 0.95 kg/L
2. Very Low Density Lipoprotetn/Pre-Beta Lipoprotein (VLDL)
I t is secreted in the liver.
ENC
19
TANt transports endogenous TAG from the liver to muscle, fat depots and peripheraí tissues.
prolonged consumption of high fat diet leads to elevated TAG in the VLDL particles.
Major composition: 65% TAG ( fasting plasma) + 6-10%
Apolipoproteins: Apo B-100, Apo Cand Apo E protein + 16% CE
Density: 0.95-1.006 kg/L
3. High Density Lipoprotein/Alpha
It is Lipoprotein
the smallest lipoproteins but the (HDL)
most dense
It isproduced in the liver and intestine (nascent (5-12nm).
It transports excess disk-shaped
cholesterol from the tissues and return it particles).
transport)- HDL maintains the equilibrium of cholesterol in to the liver (reverse cholesterol
cholesterol transport pathway. peripheral cells by the reverse
HDL transports effectively the
The
lipids to the liver and more cardioprotective.
phospholipid content of HDL is more important
than cholesterol
apolipoprotein. or even
CDC reference method:
ultracentrifugation, precipitation with heparin-MnClz and Abell-
Kendal assay (3-step method)
Major composition: 30%
phospholipid+
Apolipoproteins: Apo A-I, Apo A-L, Apo 45-50%
C protein+ 20% CE
Reference value: 40 mg/dL (cutoff level)
Interpretation: < 35 mg/dL - high risk for CHD
260mg/dL high HDL (protective)
Density: 1.063-1.21 kg/L
4. Low Density Lipoprotein/Beta Lipoprotein (LDL)
It is synthesized in the liver.
t is the major end product from the catabolism of VLDL
It constitutes about 50% of the total LPP in
tissues. plasma the major source of
-

cholesterol for
It transports cholesterol to the peripheral tissues i t
cholesterol and transports cholesterol to carries most of the
hepatic and
extrahepatic circulating
up by
LDL-receptor-mediated endocytosis. tissues, where it is taken
t is the
most cholestero-rich of the
samples, LDL contains the choleesterol lipoprotelns and most
atherogenic in fasting plasma
it is the primary
that is not present in HDL
or VLDL
target of cholesterol lowering therapy,
tis Important in primary
assessing patients with or without coronary heart marker for CHD risk.
disease (CHD).
80
 Major compostion: 50% CE+18% protein and phospholipid
Apolipoproteins: Apo B-100 and Apo E
Research method: Beta Quantification
Reference value: Optimal = <100 mg/dL
Interpretatio: Near/Above Optimal = 100-129 mg/dL
Bordeline 130-159 mg/dL
High 160-189 mg/dL
Very High 2 190mg/dlL
Density: 1.019-1.063 kg/L

CHEMICAL cOMPOSITION OFMAJORLIPOPROTEINS

TAG CholeEsterFree CholePhosphollpid Proteln
Chylomicrons 80-95% 2-4% 1-3% 3-6% 1-2%
VLDL 45-65% 16-22% 4-8% 15-20% 6-10%
LDL 4-8% 45-50% 6-8% 18-24% 18-22%
HDL 2-7% 15-20% 3-5% 26-32% 45-55%

Minor Lipoproteing
1. Intermediate Density Ipoprotein (IDL
t is a product ofVLDL catabolism- VLDL remnant."
it is converted to LDL-"subclass of LDL"
t migrates elther In the pre Bor B region (electrophoresis).
Defective clearance of IDL In type 3 hyperlipoproteinemia is probably due to deficlency of Apo E-II.
Malor opoliporotein: Apo B-100
Density: 1.006-1.019 kg/l = causing it to float on the 1.063 density potassium bromide solution

2. Lipoproteln (a)/lp (a)

It is similar to LDL (density and composition) LDL-ike particles (LDL variant) that have a
c i molecule of apo (a) linked to apo B-100 by a disulfide bond.
It has varlable migration = pre-B, or sometimes between LDL and albumin.
tis known as"sinking pre-B lipoprotein-due to electrophoretic mobility same as VLDL but
density like LDL
t ls isolated in the LDL-HDL density range by ultracentrifugz*i
I t s compiex structure is also similar to plasminogen. Lplat0-tpla)ngs
increased levels may indicate premature coronary heart dis
Independent risk factor for atherosclerosis.
Major opoliporotein: Apo B-100 and Apo la)
- L-9 {0ape
Densty: 1.045-1.080 kg/L

Abnormal Lipoproteins:
1. Lupoproteln X
t is an abnormal lipoprotein found in obstructive jaundice& LCAT deficiency.
t is a specific and senstive indicator of cholestasis.
The lipid content is mostly phosphollpid and free cholesteroi (90%l
a
t contains Apo Candalbumin.

81
2.-VLDL (foating Blipoprotein)
I t is known as "abnormally
migrating B-VLDL" has the density of VLDby ultracentrifugation but
-

migrates with LDL in the Bregion during electrophoresis.

It is found in 3 type hyperlipoproteinemia or dysbetalipoproteinemia.
It is also known as the "VLDL rich in
cholesterol"'due to defective catabolism of VLDL
There is an accumulation of IDL because of failure to fully convert VLDLto LDL.
Density: < 1.006 kg/L

Lipoproteln Metthodologles:
Generally,total cholesterol, TAG, and HDL-C can be
and LDL-C concentrations can be satisfactorily analyzed in frozen samples,
estimated with the Friedewald equation.
Lipemic samples occur in nonfasting blood, in patients
with
parenteral nutrition (TPN) therapy. hyperlipidemia or receiving total
Lipemic samples may be pretreated by
lipids because it can interfere with
ultracentrifugation or enzymatic cleavage to remove
spectrophotometric,
Lipoproteins are differentiated based on electrophoresis
turbidimetric and immunoassays.
and buoyant density
(ultracentrifugation).
Non HDL-C is computed as: TC-HDL

Specimen requirementt
Sample collected using serum separator tubes is the preferred
Plasma or serum is used for cholesterol, sample.
TAG, and HDL-C and LDL-C.
EDTA Plasma is prefered for
ultracentrifugal or electrophoretic methods because the
can be cooled to 4 C
immediately to prevent changes that can occur in the lipoproteins samples
at room
temperature,and research studies.
EDTA is the preferred anticoagulant even
though cholesterol and triglyceride concentrations are
about 3% lower than in serum.
Heparin, because of its relatively high molecular weight, has little effect
can alter the
on plasma volume but
electrophoretic mobilities of the lipoproteins.
Patient Preparation:
1. Fasting: 10-12 hours
When TAG and LDLC are being measured, fasting becomes a
In the fasting state, most plasma TAG is
requirement.
present in VLDL; nonfasting state in chylomicrons.
f testing is done in nonfasting samples, only total cholesterol (TC) and HDL-C can be
measured.
2. Diet
Concetrations of LDL- and HDL-cholesterol (LDLC and HDL-C) decine
in part as a consequence of CETP-mediated temporarily after eating,
compositional changes that occur during the
catabolism of CMs.
3. Posture
Current NCEP guidelines recommend that patients be seated for 5 minutes before
prevent hemoconcentration. sampling to
When a standing patlent reclines, extravascular water transfers to the
vascular system and
dilutes nondiffusible plasma consttuents decreases
of as much as 10% in the concentrations of

82
 TC, LDLC, HDL-C, apoA-, and apo8 have been observed after a 20-minute period of
recumbence.
Postural changes are reversible when the patient resumes the standing position-the postion of
the patient therefore should be standardized for venipuncture, preferably to the sitting position,
which is most commonly used.

Recommendaton:
Intiol screening (age 20 or older): total cholesterol, HDL-C, LDL-C and TAG.
Testing should be repeated at least once every 5 years.
t is recommended that lipoprotein measurements be made no soonerthan 8 weeks after any
fom oftrauma or acute bacterial or viral infection, and3 to 4 months after childbirth.

1.Utracentrtfugatton (densty gradient)

t s the reference method for quantitation of lipoproteins (LPPs).
t is based on the protein and triglyceride contents of lipoproteins.
t is expressed in svedverg (s) units.
Lipids have density of 1.0 g/mL while proteins have 1.4 mg/L.
Frozen sarmples are not appropriate for utracentrifugal analysis because the triglyceride-rich
ipoproteins do not withstand freezing.
Reagent: Potassium bromide solution with 1.063 density

2. Electrophoresis
Electrophoretic patterm: HDL, VLDL, LDL, Chylomicrons
PreferredSupporting medum: agarose gel-speed; sensitive; resolves lipoproteinclasses
Lipid-staining dyes: Oll Red 0, Fat Red 78, or Sudan Black B- these stains react primarily with
the ester bonds in trigycerides and cholesteryl esters
VLDL migrates with the a-2 globulin (pre B)
Chylomicrons, f present, remain at the origin.

3. Chemical prectpitaton
t uses polyanlons (hepartn sulfate, dextran sulfate and phosphotungstate) and divalent cations such
as magnesium, calcium and manganese.
Enzymatic with coupled detergent precipitation is the most useful lipoprotein tests.
Most consistent analytical ermrOr involved in the HDL-C assay is due to the presence of a small
amount of apo-B containing LPPs.

a. HDL
t uses dextran sutfate (synthetic heparin) with magnesium (precipitants).
CoCreference 3-step methodunltracenirifugation,heparinmanganese precipitatlon and Abell-
Kendallassay
Homogeneous assays are the most popular method for measuring HDL-C these fully
automated two reagent procedures do not require off-line pretreatment (precipitation) and
separation (hence the term "homogeneous") and can be adapted to most chemistry analyzers.
b. LDL
EDTA plasmais the sample preferred for betaguantification
Betaquantification combines ultracentrifugation and chemical precipitation.
Polyanions remove apo B-containing lipoproteins.
 Beta Quantificotion
Assumes that virtualy all cholesterol is
contained in the three major lipoprotein classes (i.e.,
VLDL, LDL, and HDL).
In this method,plasma is ultracentrifuged for at least 18 hours at 105 Kx
g; VLDL and CMs
accumulate as afloating layer, leaving
predominantly
solution is measured for cholesterol and is
LDL and HDL in solution (Rifai,
2000). The
LDL-C is calculated according to the
precipitated for LDL lipoproteins.
difference between these two neasurements.
Homogeneous Direct LDL-C Method
I t is useful when
triglycerides are elevated because
triglycerides even at relatively high concentrations (600they subject to interference
are not
by
It uses combination of two reagents. The first
a
mg/dL.
reagent usually selectively rernoves non-LDL
lipoproteins (and/or stabilizes or inhibits LDL from
reagent releases cholesterol from LDL so that it can be reacting
with enzymes), and the second
rneasured enzymatically.
4. Chromatographic methods
t utilizes either Gel Chromatography or
Affinity Chromatography.
5. Immunochemical methods
t uses antibodies specific to epitopes on the
apolipoproteins.
6.
Immunoassay or Immunonephelometry Apolipoprotein assay
t is based on the
measurement of the turbidity created by
Studies have shown that apo A-1 and apolipoprotein-antibody complexes.
apo B may be better indicators
than lipoprotein assay. of atherosclerotic disease
Lipopratein (a) is measured by immunoturbidimetric assay.
Formula for LDL- Cholesterol (LDL- -Total
LDLCis currently considered the most Cholesterol-HDL- VLDL
important value in assessing cardiac risk and
therapy. directing
LDL-C can be measured directly but is
usually calculated using the Friedewald formuia.
Calculated values require evaluation of fasting
samples.
Friedewald Method (Indirect Method):&
This method is unsuitable for nonfasting samples that contain
contain -VLDL chylomicrons or samples that
VLDL (mmol/
Plasma Trighvceride
2.175
VLDL (mg/d1)
Plasma Trighvceride
5.0
De Long Method CIndirect method):
VLDL (mmol/)
PlasmaTriglyceride
2.825
VLDL (mg/d
PlasmaTrigteceride
6.5

84
 SIGNIFICANT HUMAN APOLIPOPROTEINS

Plasma concentration|
Apollipoprotein Major Iipoproteins Mr (kDa)|Amino acids Chromosome (umol/L) (mg/dL)
A- HDL 29 243-245 11 32-46 90-130
A-I1 HDL 17.4 154 1 18-29 30-50
A-IV 44.5 396 11

a) Lpa) 350-700 Variable 6

B-100 VLDL, IDL, LDL 512.7 4536 2 1.5-1.8 80-100
B-48 CM 240.8 2152 2 0.2 <5
C CM, LDL 6.6 57 19 6.1-10.8 4-7
C- CM, LOL 8.9 78 or 79 19
3.4-9.1 3-8
C-II CM 8.8 |79 9.1-17.1 8-15
D
HDL 19 169 3

CM, LDL, 1DL 34.1 299 19 0.8-1.6 3-6

Source: Henry's Clinical Diagnosis and Laboratory Management, 22nd ed, 2011.
Mr-relatlve molecular mass

APOLIPOPROTEIN FUNCTIONS AND SIGNIFICANT CHARACTERISTICS

Main
Apolipoprotem distribation Frunction (if known) Comments

Activates LCAT that esterifies

A-I HDL cholesterol in plasma Synthesized in liver and intestine; HDL
Ligand for ABCA1 biosynthesis
May inhibt lipoprotein and hepatic lipases
A-I HDL and increases plasma TAG
HOL, CM, and May be acofactor for LCAT; increased during
A-IV free in plasma fat absorption; HDL biosynthesis
Carboxy-terminal recognition signal Very large structural proteln, synthesized in
B-100 VLDL and LDL targets LDL to the LDL fapoB, E) the liver with lipids of endogenous origin
receptor ie., not chylomicrons)

CM Not recognized by LDL receptor Synthestzed in intestine, encoded by same

B-48 gene and same amino terminus as apoB-100.
CM and VLDL May inhibit hepatic uptake of VLDL and CETP
C
Activates poproteln Hpase Deficiency causes reduced clearance of
CM and VLDL
Stimulates hydrolysis of TAG triglyceride-rich lipoprotelns.
Inhibits lipolysls of TAG-rich Deficiency causes reduced clearance of TAG
C-I! LDL, HDL lipoproteins; decreases clearance rate rich Ripoproteins.
of remnant particdes
HDL Activates LCAT
E-4 is associated
CM, VLDL, DL Recogrition factor that targets CM and E-2, E-3, and E-4 isoforms;
risk of C and
remnats and VLDL remnants to hepatic receptor, with high LDL-C, hligher
85
 Main
Apolipoprotein distribution Function (if known) Comments
HDL also binds to cell surface LDL receptors Alzheimer's disease; E-2 associated with type
and proteogtycans 3 hyperiipoproteinemia
HDL, LDL VLDL |Regulates CETP function

Antlbodies against apoH or Br-glycoprotein-1

VLDL Related to activation of LPL;
trigtyceride metabolism
Cell-aggregating factor in Sertoli cells;
inhibitor of the C5b 7 complement
complex; beta-amyloid clearance in
glial cells; cholesterol trafficking in
brain
|HDL
M HDL CM, LDL
VLDL
are a subset of antiphosphollpid antibodies,
and may be associated with hyperthrombosis
and stroke.
Involved in apoptosls; linked to neurologic
diseases like Pick's and Alzheimer's; also
known as clusterin
May be linked to reverse cholesterol
transport
Not present in plasma
May be linked to HDL remodeling

Apofa) Homologous to plasminogen, may be

Lpla) prothrombotic; bound to apo8-100 by
Source: Henný's Clinical
Diagnosis and Laboratory Management, 22d ed, 2011.
disulfide inkage
ABCA1, ATP-binding cassette
HDL high-density lipoprotein;transporter,
CETP, cholesterol ester transfer
IDL, intermedisto-density lipoprotein; LCAT,protein pathway; CHD, congenital heart disease; CA1, chvlomicror;
lecithin/chclesterol
Lpla), lipoprotein A; LPL, lipoprotein lipase; VIDL, acyltransferase, LDL, low-density lipoprotein;
LDIC, low-density lipoprotein cholesterol;
very-low-densitylipoprotcin.

Disorders
1. Familial
Associated with Lipids and Lipoproteins:
Hypercholesterolemia (Type 2a)
It is autosomal dominant disorder caused by
an
defective or
Defective LDL receptors cannot bind or clear LDL TC and deficient LDL receptors.
=
LDL-C 2-3x above
Clinical findings: (+) xanthelasma and planar (tendon) xanthomas ( normal)

2. Familial Dysbetalipoprotelnemia (Type 3

I t invoves accumutation
Hyperlipoprotelnemia)
of plasma VLDL rich in cholesterol and
chylomicron remnants.
It is also associated with the
presence of apo E2/2 (rare form of apo E).
It involves both the endogenous and
exogenous pathway of lipoprotein metabo!isn.
The VLDL fraction migrates abnormally in the
B region("3-VLDL") creating broad
electrophoretic pattern. ß band ( pre B)
Lab resuts: equal elevations of cholesterol and TAG and
presence of B-VLDL
Clinical findings: xanthomas and premature vascular disease (coronary heart disease and
peripheral artery disease)
Pathognomonic feature: broad abnomal band between VLDL and LDL (
Screening test: measurement of the VLDL-C/triglyceride ratio B-VLDL)
Reference volue VLDL-C/trigtycerlde ratio: 0.2 (Type 3 patlents have a ratio
>0.3)

86
3. Abetalipoproteinemia (Bassen-Kornzwelg
Syndrome
I t is an autosomal recessive disorder, defective apo B synthes/s.
VLDL, LDL and chylomicron are all not found in plasma.
Cholesterol and TAG levels are both decreased.
t is associated with defects in absorption fat-soluble Vitamins AEK deficient fat soluble
vitamins.
Vitamin D does not require chylomicrons for absorption and therefore is typicaly not deficient.
Because both vitamin A and vitarnin K have transport systems independent of lipoproteins,
clinical deficiency is not as severe as that seen with vitanin E, which not only depends upon
chylomicrons for absorption but relies upon VLDL and LDL for delivery to tissues.
It is also assoclated with deficiency of microsomal triglyceride transfer protein (MTP).
It ls characterized by cerebellar ataxia, acanthocytosis, fat malabsorption

4. Hypobetalipoprotelinemia
It is due to apo-B deficiency
resulting from point mutation in apo-B.
Decreased:-LDL-Chole &total cholesterol
Decreased or normal levels: VLDL-Chole& Total TAG

5. Niemann-Pick disease (upid storage dtsease)

tis an inherited disorder of lipid metabolism, in which there are accumulations of spingomyelin
in the bone marrow, spleen and lymph nodes.
t involves deficiency of enzyme responsible for removing phosphorylcholine from spingomyelin.

6. Tangier Disease
s a rare autosomal recessive disorder characterized by complete absence of HDL due to a
mutatlon in the ABCA1gene on chromosome 9
The mutation leads to an inability to effectively transfer cholesterol and phospholipids from
within the cel onto nascent apoA1 proteins in plasma. The resulting buildup of cholesterol
within the cell becomes toxic-LDL-C is reduced.
HDL is also abnormal and significantly reduced due to increased HDL catabolism.
Is also associated with deficiency of ATP-binding cassette protein A1 (ABCA1).
Lob results: low bood cholesterol
Clinical findings: orange or yellow discoloration of the tonsils and pharyx
ABCA1 protetnenables cholesterol to ext the cell, upon which It combines with apoA-l to form
the HDL

7. Lipoprotein Upase (LPL) Deficiency

I s a rare, autosomal recessive disorder that presents in childhood with abdominal pain and
pancreatitls.
t results to inabilty to dlear chylomicron particles, creating the dassic "type 1
chylomicronemia syndrome
Labresuts: 1AG= 10,000 mg/dL or 113.0 mmol/L (postprandial result) implying that
Patients with this condition do not develop premature coronary disease,
chylomicrons themselves are not atherogenic,
LPLsessential for hydrolysis ofTAG and conversion of chylomicrons to chylomicron remnants.
Deficiency of Apo C-lalso results to chylomicronemia.

87
8. Lecithin Cholesterol Acyl Transferase (LCAT) Deficlency
I t is due to mutation in the LCAT gene.
Lab results: HDL-C <10 mg/dl, TC is normal or high
Clinical findings: corneal opacities, normochromic anemia, and renat fallure in youngadults
Fish-eye disease is milder form of LCAT deficiency.

9. Tay-Sachs disease
I t is an inherited neurodegenerative
disorder
of lipid metabolism characterized by a deficiency
of the enzyme hexosaminidase A, which results in the accumulation of spingolipids in the brain.

10. Chylomicron Retention Disease (Anderson's disease)

This syndrome is distinct from
I t is characterized by
abetalipoproteinemia, as only apoB-48 appears to be affected.
hypocholesterolemia, chronic diarrhea, fallure to thrive, and deficiency of
fat-soluble vitamins (vitamin E in particular); the latter can lead to
Clinical findings: fat malabsorption and low levels of neurologic deficits.
plasma lipids.
11. Sitosterolemia
I t is a recessive disorder wherein
plant sterols are absorbed and accumulate in plasma and
peripheral tissues.
It results from mutation in the ABCG8 or ABCG5
gene, both located at chromosome
Lab resut: high serum LDL-C during childhood 2p21

FREDRICKSON CLASSIFICATION
Types
1. Type 1Hyperchylomicronemia
TAG CHOL LDL VLDL CM Feature
High N N
Familial LPL Deficiency NHigh Low cardiac risk; eruptive
xanthoma; recurrent
2. Type 20
N pancreatitis
Familial Hypercholesterolemia HighHigb| N N High cardlac risk;
xanthelasma; tendon
xanthoma; corneal arcus,
hypothyroldism
Type 2b-Mked Defect High High High
and nephrotic syndrome
High N
Fomilial Combined Hyperlipidemia High cardlac risk
3. Type 3
High High High N
Familial Dysbetalipoproteinemia eruptve and palmar
4. Type 4
High N Xanthomas
Fomillol Hypertriglycedemio High N Low cardiac risk
5. Type 5
High High
High High Low cardiac risk;
eruptive xanthoma,
may be associated
N-Normal; CM-chylomicron
with paficreatitis

88
Fredrickson Classiication
1. Type 1-Hyperchylamicronemia/ Familial LPL Deficiency
> A block in the progresslon from chylomicron (CM) to chylomicron remnants results in the
accumulation of CM in types 1 and 5.

2. Type 2 Hyperlipoprotelnemia
I n type 2, a block in LDL metabolism and defective apo B proteln that does not bind to LDL
receptor or mutant LDL receptor that does not recognize apo B are observed.
Familial combined hyperlipidemia (type 2b) is the most common primary hyperlipidemia.

3.
Type 3 Dysbetalipoproteinemia
The presence of floating B-VLDL in type 3 dysbetalipoproteinemia is due to failure to convert
VLDL to LDL causing IDL to accumulate.
The ratio of VLDL-C to piasrna triglycerides may be useful in the evaluation of type 3
hyperlipoproteinemia.
4.
Type n4HyperilipoproteinemBa/typertrighyceridemia
type 4, a block In the conversion of VLDL to IDL and LDL results to elevated TAG and VLDL, but
LDL is normal.
The production of excess insulin and used of antischizoprenic drugs (olanzapine) lead to
bypertrighyceridemia also seen in Type IV.

5. Type 5 Hyperipoproteinemia
>Types 1,4,5: LPL deficiency; inability to breakdown TAG.

Noles to Remember
The main function ofthe apo B-containing lipoproteins (LDL, VLDL and chylomicrons) is to
delver cholesterol and triglycerlde to various tissues.
Lipemia in plasma is cleared as it passes through various tissues by the enzyme, lipoprotein
lipase (LPL) which is present in the capillary walls of many tissues mainly adipocytes, skeletal and
cardlac muscles, spleen and lungs
High HDL-C: intakeof alkcoholanddrugs (phenytoin, rifampicin, estrogen) and exercise
LowHDL-Cphysicalinactivity, obesity, high CHO diet andintake ofdrugs (beta blockers,
progesterone, anabolic steroids)
High levels of cholesterol in the HDL fraction are negatively assoclated with cardiovascular
disease, while elevated levels of cholesterol in the LDL fraction are positively associated with it.
High LDL-C and low HDL-C are risk factors for atherosclerotic disease.
Medical disorders that lead to secondary dysllpoprotelnemla Inchude thyroid, hepatic, and
kidney disease.

89
 PROTEINNS

Protein comes from the greek word

proteis, meaning "first rank of importance."
These are synthesized in the liver and secreted theby hepatocyte into the circulation except
immunoglobulins (plasma cells).
These are macromolecules composed of polymers of covalently linked amino acids that are
involved in every cellular processes.
Proteins can bear positive and
negative charges (omphoteric) because of their acid and basic
amino acid compositions.
They can be both a weak base or weak acid, hence,
Their solubility is due to high dielectric
proteins effective blood buffers.
are
property.
They are very effectve antigens due to their molecular mass,
tyrosine content and their
specificity.
They provide 12 to 20% of the total daily body energy
Proteins are 50% to 70% of the cell's dry requirement.
weight.
Functdons$
1. Repair body tissues
2. Important in blood
coagulation and immunologic function
3. For transport of metabolic
substances
4. Maintenance of osmotic pressure
5. Maintenance of blood pH (buffers)
6. Biocatalysts

Four Strucures of Protelns

1. Primary structure
t is the linear sequence
of thee amino acid.
It determines the identity of protein, molecular structure, function binding capacity and
recognition ability.
Any change in the amino acid composition can significantly alter the protein.

2. Secondary structure
It invohves
the winding of the polypeptide chain.
It refers to
spectfic 3-dimensional conformations alpha helix, beta pieeted and bend form.
3. Tertiary structure
I t is the actual 3-dimensional configuration; the folding
pattern of the
protein.
It is responsible
for many of the physical and chemical properties of the
protelns.
tt is maintalned by electrovalent linkages,
hydrogenbonds, disulfide bridges, Van der waals
forces and hydrophobic interactións.

4. Quaternary structure
Itis the association of 2 or more polypeptide chains to form a functional protein molecule.
Albumin has no quaternary structure (single polypeptide chain)
Example: hemoglobin, LDH, CPK

0
Notes to Remember.
Glucogenic amino acids are those that generate precursors of glucose such as pyruvate or citric
acid cycle intermedlates.
Example of glycogenic amino acid: alanine (pyruvate), arginine (a-ketoglutarate), aspartate
Ketogenic amino acidsare degraded to acetyl-CoA such as leucine or lysine.

Classtfication of Protsins
1. Simple Proteins
They contain peptide chains which on hydrolysis yield only amino acids.
These may be fibrous (flbrinogen, troponins, collagen) or globular (hemoglobin, plasma proteins,
enzymes, peptide hormones) in shape.
These are globular proteins are compact and have little or no space for water in the interior of
the molecule; they retain their biological activities within narrow ranges of temperature and pH.

2. Conjugated Proteins
These are composed of a protein (apoprotein) and a non protein moiety (prostheticgroup)
These proteins impart certain characteristics to the proteins.
1. Metalloproteins ferritin, ceruloplasmin, hemoglobin and flavoproteins
2. Lipoproteins- VLDL, HDL, LDL, chylomicrons
3. Glycoproteins- haptoglobin, ay-antitrypsin (with 10%-40% CHO)
4. Mucoproteins or proteoglycans mucin (higher CHO content than CHON)
5. Nucleoproteins chromatin (combined with nucleic acids)

Ntrogen balence is a balance between anabolism (synthesis) and catabolism

a. Negative ntrogen balance
It is when a protein catabolism exceeds.anabolism.
-It is characterized byexcessive tissue destruction such as burns, wasting disease, high fever
and starvation.
b. Positive nitrogen balance
- It is when anabolism is greater than catabalism.
-Example: growth, pregnancy and repair processes.

PLASMA PROTETNS
1. Prealbumin (Trensthyretdin)
t migrates ahead ofalbúmin.
t has a half-Ife of only 2 days
It is rich in tryptophan and contains 0.5% carbohydrate.
t has considerable -pleated sheet conformation.
It serves as transpört protein for Ta and retinol (vitamin A) - by complexing with retinol-binding

protein.
it is used to detect malnutrition and individual's response to dietary supplermentation.
Itis used aslandmarkto confirm that the specimen is realy cSF it crosses möre easily into the
CSF than other proteins.
Increased: alcohollsm, chronic renal falure, steroid treatment
Decreased: poor nutrition
Reference vatue: 18-45 mg/dL ("CF: 10-mg/

91
2. Albumin
I tisthe protein present in highest concentration in plasma (% the plasma protein mass).
It is synthesized in the liver.
A general transport protein (binds various substances in the blood).
It maintains osmotic pressure.
It is an indicator of nutritional status.
It serves as circulating reservoir of amino acids.
t ls a sensitive and highly prognostic marker In cases of cystic fibrosis.
t is a "negative acute phase reactant" it decreases in acute
-

inflammatory processes.
Lowest plasma albumin levels are seen in active nephrotic syndrome.
Reference value: 3.5-5.0 g/dl (CF: 10-g/L

3. Globulin
I t is a group of proteins consists of a, ,
B, and yfractions.
I t is usually measured by
subtracting the vaiue of serum albumin from the total protein
concentration.
Elevated concentration in early cirrhosis will balance
total protein.
loss of alburnin resulting to normal levels of
Measurement: Total Protein Albumin =Globulin
Reference value: 2.3-3.5 g/dL (23-35 g/L)
a a-Antitrypslin (AAT)
I t is an acute-phase reactant; a gycoprotein.
t neutralizes trypsin-ike enzymes (like neutrophil
elastase) this enzyme is released from WBCs
combat infection but it can also destroy alveoli which can lead to t

t is a major inhibitor of protease emphysema.

activity prevents self-destruction of tissues.
-

t comprises 90% of the ai-globulin band.

Increased: inflammatton, pregnancy and contraceptive
use
Deficiency: emphysematous pulmonary disease and juvenile hepatic cirrhosis
Reference value: 145-270 mg/dL (CF: 0.01-g/L)
b. a-Fetoprotein (AFP)
t i s a gycoprotein; migrates between albumin and a-1
giobulin band.
t i s synmthesized initially
by the
fetal yolk sac and then the
by fetal parenchymal cells of the liver.
I t i s the most abundant protein in fetal serum.
t peaks in the fetus at 13 weeks of
gestation.
.
t is detectable in the maternal blood up to the 7or 8" month of
AFP is increased in the presence of twlns pregnancy maternal serum
(transmitted across the placenta).
t has no known functlon in adults.
Specimen: maternal serum; amniotic fluid; serum (for cancer screening8)
Method: immunochemical test (using
monocional antibodies against AFP), RIA and EIA
Reference value: 5 ng/mi (adut and children sera)

92
Diagnostic sianifcance
Maternal serum AFP is used as a screening test for any fetal conditions- it detects neural tube
defects (NTDs) and Down syndrome (0S).
Maternal AFP screening for NTD and DS is done between 15 and 20 weeks' gestational age when
the maternal serum Increases gradually.
AFP is also used as a tumor marker (hepatic and gonodal cancer).
Elevated serum AFP levels postnatal occur only with conditions of abnormal cell multiplication
such as cancer.
Increased: hepatoma (> 1000 ng/nL), NTDS,anencephaly, spina bifida, atresia of the GIT, fetal
distress, ataxiatelangiectasia, tyrosinosis, and hemolytic disease of the newborn (HON)
Decreased: Down syndrome and trisomy 18

c. a-Acid Glycoprotein /orosomucoid (AAG)

t contains high percentage ofCHO and sialic acid (45% CHO+11-12%sialic acid).
thas low pl (2.7; t is negatively charged even in acid solution.
It has greatest affinity for progesterone; binds quinidine (cardioactive drug).
I t i s a useful diagnostictool in neonateswith bacterial infections.
Its amino acid sequence is similar with immunoglobulin.
Increased: pregnancy, cancer, pneumonia, rheumatoid arthritis (RA) and cell proliferation
Reference value: 55-140 mg/dL (CF: 0.01-g/

d. a-Antichymotrypsin (a-)
I t is a serine proteinase with cathepsin G; acute phase reactant.
I t migrates between the a and aa zones; synthesized in the liver.
I t binds and inactvates prostate-speciñic antigen (PSA) - PSA complexed with a-xis
I t is the majorform of PSA found in human sera.
It is assoclated with the pathogenesis of Altheimer's disease dxis a vital component of the
amyloid depostts found in persons with such disorder.
Increased: infection, malignancy, burn, AMI and Alzheimer's disease
Deficiency: iver disease
Reference value: 30-60 mg/dL (CF: 0.01-g/)

e.Hemopexin
I t binds heme released by degradation of hemoglobin has the strongest affinity for heme.
I t helps in the diagnosis of early hemolysis.
I t migrates in the beta region during electrophoresis.
Reference value: 50-115 mg/dL (CF: 0.01-g/)

f.Group-specific component (Gc)-Globulin

it exhibts affinty with vitamin D and actin (vitamin D- binding protein).
It migrates in the a-1 and a-2 Interzone during electrophoresis.
Method: Radial immunodiffusion
Reference value: 20-55 mg/dL (CF:0.01-g/)

93
8. Haptoglobin
t is an a gtycoprotein; an acute phase reactant.
It is synthesized in the hepatocytes.
It has two heavy chains and two light chains linked by disulfide borids in anaiogy to the basic structure
of immunoglobulins.
It binds free hemoglobin by its a chain.
t preventsthe loss of hemoglobin and its constituent iron into the urine.
t i s a useful measurement for serially monitoring patients who have a slow but steady rate of
red cell breakdown such as by mechanical heart valves, hemoglobinopathies, or exercise-
associated trauma.
I t evaluates degree of intravascular
hemolysis (HTR& HON).
I t s plasma level is slightly decreased after blood transfusion.
I t is also one of theproteins used to evaluate rheumatic diseases.
Increased: stressful conditions, myoglobulinuria
Decreased: intravascular hemolysis and hemoglobinuria
Method: Radial immunodiffusion and immunonephelornetry
Reference value: 26-185 mg/dl (CF: 0.01-g/
h. Ceruloplasmin
t i s a copper-binding
a glycoprotein that has enzymatic activities.
Itis synthesized in the liver, where 6 to 8 atoms of
copper are attached.
I t imparts a blue color proteln.
to
I t is a marker for Wlson's disease
(0.1g/L of ceruioplasmin).
Cinical features of Wilson's disease: deposition of copper in skin, liver, bralin
and cornea (Kayser-Flelsher rings)
Increased: infammation, cancer and pregnancy
Decreased: Wilson's disease, malnutrition, malabsorption,
nephritic disease and
Menkes'klnky-hair syndrome
Method: Copper oxidase activity
Reference value: 18-45 mg/dL (CF: 10- mg/L
i. a-Macroglobulin (AMG)
I t is the largest major
nonimmunoglobulin protein in plasma.
Itis found principaly In the intravascular
spaces; it does not diffuse from the plasma
space; lower amounts can also be found in CSF.
It inhibits proteases such as
trypsin, pepsin and plasmin.
t Increases 10x In
nephrosis- the loss of AMG Into urlne is prevented by ts
t forms a complex with
prostate-specific antigen (PSA). large size.
Increased: nephrotic syndrome, diabetes and liver disease
Method: radial immunodiffusion,
Reference volue: 150-420 mg/dL (CF: immunonephelometry, ELISA and latex agglutination
0.01-g/L

94
i.Br-Microglobulin
t is a light chain component of the major human leukocyte antigen (HLA).
t is found on the surface of most nucleated cells; present in high concentration on lymphocytes.
t is needed in the production of CD8 cels.
It is freely filtered at the glomerulus, and then is reabsorbed and metabolized completely by the
proximal tubule elevated plasma levels are the result of impaired clearance by the kidneys or
overproduction of proteins as seen in inflammatory diseases (RA and SLE).
it appears in the urine when
reabsorption is incomplete because of proximal tubulardamage, as
in acute kidney
injury.
i t has a tendency to fold into a
Bsheet configuration, resulting to amyloid formation -

it is a
common cause of dialysis-associated amyloidosis.
Increased: renal falure, multiple myeloma, rheumatoid arthritis
(RA), systemic lupus
erythematosus(sLE), and HIV
Method: immunoassay
Reference vaBue: 0.2-2.8 ug/dt

k. Transferrin (Siderophilin)
I t is a major component of the
It is a gtycoprotein,
B> globulin fraction (electrophoresis).
synthesized in the liver; also a negative acute phase reactant.
It transports iron to lts
storage stes; CSF contalns small amount.
It is used to determine the cause of
anemia, to measure iron metabolism and determine the
iron-carrying capacity of the blood.
It
prevents loss of Iron through the kidneys.
In iron
defliclency anemia, its concentration is normal or increased.
A n elevated plasma level appears as
"paraprotein" pseudoparaproteinemia severe iron
-

in
deflclency anemia
Low plasma transferrin can impair hemoglobin
production and lead to anemia.
Deficiency of transferrinmay result in the accumulation of iron in apoferritin or in
preclpitates In tissues as hemosiderin. histiocytes, or
Increased: hemochromatosis (bronze-skin), IDA
Decreased: liver disease, malnutrition, nephrotic syndrome
Method: Immunodiffusion and immunonephelometry
Reference value: Male 215-365 mg/dL (CF: 0.01-g/L)
Female 250-380 mg/dL (CF: 0.01-g/L)

. Immunoglobuilns
These are synthesized in the plasrna cells.
These are ghycoprotelns composed of 82% to 96%
protelns, and 4% to 18% carbohydrate
produced by white blood cells known as B cells, that confer humoral immunity.
Examples: IgS, lgA, IgM, igD and IgE
gG Is the most abundant antibody found in plasma and hymph; igA is the main
mucous secrettons; igM Is the first antibody found in
antibody that appears in response to antdgenic stimulation;
IgD is present mostly on the surface of 8 cells; and
anaphylactic reactions. lgE is antibody associated with allerglc and
Increased: hepatic disease, infections, toxoplasmosis,
herpes, syphilis, allergic reactions, multiplecytomegalavirus infections, rubella,
Method: nephelometry, turbidimetry, radial immunodiffusion myeloma
and immunoassay

95
m. Lipoprotein
i t binds with proteins and lipids forming LDL, HDL, VLDi. and chylomicrons.
i t transports cholesterol, TAG and phospholipids.
n. Fibrinogen
it is one of the
largest proteins in the blood.
t i s synthesized in the lver; classified as glycoprotein (has high CHO content).
It is the most abundant
of the coagulation factor it forms a fibrin clot when activated by
thrombin.
t appears as a distinct band between
Band y-globulins (electrophoresis).
It is one of
the
acute-phase reactants markedly increased in inflammatory process.
t may serve as a marker for
long-term prognosis of cardiovascular disease.
High levels in plasma may cause elevated erythrocyte sedimentation rate
cells and allowing them to sediment faster in (ESR) by coating the
-

clumps.
increased: infammatory disorders,
pregnancy and used of birth contro! pills
Decreased: extensive coagulation
Reference value: 200-400mg/dL (2.0-4.0 g/L)
o. Complement
tis one of the natural defence mechanisms
that protects the human body from infection
t participates in the immune reaction and
serve as a link to the
t circulates as non inflammatory response.
functional precursors.
Complement C3 is the most abundant form in serum i t is also important in the pathogenesis
of age-related macular
degeneration.
3 (and also CA) concentration is a convenient
marker forassessing disease activity in rheumatic
disorders such as RA and SLE.
Increased: inflammatory conditions
Decreased disseminated intravascular coagulation (DIC),
Method: immunonephelometry and turbidimetr
hemolytic anemia and mainutrition

p.C.reactive protein (CRP)

it is a member of pentraxin protein family.
CRP was so named because itbinds to the C-polysaccharide
of
precipitates with the C substance, a polysaccharide of pneumococci. the pneurnococcus it
t is a general scavenger molecule;
gamma-migrating protelin.
it may be undetectable in the blood of
healthy individuals.
t appears in blood of the patients with diverse
inflammatory diseases.
t is a cardiac marker used as an early warning test to persons at risk of
disease. coronary artery
t is an infammatory marker that
appears to reflect the severity of CHD and may contribute to
its pathogenesis.
tis used also as a rapld test for presumptive diagnosis of bacterial versus viral infection.
increased: acute rheumatic fever, myocardial infarction (MI), RA, gout, bacterial and viral
infection
Reference vafue: <1.0 mg/dL
CF Conversion factor

g6
 MISCELLANEOUS PROTEINS: c/s ACUITC wYoCARD\HL NFAR(TIDN
1. Myoglobin
t is a small heme protein found in skeletal and cardiac muscles.
transports and stores oxygen from hemoglobin to intracellular respiratory enzymes of
contractile cells.
t has higher affnity for oxygen than does hemoglobin.
It is approximately 2% of the total muscle protein.
t has a molecular weight of only 18 kDa, thus,
myoglobin apparently leaks from damaged cells
more rapidly than other proteins.
t is a potential nephrotoxin it has to be excreted when plasma concentration exceeds
reference ranges.

DlagnasticIt isSkantisance:
one of theprotein markers to diffuse out of ischemic muscle cells.
t is a marker for chest pain (angina) and
early detection of acute myocardial infarction (AMI).
In AMI (screening test), the onset is 1-3 hours, peak level 5-12 hours, normalize in 18-30 hours.
It is a useful marker for monitoring the success or failure af
reperfusion.
Increased: AMI, angina, rhabdomyolysis, muscle trauma, extrenous exercise, intramuscular
injection and acute renal failure

Methods:
It is measured in serum by immunoassay.
At high levels in urine (myoglobimurial, it produces a positive dipstick reaction for occult blood
due to pseudoperoxidase activity.
AMI value:100 ug/L

2. Troponins (Tn)
These are complex of three proteins (regulatory proteins) that bind to the thin filaments of
cardiac muscles.
These are regulators ofactin and myosin.
CTnT and cTnl are nearly absent from normal serum many heathy individuals have
undetectable levels.
TnC, Tnl, TnT = present in both cardiac and skeletal muscles
TnC binds calclum ions that regulate muscle contractions.
Tni and TnT are almost absent in normal serum.
Reference value« 0.1 ng/ml («0.1 ug/L

Diagnastic Skgniicance
The most important marker for cardiac injury (AMI)-they are derived from heart muscles.
The tevels in blood may elevate after AMi attack in the absence of Ck-MB elevations.

97
Cardiac Troponins:
a. Troponln T
(TnTTropomyosin-binding subunt
valuable tool in the diagnosis of AMI
t is a
I t is useful for assessment of early and late AMI; elevated also in renal diseases and muscular
cd dystrophy.
t i s a sensitive
t is useful in
markerfor the diagnosis of unstable angina (angina at rest).
monltoring the effectiveness of thrombolytic therapy in AMI patlents.
In AMI, it rises within 3-4 hours after
onset of myocardiat damage, peak level is at 10 to 24
hours, return to normal in 7 days (but may remain elevated for 10-14
Serum levels at or above 1.5 ng/ml are considered to be
days).
suggestiveof AMI.
b. Troponin I
(Tni)/Inhilbitory subunt or Actin-blnding unit
It is only found in the myocardium-greater cardiac specificity than TnT.
t is highly specific for AMI not elevated in renal failure patients and no detectable amount in
the skeletal muscles.
t is 13x more abundant in the
myocardium than CK-M8 on a weight basis.
It is a very sensitive indicator af even minor amount of
cardiac necrosis.
In AMI, levels begin to rise 3-6
hours, peak in 12-18 hours, return to normal in 5-10 days.
Methods:
They are measured in serum by immunoassay.
Reference values of Tnl and TnT as cardiac markers depend on the antibodies and calibrators
used in the immunoassay.

3. B-type natrluretic peptide (BNP)

t i s a cardiac marker.
t increases in response to peptide (BNP) ventricular systolic and diastolic dysfurction and is
diagnostic of congestive heart failure
hcPOrinlo plasa chaice scun (usto)
4.CystatinC
It is a low molecular weight protein and a cysteine
proteinase inhibitor.
It is freely filtered by the glomerulus and completely reabsorbed and
catabolized by the
proximal comvoluted tubule-it is produced and destroyed at a constant rate.
t has been included in the list of endogenous renal marker
owing for its sensitivity for
detemining the glomerular filtration rate.
t has been proposed as an alternate test for serum creatinine and
creatinine clearance test to
screen and monitor kidney dysfunction.
t not affected by physiologic factors like age, diet, gender and muscle
mass, unlike creatinine.
It may be especialty useful in those cases where creatinine measurement is not
appropriate,
such as in liver disease, obesity or those with muscular disease.
Increased: renal disease
Method: particle-enhanced immunoturbidimetry, immunonephelometry

98
PROTEINS IN BODY FLUIDS
1. Urinary Protelns
Majorty of proteins found in the urine arise from the blood.
The presence of urine albumin is generally considered abnormal even in trace amounts.
Only a small amountof protein is present in normal excreted urine (< 20 ug/min-
normal albumin excretion rate).
Some heathy Individuals exhibit albuminuria folliowing intense exerclse.
Urine protein of 26 mg/dl produces color change on urine dipstick.
Protelnuria (>0.5g/day) results from either giomerular or tubular dysfunction.

Tpesof Proteinuria:
a. Glomerular proteinuria
t is the most common and serious type of abnormal proteinurla.
tisoften called albuminuria.
b. Tubular proteinuria
t i s the appearanoce of low molecular mass proteins in the urine due to defective reabsorption.
Urine albumin excretion: slightly increased
.Overload proteinuria
I t includes hemoglobinuria, myoglobinuria and Bence-Jones proteinuria.
d. Postrenal proteinuria
It refers to protein coming from the urinary tract caused by infection, bleeding or malignancy.

Microalbuminurla
It is an earty indicator of glomerular dysfunction and precedes nephropathy associated with type
1 diabetes.
It is albumin excretion of 30 ug/mg creatinine to 300 g/mg creatinine (albumin-creatinine
ratio).
Positive Microalbuminuiria: 2 out of 3 specimens submitted for testing within a three- to six-
month period are with abnormal findings
Physiologic increase: physical exercise during the previous 24 hours
tncreased: diabetic nephropathy, fever, infection and hypertension
Specimen: random urine (reject bloody specimen)
Method: random-spot albumin-creatinine ratio
Reference value: 0-29 ug/mg creatinine
Microalbuminuria: 30-300 ug/mg creatinine
>300 ug/mg creatinine
Clinical albuminuria:

2. CSF Protein
CSF is an utraflitrate of plasma formed in the choroids plexus of the ventricles of the brain
The CSF normally contalns very little protein because the protelns In the blood do not cross
easily in the blood brain barrier
For CSF glucose and proteins analyses, it is recommended that a blood sample is analyzed
concurrently.
Method: TCA, SSA, Coomassie Briliant Blue (dye), Lowry and Kinetic Biuret Reaction
Increased: bacterlal, virat and fungat meningitis, traumatic tap, mutiple sclerosis, intracerebral

99
 hemorrhage, myxedema, drug toxicity
Decreased: intracranial hypertension, hyperthyroidism, leakage of CSF due to trauma
Reference value: 15-45mg/dL
CSF albumin: 10-30 mg/dL (2/3 of the CSF total
protein)
Notes to Remembe
1. CSF Oligoclonal banding
I t i s the presence in CSF of 2 or more IgG bands in the y region; seen in multiple sclerosis.
I t i s an indication of antibody production within the CNS.
Approximately 90% of multiple sclerosis patients show oligoclonal bands in the gamma
region of the protein electrophoresis.
Primary purpose of CSF protein electrophoresis: to observe this tyoe of
bandingrepresenting inflammation within the CNS
()result for CSF: 22 bands in the CSF not present in serum- multiple sclerosis
Other disorders with 2 or more bands in the CSF:encephalitis, neurosyphilis, Guillain-Barre
syndrome, neoplastic disorders
Disorders with serum banding appearing in CSF: leukemia, tymphoma and viral infections
Supporting medium and stain: Agarose gel and Coomassie Brilliant Blue

2. Aminoacidopathies
t i s inherited disorders of amino acid metabolism.
t exist in elther the activity of a specific enzyme in the metabolic pathway or in the membrane
transport system for amino acids.

a. Alkaptonuria
t is an inborn error of metabolism characterized by the absence of homogentisate oxidase in
the tyrosine pathway.
Clinical feature: ochronosis (tissue pigmentation)
>Diagnostic indicator: darkening of urine upon standing at room temperature

b. Homocystinurta
t i s characterized by impaired activity of cystathionine B-synthetase.
t results to elevated levels of homocysteine and methionine in blood and urine.
Cinical features: physlcal defects, thrombosis, osteoporosis, eye lens abnormality, mental
retardation
Screening test: Modified Guthrie Test ( L-methionine suifoximine antagonist)
-

c. Maple Synup Urine Disease (MSUD)

t i s characterized by markedily reduced or absence of a-ketoacid decarboyiase.
t results to accumulation of branched-chain amino aclds (leucine, isoleuclne and
blood, urine and CSF.
valine) in
Clinical features: fallure to thrlve, muscular rigidity, mental retardation, hypogtycemia
Screening test: Modified Guthrie Test (4-azaleucine- antagonist)
Diognostic test: Amino acid analysis (HPLC)
Indicotor: 4 mg/dL of leucine is indicative of MSUD
d. Phenylketonurta (PKU)
t is an autosomal recesslve trait characterized by the deficiency of the enzyme phenylalanine
hydrolase (PAH)/phenylalanine-4-mono-oxygenase, which catalyzesthe conversion of
phenylalanine to tyrosine.
t is charactertzed by the presence of phenylpyruvic acid (prime metabolite) in both blood and
urine in elevated concentration.
Deficiency of tetrahrydrobiopterin (BH also results in elevated blood levels of phemylalanine.
Reference value for serum phenylailanine: 1.2-3.4 mg/dl (70-200 umol/)
Clinical features: retarded mental development (infants and children)
Diagnostic indicators: »1200 Humol/L of phenylalanine in blood; "musty odor" of urine
Screening test Guthrie Bacterial inhibition Assay (8acilussubtlis spores &Pr-thienyiaanine antagonist)
() result = bacterial growth f the phenylalanine is > 4mg/d

e. Tyrosinemia
t is characterized by the deficiency of either of these enzymes: tyrosine aminotransferase
FAA
(tyrosinernia !)}; 4-hydroxyphenylpyruvic acid oxidase (tyrosinemia l); fumarylacetoacetate
hydrolase (tyrosinemia l).
t is accompanied by elevated methionine and p-hydroxyphenylpyruvic acid in blood.
Deficiency of these enzymes would lead to liver damage or cirrhosis.

101
 KIDNEY FUNCTION TESTS
The kidneys are paired, bean-shaped organs located retroperitoneally on either side of the
spinal column.
There are 2 regions of the kidneys an outer region called the cortex and an inner region called
the medulla.
Nephrons, the functional unit of each kidney, are composed of 5 basic parts namely, giomerulus,
proximal convoluted tubule, loop of Henle, distal convoluted tubule and collecting duct.
The kidneys conserve fluids by building high sodium chloride
gradlents in the Interstitlal space
between the descending and ascending limbs of the oop of Henle, using the countercurrent
multiplier mechanism.
The proximal convoluted tubule is responsible for the reabsorption of sodium, chloride,
bicarbonate, and other ions; glucose; amino acids and proteins; urea and uric acid.
About 25 liters of dilute urine is delivered to the ascending limb of Henle. Because the tubules
from this point to the beginning of the cortical collecting duct are water impermeable, the
volume remains unchanged at 25 liters, but the osmolality decreases progressively to about 60-
80 mOsm/L
Collecting duct is the final site for either concentrating or diluting urine.
Plasma contains 20-35 mg/dL of non-protein
nitrogenous compounds.
Nonprotein compunds: urea 45%, amino acid 20%, uric acid 20%, creatinine 5%, creatine 1-2%
and ammonia 0.2%
Renal function panel: glucose, BUN, creatinine, sodium, potassium, chioride, phosphorus,
calcium, albumin and CO2

Functions
1.
of the IKdneysi
Elimination of waste products
2. Maintenance of blood volume
3. Maintenance of electrolyte balance
4. Maintenance of acid-base balance
5. Endocrine function (erythropoetin secretion)
L TESTS FOR THE GLOMERULAR FILTRATION RATE
Glomerular Fltration Rate
I t is a measure of the clearance of normal molecules that are not
bound to protein and are
freely filtered by the glomeruli neither reabsorbed nor secreted by the tubules.
I t is considered the best overall indicator of the level of
kidney function.
150 liters of glomerular fltrate is produced daily.
GFR decreases by 1.0 ml/minute/yearafter age 20 to 30 years
About 180 liters of water is filtered daily; 150 liters is reabsorbed in the
proximal tubule, and
about 5 liters in the descending limb of Henle of cortical nephrons.

1. Clearance
I t is the removal of the substance from plasma into urine over a fixed
time.
It represents the volume of plasma that would contribute all the solute
excreted.
it is expressed in
milliliter/minute, representing the volume of plasma that would be totally
cleared of the solute in one minute.
Plasma concentration and clearance is inversely proportional as clearance of a substance
declines, ts concentration in plasma increases.

102
Formulafor Clearance: x Volume Iml x 1.73
Clearance
P minutes A
(mL/min)
Where:
U concentration of the analyte in urine
concentration of the analyte in plasma
Volume volume of urine in milliliter in 24 hours
minutes time required to collect urine (1440 minutes)
1.73 constant value; average body surface of an adult individual
and
A body surface of the patient (obtained from a nomogram; height
weight are taken)

a. Inulin dearance (Reference method)

t is not routinely done because of the necessity for continuous IV infusion requires an

intravenous infusion and timed urine collections over many hours

t has highervalues in male due to larger renal mass.
Priming dose: 25 ml of 10% inulin solution
Continous infusion: 500 mL of 1.5% inulin solution
Reference Values: Male 127 ml/min
Female 118 ml/min

Aternatives toInulin(OtherExogenousSubstances:
1. Radioactlve markers125Hothalamate and 99mTe-DTPA (metastable technetium99-labeled
diethylene triamine pentaacetic acid)
2. lohexol and Chromium514abeled ethylenediaminetetraacetic acid (51Cr-EDTA)
The advantage of these substances is that it does not require urine collection.
3. Nonradiolabeied iothalamate

b. Creatinine Clearance
I t provides an estimate of the amount of plasma that must flowed through the kidney
glomeruli/minute.
It is an excellent measure of renal function-creatinine is freely filtered by the glomeruli but not
reabsorbed.
t is a measure of the completeness of a24-hour urine collection
Production and excretion of creatinine is related directly to muscle mas -when renal function
is normal and stable, creatinine excretion is almost equal te its production, which depends
primarly on muscle mass./
The amount of creatinine generated from creatine tumover tends to remain constant for 24 hours.
Excretion of creatinine ls not routinely afected by diet-1.2-1.5 g creatinine excreted/day,
Major limitoation: accurate urine collection
Reference Volues: Male 85-125 ml/min
Female 75-112 ml/min

103
increased Creatinine Clearance DecreasedCreatinine Clearance
1. High cardiac output
1. Impaired kidney function
2.Pregnancy 2. Shock, dehydration
3. Burns
3. Hemorrhage
4. Carbon monoxide poisoning 4. Congestive heart failure

c.Urea Cearance
t can demonstrate
progression of renal disease or response to therapy.
It does not give reliable estimates of the
GFR since irea is freely filtered by the glomeruli but
variably reabsorbed by the tubules - in the presence of high urine osmolality and high urea
concentration, the amount reabsorbed in the inner medullary collecting duct is increased.
t is about 50% of creatinine clearance
(50% GFR), in the presence of normal renal function
without volume depletion, but in the presence of severe volume
be as little as 10% of creatinine clearance.
depletion, urea clearance could
In advanced renal failure, urea clearance
approaches unity with GFR, and is a better predictor of
GFR than creatinine clearance - as renal function
declines, the fraction of urea reabsorbed
declines progressively, whereas the tubular secretion of creatinine increases
progressively.
Volume depletion decreases creatinine clearance only by reduced filtration, and urea ciearance
by both reduced filtration and increased reabsorption.
The faster the rate of urine flow, the less urea is reabsorbed and vice versa.

2. Cystatin C
Itis a low molecular welght protease inhibitor and produced at a constant rate by all nucleated
cells.
itis freely filtered at the glomerulus, not secreted by the renal tubules but reabsorbed.
t is completely reabsorbed and catabolized by the proximai convoluted tubule, hence its
presence in urine denotes damage of that tubule - serum level is an indirect estimate of GFR.
It is not affected by muscle mass, age, diet and gender.
t increases more rapidly than creatinine in the early stages of GFR impairment.
Plasma level increases when glomerular filtration decreases, however, renal clearance of this
substance cannot be measured because it is completely reabsorbed.
Advontoge: to assess GFR among pediatric and elderly patients, and renal transplant patients.
Specimen: serum or plasma (fasting is not required)
increased: acute and chronic renal fallure, diabetic nephropathy
Reference value: 0.5-1.0 mg/L (adults)
0.9-3.4 mg/L(> 65 years old)

Modified Cystotin CEquation:

GFR (ml/min) = 84.69 x Cystatin C (mg/L) x 1.384

3. Trace Protein
I t is a low mglecular weight glycoprotein.
It belangs to the lipocalin protein family and functions as prostaglandin D synthase.
I t is isolated primarily from CSF plasma BTP originates from the brain and is freely filtered at
the glomerulus, then is reabsorbed completely and catabolized by the proximal tubule.
Increased:renal disease (because of reduced filtration In the presence of constant production)

104
 White Formuia usina6 traceprotein:
GFR1 (mi/min) = 112.1x BTP-2657 x urea280 x (0.880 if female)
GFR2 (ml/min) = 167.8 x BTP4.758 x creatinine0.204 x (0.871 if fernale)

PogeFormula using BT2

GFR1 (ml/min) = 89.85 x BTP5541 x urea2018
GFR2 (ml/min) 974.31x BTP023* x creatinine547
=

II TESTS FOR RENAL BLOOD FLOW

A. Blood Urea Nitrogen
amino acid catabolism 45% of the total
I t is the major end product of protein (dietary) and

NPN
It is approximately 80% of the nitrogen excreted.
I t is synthesizedin the lver from COz and the ammonia from the deamination of amino acids
(Ornithine or Kreb's Henseleit cycle).
in the proximai convoluted
t is freely filtered at the glomerulus but is reabsorbed substantially
of urea in the proximal tubule
tubule and the inner meduliary collecting duct reabsorption
whereas in
OcCurs passively through the lipid membrane without the help of urea transporters,
the inner medullary collecting duct, urea reabsorption is mediated by urea transporters.

It is the first metabolite to elevate in kidney diseases.

t is easily removed by dialysis.
good indicator of nitrogen intake and the state of hydration
t is a
About 258 of urea is excreted daily, in stable nitrogen balance.
content of urea.
The concentration of urea is expressed only by the nitrogen
To obtain the concentration of urea from BUN: 2.14 x BUN = urea (mg%)
Reference value: 8-23 mg/dL{2.9-8.2 mmol/L)
BUN:Creatinine ratio: 10:1-20:1

Methods:
Fasting sample is usualy not required.
Fluoride or citrate wil both inhibit urease.
Thiosemicarbazide and ferric ions are added to enhance color development.
measurements may be used for calculation of nitrogen
balance.
Urinary urea

2.Chemical Method(Direct Method)

Diocetyl Monoxime Method
Urea+DAM Yellow Diazine Derivative

2. Enzymatic Method (indirect Method)

a. Hydrolysis of Urea by Urease
Urea+Urease NH3 +C02

rease is prepared from jack beans.

After urease reaction, the ammonia produced can be treated with Berthelat reagents.
caiculate the concentration of
Ammonla and COa produced are measured by various methods to
used.
ureatn the original sample. Measurement of ammonia is most often

105
 b. Coupled Urease/Gutamate Dehydrogenase (GLD) method- UV
Urea + urease enzymatic methhod
NH CO

GLD
NH+2-oxoglutarate + NADH Glutamate NAD H0
3. Isotope
Dilution Mass Spectrometry (IDMS) Reference method
increased BuN
1. Chronic renal Decreased BUN
disease 1. Poor nutrition
2. Stress
2. Hepatic disease
3. Burns
3. Impaired
4. High protein diet absorption (celiac disease)
4. Pregnancy
5. Dehydration

Notes to Remember
Clinically, BUN riss in response to renal
dysfunction.
Low BUN not
are
generally considered abnormal renal function.
Serum urea
levels drop in severe hepatic disease because of a decline in the
to generate urea from ammonia. capacity of the liver

B. Creatinine
i t is the end product
of muscle metabolism derived from creatine (a-methyl
It is also produced
by three amino acids such as methionine, guanidoacetic acid).
t is partially secreted arginine and lysine.
by the proximal tubutes via the organic cation
t is not affected by protein diet; not easily removed transport pathway.
It is not reused in the by dialysis.
body's metabolism, solely as a waste product.
t is commonly used to monitor
renal function; an index of overall renal
It is a measure of the function.
I t is also used to
completeness of 24-hour urine collection (urine creatinine).
evaluate fetal kidney maturity a s gestation progresses, more creatinine is
excreted by the fetus into the amniotic fluid (2
The amount generated in an individual is mg/dL).
The generation remains fairly constant- proportional to the mass of skeletal muscle present.
plasma levels are constant.
Reference value: Male= 0.9-1.3 mg/dL (80-115 umol/L)
Female 0.6-1.1 mg/dL
umol/1)(53-97

New Classification for Acute Kidney Injury (AKI)

Stage Serum creatinine criteria
>0.3 mg/dL (26.4 umol/L) or >150%-200% Urine output criterla
>200%-300% c0.5 mi/kg for >6 hr
300%, 4 mg/dL (354 umol/L), or acute increase of <0.5 mL/kg for >12 hr
<0.3 ml/kg for >24 hr or
0.5 mg/dL anuria >12 hr
Souroe: Henry's Clinical
Diagnosis, 22 ed, 2011.
>AKI as "functional or structural abnormalties or
markers of kidney
abnermalities in blood, urlne, or tissue tests or imaging studies present damage including
months." for less than three

106
 AKI is associated with retention of creatinine, urea, and other metabolic waste products that are
normally excreted by the kidney.
Although severe AKI may result in ollguria or even anuria, urine volume may be normal or even
increased.

Methods:
Fasting sample is not required.
Hemolyzed and icteric samples should be avoided.
Both serum and urine creatinine have been used as indices of renal function because of the
constancy of creatinine forrmation.
A 24-hour urine sample with « 0.8 8/day of creatinine indicates that some of the urine was
probably discarded.
Patients taking cephalosporinantibiotics may have falsely increased results when the Jaffe
reaction is used.

1. Chemical Method-DirectJaffe Method

Principle: A red-orange tautomer of creatinine picrate is formed when creatinine is mixed with
aikaline picrate reagent.
Interferences: ascorbate, glucose, uric acid and a-keto acids false increased levels
bilirubin and hemoglobin false decreased levels
a. Folin Wu Method-a sensitive but nonspecific method
b. LIoyd or Fuller's Earth Method
It is a sensitive and specific method.
Adsorbent: loyd's reagent (sodium aluminum silicate)
Fuller's earth reagent (akuminum Mg' silicate)
Adsorbent removes interferences present in the specimen, and elution techniques are then
utilized to separate the creatinine from the adsorbent which is then made to react with the
freshly prepared Jaffe reagent.
Disadvantage: it is a time consuming method and not readily automated; not routinely performed.

Jaffe Reagent(Alkaline Picrate):

a. Saturated Picric Acid
b. 10% NaOH

2.Kinetic Jaffe Method

I t requires automated equipment for precision.
Itis a popular, inexpensive, rapid and easy to perform method.
rate of change in
Principle: Serum is mixed with alkaline picrate solutlon and the
absorbance is measured between 2 points.
DHfferential rate of coior development of noncreatinine chromogens, thus allowing a rate
dependent separation of creatinine from interfering substances.
of picric, to form
carbony oxygen of creatinine may attack the carbon
a
In this reaction, the
covalent adduct (Janovsky-like reaction).
Interferences: a-keto acids and cephalosporins-false increased levels

107
 3. Enzymatic Method
I s used to eliminate
nonspecificity of the Jaffe reaction; specific than Jaffe test.
Interference by glucose and other Jaffe
chromogens in creatinine measurement does not occur
with enzymatic methods.
However, enzymatic methods have certain interferences
interference from bilirubin and of their own (e.g., possible negative
catecholamines).
a. Creatinine Aminohydrolase-CK Method
I t requires a large volume of pre-incubated sample; not widely used.
Creatinine + H20
creatinine aminohydrolase Creatine
Creatine+ ATP Creatine PO + ADP
ADP+ Phosphoenol pyruvate Pyruvate inase ATP +
Pyruvate
Pyruvate + NADH Lactate + NAD'

b.
Creatinase-Hydrogen Peroxide Method
It has potential to
replace Jaffe method (specific than Jaffe method).
Without interference from acetoacetate or
Creatininase is also known as creatinine cephalosporins
aminohydrolase.
Creatinine + H20 Creatininase Creatine

Creatinase
Creatine+ H;0 Sarcosine + Urea
Sarcosine oxidase
Sarcosine+H:0 02 Glycine + HCHO+H0h
Peroxidase
H202+phenol+4-aminophenazone Benzoquinonemine dye (red)
4. Isotope Dilution Mass Spectrometry (IDMS) Reference method

Increased
1.
SerumCreatinine Decreased Serum Creatinine
Impaired renal function 1. Decreased muscle
2. Chronic nephritis mass

3.
2. Advanced and severe liver disease
Congestive heart failure 3. Pregnancy
4. Inadequate dietary protein
Notes to Remember
.Elevated plasma creatinine concentration is associated with abnormal renal
asit relates to glomerular function. function, especially
Plasma creatinine is a relatively
insensitive
renal function has deteriorated more than marker
and may not be
measurably increased until
In muscle disease such
50%
as muscular dystrophy,
plasma creatine and urinary creatinine are elevated.poliomyelitis, hyperthyroidism, and trauma,

108
 In the presence of normal renal function, plasma creatinine concentration is usually within
reference limit or normail in muscular diseases.
However, in severe muscle wasting, production of creatinine could be reduced to less than 25%
of the amount predicted from thee body weight.

Low RatioBun:Creal s10:1 High Ratio /BUN:Crea)>20:1with normal creatinine

1. Low protein diet 1. Prerenal azotemia 4. Gl hemorrhage
2. Acute tubular necrosis 2. Dehydration 5. High protein diet
3. Repeated dialysis 3. Catabolic states
4. Hepatic disease

High Ratio(eUN:Crea) 220:1 with_increased creatinine

1. Postrenal azotemia
2. Prerenal azotemia with renal disease
3. Renal failure

C. Blood Urnc Acd

It is the major product of purine (adenine and guanine) catabolism.
It is the final breakdown of nucleic acids catabolism in humans.
t is formed from xanthine by the action of xanthine oxidase in the liver and intestine.
t is freely filtered, partally reabsorbed and secreted in the renal tubules.
tis aweak acid; at pH 7.4, >95% exists as monosodium urate.
Derived from 3 sources: catabollsm of ingested nucleoproteins, catabolism of endogenous
nucleoprotelns and direct transformation of endogenous purine nucleotides.
About 1 gram of uric acid is excreted normally.
Reference value: (Uricase ) Male = 3.5-7.2 mg/dl (0.21-0.43 mmol/L
Fernale = 2.6-6.0 mg/dL (0.16-0.36 mmol/L)

Disease Correlation:
A. Hyperuricermia
1. Gout
t is a disease found primarily in males and first diagnosed between3 and 5 decade of life.
There Is pain and inflammation of the joints (acute inflammatory arthritis).
t is characterized by the presence of "birefringent crystats in the synovial fuid" definitive
diagnosis.
Persons with gout are highly susceptibie to nephrolithiasis.

2. Increased nuclear metabolsm

t is seen in leukemia, lyrnphoma, multiple myeloma or polycythemia, hemoiytic and
megaloblastic anemias.
Monitoring is important to avoid nephrotoxicity
For treatment:alHopurinol drug

3. Chronic renal disease

tisdue to decreased GFR and tubular secretion.
BUA: >10 mg/dL (can cause production of urinary tract calculi)

109
4. Lesch-lyhan syndrome (inborm errors of purine metabolism)
tis hypoxanthine-guanine phosphoribosy! transferase
deficiencyof (HGPRT)
Other Couses of Hyperuricemia:
1. Secondary to gycogen storage diseases
2. Toxemia of pregnancy and lactic acidosis
3. Increased dietary intake
4. Ethanol consumption

B. Hypouricemia
1. FanconY's syndrome-renal-type aminoaciduria
2. Wilson's disease
3. Hodgkin's disease

Methods:
Fasting may not be required, but for diagnostic purposes, fasting sample is preferred.
High plasma uric acid is observed in newborns which eventually decreases in the succeeding
years.
Uric acid is stable in both serum and urine for 3 days at room temperature.
Potassium oxalate (anticoagulant) cannot be used.
Ascorbic acid and bilirubin are the major interferences.

1. Chemical Methods
Principle: Reduction-Oxidation (Redox} Reaction

NaCN/ NacoOs
Uric Acid + Phosphotungstic Acid Tungsten Blue +Allantoin +CO

a. Sodium cyanine (NaCN) Folin Brown

Newton Benedict
b. Sodium carbonate (Na,cos) Archibald Caraway
Henry
Logphase - is the incubation period after the addition of an alkali (NaCN/Na>CO») to inactivate

non-uric acid reactants

2. Enzymatic Methods
Uricase Method
I tis a specific method.
Uric acid has a UV absorbance peak at 293nm, allantoin does not have UV
a peak at that
wavelength.

Principle: The enzyme uricase oxidizes uric acid to form allantoin. Uric acid has a maximum peak of
absorption of 293mm. The resutant product (allantoin) has no absorptlon at this wavelength. Thee
decrease in the absorbance isproportional to the concentration of uric acid present in the sample.
Uricase

Uric Acid+02 Allantoin+ CO2+ H0

110
3. Isotope Dilution Mass Spectrometry (IDMS)- Reference method

Disease Comelatien:
1. Azotemia
substances like urea and creatininein blood.
Itis eevated concentrations of nitrogenous

Types of Azotemig:
a. Pre-renal azotemla
it is diminished glomerular filtration with normal renal function (GFR ). R°ni fs
t is characterized by decreased renal blood flow GFR decreases and tubular reabsorption
increases leading to slower filtrate flow.
Couse: dehydration, shock and congestive heart failure
creatinine is normal.
Dehydration should be considered when BUN is elevated but the plasma

b. Renal azotemia
it is characterized by damaged within the kidneys (GFR).
Lab result: BUN 100 mg/d
Creatinine 20 mg/dl
BUA 12 mg/dl
imbalance.
slowly rising plasma creatinine, anemia and electrolyte
There is striking BUN Ievel,
Couses: acute/chronic renal disease, glomerulonephritis
Complications: coma and neuropsychiatric changes

c. Post renal azotemiaa

I t is usualy the result of urinary tract obstruction (GFR).
increased
Urea level is higher than creatinine due to back-diffusion of urea into the circulation;
urea and creatinine in blood.
Causes: renal calculi (nephrolithiasis), cancer or tumors of genitourinary tract

2. Uremia
t is a cinical syndrome comprised of a marked elevation in plasma urea and other nitrogenous
imbalance (K* elevation).
waste products, accompanied by acidemla and electrolyte
t is characterized by anemia (normocytic normochromic), uremic frost (dirty skin), generalized
edema, foul breath and sweat is urine-like.
The kidneys fail to eliminate waste products of
metabolism.
with burr cells (echinocytes) and
This condition is responsible for changes in red cell shape,
blood films presence of burr cells during the
elipsoidal cels commonly preset on peripheral
-

course of ilness may signal the development

of renal dysfunction.

111
 Im. TESTS MEASURING TUBULAR FUNCTION
A.Excretion Tests
1. Para-Amino Hippurate Test (Diodrast
It
Test)
measures renal plasma flow.
This method requires clearance of the
dye.
Reference value: 600-700 m/minute
2. Phenolsulfonthalein Dye Test
I t measures excretion
of dye proportional to renal tubular mass.
Dose:6 mg of PSP is administered IV.
Reference value: 1200 mL blood flow/minute
B. Concentration Test
I t reflects the function of the
t i s used to
collecting tubules and the loops of Henle.
assess the quantity of solutes present in urine, which reflects the
ability cf the
kidneys to produce a concentrated urine.
It can detect renal
damage that is not yet severe enough to cause elevated plasma urea and
creatinine levels.
Monitoring the concentration of chloride and sodium in urine reveals the
concentrate the ultrafiltrate in tubules. ability of the kidney to
3 most prevalent solutes
excreted: urea, chloride and sodium
Specimen: first morning urine

1. Specific Gravity (SG)

It is the simplest test of renal
It
concentrating ability.
compares the weight of a fluid with that of distilled water at a
reference temperature.
Measurement is affected by solute number and mass
Increases in large urinary solutes such as
(high molecular weight).
glucose; urea and protein increase the "true specific
gravity."
"Fixation" of SG at 1.010 indicates severe loss of
concentrating ability of the kidneys.
Specific gravity of 1.010 is equal to SG of ultrafiltrate in Bowman's
plasma)
space (same as protein-free
High molecular weight substances: x-ray dye and mannitol yield high SG 1.050).
Reference value: 1.005-1.030

2. Osmolality
It is an expression of oconcentration in terms of the total
number of solute particies
of solvent (moles/kg solvent). present/ kg
t is affected only by the number of solutes
present, thus nore accurate thanspecific gravity in
assessing renal tubular function (concentration ability).
Urine osmolality is due primarily to urea; serum osmolality is
primarily due to sodium and
chloride.
Measurements of serum osmolality is useful for assessing water deficit or excess.
Values are not affected by high molecular weight substances as
opposed to specific gravity
Proteins and lipids do not contribute to osmolality.
Normal ratio of urine osmolality to serum osmolaBity is 1:1.

112
Methods
Osmolalty is determined by measuring a colligative property of the sample (urine or
serum)such as freezing polnt, vapor pressure, osmotic pressure, bolling polnt.
An Increase In osmolallty (solute) increases thee osmotic pressure and boiling point.
If a patient is on a fluld-restricted diet, the osmolality of urine should be significantly higher
than the osmolality of plasma.
Sample: serum or urine
Reference value: Serum 275-295 mOsm/kg
24-hour urine = 300-900 mOsm/kg

1. Direct ethod: Freezing point osmometry(popular method)

Vapor pressure Osmometry (Seebeck Effect)
An increase in osmoiality (solute) decreases the freezing point and vapor pressure.

2. Indirect Method: Formula for Computing Serum Osmolality

To use glucose or urea in osmolality. calculations must be converted from milligram units to
molar units.
Serum Osmolality =1.86 x Na' Glucose imR/dl)+BUN (mg/dl)
18 2.8
Interpretation of Resuts:
Concentrated urine: 1.025 SG and > 800 mOsm/kg
Lost of renal concentrating ability: 1.2:1; diabetes insipidus = < 1:1
f the ratio of urine:serum osmolality is > 1:1, it is glomerular disease and presence of increase
solute in the urinary fitrate.
f the serum osmolality is > 2.1-2.3x the value of serum sodium, it may be due to hyperglycemia,
uremia and anion gap acidosis.
In DM, 5000 mOsm/day of solutes requires significant amount of water for elimination
(polyuria), thus requiring excessive water replacement (polydipsia).
An increase in plasma osmolaility causes a decrease in urine flow due to the reabsorption of
more water by vasopressin from the glomerular filtrate as it passes through the tubules, and as
result lowers plasma osmolality.
With reral loss of water, the urine osmolality is decreased or normal.

Notes to Remember
Urea is the only ineffective osmol that has substantial concentration in the normal plasma, 5
mOsm/L
For the contributions of urea nitrogen and glucose for serum osmolality, their values in mg/dl
are divided by one tenth of each (2.8 and 18) of their molecular weights (28 and 180), because
osmolality is expressed as rmOsm/, not mOsm/dt.
Hyperphosphatemia and hypocalcemia, in the face of elevated BUN and creatinine, indicative of
renal disease, strongly suggest tubular failure.
Non-electrolyte solutesthat accumulate abnormaly in the serum (eg, ethanol, ethylene gycol,
methanol, mannitol) will cause the measured osmolality to exceed the calculated asmolality,
producing an osmolal gap.
Osmolal gap is the the difference between measured and calculated plasma osmolalty.
An osnolal gap > 12 mOsm/kg is significant as seen in DKA, drug overdose and renal failure.
Osmolal gap is also a sensitive indicator of alcohol or drug overdose, causing a large "osmolal
gap" in ethanol intoxication.

113
TPAG n inportont Fn t
GuoDtokt scvcrity (a% LIVER FUNCTION TESTS
The liver is the chief metabolic
organ in the body.
It receives 15 mlL of blood
per minute.
I t is composed of 2 types
of
The celts are arranged into the
cells, hepatocytes and kupfer cells (phagocytic).
lobule, the anatomic urnit of the lver.
The liver has a unique capacity to
regenerate by cell division, and hypertrophy of the remaining
tissue in case of tissue injury due to
biliary obstruction or toxic exposure
Severe loss of hepatic functions may result to
the functions of excretion, detoxification and
diagnostic changes in synthetic capacities and in
metabolic activity that are reflected in multiple
standard and specialized tests.
To abolish liver tissue function, more than 80% of the liver must be destroyed.

Functions of the Liver

1. Synthetic Function
Liver secretes plasma proteins, carbohydrates, lipids, lipoproteins, clotting factors, ketone
bodies and enzymes.
The normal liver produces about 12
grams of albumin daily.
I t is aiso involved in the metabolism
2. Conjugation Function of cholesterol
into bile acids.

I t is Involved in the metabolism

of bilirubin.
200 to 300mg of bilirubin is produced
3. Detoxification and daily in the healthy adult.
Drug metabolism
Liver serves to protect the body from
potentially injurious substances absorbed from the
intestinal tract and toxic by-products of metabolism.
Ammonia (toxic by-product) is converted to urea in the liver.
4. Excretory and Secretory Function
Excretion of bile involves the elimination of bile acids or
salts, pigments, cholesterol.
Bile acids (cholic acid and
chenodeoxycholic acid) are conjugated with the amino acids glycine
and taurine to form bile salts.
5. Storage Function
Storage site for all fat-soluble and water soluble vitamins.
Liver is also the storage depot for glycogen, which are released
when glucose is depleted.

I. TEST MEASURING THE HEPATIC SYNTHETIC

FUNCTION
I t is useful for
quantitating the severity of hepatic dysfunction.
Serum albumin and the vitamin K-dependent
coagulation factors provide the most useful indices
for assessing severty of liver disease.

A. Total Protein
In measuring total proteins in serum, fasting may not be required.
Analysis of proteins is important for assessing nutritional status and presence of severe
involving the lver, kidney and bone marrow. diseases
Total protein and albumin are about 10% higher in
ambulatory individuals.
Plasma levels of total protein is 0.2 to 0.4g/dt higher than serum due to
fibrinogen.
Transudates have a total protein of <3.0 g/dL (k 50% of the serum total protein);
3 g/dL
exudates has

114
 It is usually performed in serum, which has no fibrinogen and no anticoagulant that may slightly
dilute proteins in plasma.
Method).
Hemolysis and ictericia may falsely elevate the total protein (Biuret
Reference value: 6.5-8.3 g/dL

1. KJeldahl Method
tt is the reference method but not routinely use.
t is based on measurement of the nitrogen eontent of protein.
filtrate (PFF).
it uses serum treated with tungstic acid, forming protein-free
to 6.54 grams of proteins.
According to Kjeldahl, 1 gram of nitrogen is equivalent
15.1%-16.8% = nitrogen content of proteins
Reagent: HSO4 (digesting agent)
Endproduct: Ammonia

2. Bluret Method cowow / KaunwEy ircc

Federation of Clinical
tis the most widely used method,
recommended by the International
Chemist (IFCC) expert panel. which protein
in automated analyzers in
tis extensively used in clinical laboratories, particularly
concentration can be measured down to 10 or 15 mg/dL
medium to measure total protein.
t requires at least peptide bonds and an alkaline
2 violet-colored
involved in the peptide bond forming a
Principle: Cupric ions complex the groups and reflects
bonds present
chelate which is proportional to the number of peptide
the total protein level at 545nm.
Reagents: Alkaline CopperSulfate
Rochelle Salt (Nak Tartrate)
NaOH &Potassium lodide
V: 6-5-41ga 0 g
3. Folin-Cocateu (Lowry) Method
I t has the highest analyticalsensitivity
such as tyrosine, tryptophan and histidine to give a deep
Principle: Oxidation of phenolic compounds
bluecolor.
Main reagent:Phosphotungstic-molybdic
acid or phenol reagent
Color enhancer: Biuret reagent.

4. Uttravlolet Absorption Method bonds at

at 210nm is due to the absorbance of the peptide
Principle: The absorbance of proteins
specfic wavelength.
at 210nm.
Protelns absorb light at 280nm and
is due to tryptophan, tyrosine and phenylalanine.
Absorption at 280nm

(SPE) CONTRMAT70AY T9T

5. Serum Proteln Electrophoresis
an electric field.
Principle: Migration of charged particles in
of SPE is for the identification of monoclonal
The single most signficant clinical application
them from polyclonal hypergammaglobulinemia.
spike of Immunoglobulins and differentiating inflammation assoclated with tissue injury, tht
Myocardial infarction produces a pattern of acute
is, elevated acute phase reactants (AAT,
haptoglobin, alpha-1 antichymotrypsin).
albumin, al-antitrypsin, a2-macroglobulin,
Major proteins that contrlbute to electrophoresis:
fibrinogen, and immunoglobulins
haptoglobin, P-ipoprotein, transferrin, complement C3,
onoUond pko
COMPIKAMS M EMYELOM 115
6Oa Spike. nonoconat spikt
 Normal SPE Pattenm
1. Albumin (1 band)-fastest band
2. Alpha 1-Globulin (2dfostest bandh gycoproteins, AAT, AAG, thyroxine binding-globulin (TBG6)
t increases as a non-specific response to inflammation.
3. Alpha 2-6lobulin (3bandfostest band)- haptoglobin, AMG, ceruloplasmin
4 Beta-globulin (4h band) transferrin, beta-lipoprotein, hemopexin, complement (C3 and C4)
5. Gamma-globulin (5th band;slowest band)- immurioglobulin and CRP

ReferenceValues for Each53-65%

Eraction:
(3.5-5.0 g/dL) o he seweriy « iver dk. atumin Morc scVtRE
1. Albumin
2. i-globulin 2.5-5% (0.1-0.3 g/d
3. arglobulin 7-13% (0.6-1.0 g/dL)
4. B-globulin 8-14% (0.7-1.1 g/dL)
5.v-globulin 12-22% (0.8-1.6 g/dl)

Abnormal Serum Electrophoretic Patterns

1. Gamma spike - Multiple myelomaI9t
2. Beta-gamma bridgins Hepatic cirrhosis Pdt CLOa CkMAo PAT 19A
3. a-globulin band spike
Nephrotic syndrome
4. a-globulin flat curve Juvenile cirrhosis (AAT deficiency)
5. Spikes of a a and Bglobulin bands inflammation
-

The presence of free hemoglobin will cause a "blip" in the late alpha-2 or early beta zone
region.
The presence of small spikes in beta
region is due to iron deficiency anemia (transferrin).
Rheumatoid arthritis and malignancy would result to polycicnai gammopathy ciassified as
chronic inflammation.

6. Refractometry
It is an alternative test to chemical analysis of serum total
proteins.
t is based on measurement of refractive index of solutes in
serum.

7. Turbidimetrlc and Nephelometric Methods

These methods utilize sulfosalicylic acid and or trichloroacetic acid.
Measurement depends on the formation of a uniform fine
precipitate which scatters incident
light suspension (nephelometry) or block light (turbidimetry).
in

8. Salt Fractionation
Globulins can be separated from albumin by salting-out
procedures using sodium saits.
Reagent: sodium sufate salt
. The albumin that remains in solution in the
supernatant can be measured by of theany routine
total protein methods; globulin is insoluble in water but not in dilute salt solution.
 Solubilty Property of Proteins
Proten Soluble Insoluble
Albumin Water Saturated salt solution
Concentrated salt solution Highly concentrated salt solution
Hydrocarbon solvents
Globulin Weak salt solution Water
Hydrocarbonsolvents Saturated salt solution
Concentrated salt solutlon
9. Other Measuremets for Protelin
Coomassie brillant blue dye is for detection of proteins to as little as 14g
Ninhydrin, which develops a violet color by reacting with primary amines, is widely used for
detection of peptides and amino acids after paper chromatography; amino acld analyses from
ion-exchange columns; as well as for detection of drugs on toxicology screens using thin-layer
chromatcgraphy.

Increased Total Protein Decreased Total Protein:

1. Malignancy 1. Hepatic cirrhosis
2. Multiple myelorna 2. Glomerulonephritis
3. Waldenstrõm's macroglobulinemia 3. Nephrotic syndrome
4. Starvation

B. Prothrombin Time (Vitamin K Response Test) t imourtom pm te

obstructive
I t differentiates Intrahepatic disorder (prolonged protime) from extrahepatic
olcing liver disease (normal protme).
protime despite vitamin K administration indicates loss of hepatic capacity to
Migcd korProlonged
v rCendsynthesize the proteins.
:. fomik acute viral ortoxic hepatitis, prolonged protime signifies massive cellutlar damage.
Vitamin K is administered intranmuscularly, 10 mg daily for 1 to 3 days.
ntranepanc oo CiRATED PASM
pmingco
oren YiD
The concentration of this protein is inversely proportional to the severity of the liver disease.
Piasma levels of albumin decline when severe hepatocellular disease lasts more than 3 weeks.
In hepatic cireulatory disorder, albumin is used because its concentration reflects the shift of
protein and fluld into ascites and, its important contribution to the plasma oncotic pressure.
Decreased serum albumin concentratlon may be due to decrease synthesis.
Low total protein + low albumin = hepatic cirrhosis and nephrotic syndrome

Dyes used for measurement:

1. Bromcresol green (BCG) most commonly used
2. Methyl orange (MO}
3. Hydroxyazobenzene benzoic acid (HABA)
4. Bromcresol purple (BCP) most specificdye
Albumin can be measured by direct methods based on its dye-binding property.
interact with the
Albumin reversible binds many smail molecules, including dyes that do no
other serum proteins.
thus allowing direct
Dyes bound to albumin absorb maximally at slightly different wavelengths,
spectrophotometric quantitation of the albumin.

117
 BCG is used extensively in automatic analyzers for detemining serum albumin in parallel with
Biuret reagent for total protein.
The presence of drugs such as penicillin, and the use of hemolyzed and icteric samples affect
measurement of serum albumin.
o BCG is affected by the presence of
penicillin leading to faise decrease of albumin.
BCG and BCP are not significantly affected by
hemolyzed samples.
o HABA method is influenced by
hyperbilirubinemia.
BCG and BCP are catioinic dyes, and free from interference from bilirubin.

Hyperalbuminemia:
1. Severe dehydration
2.Prolonged tourniquet application- artifactual hyperalbuminemia
Hypoalbuminemia:
1. Reduced synthesis
a. Chronic liver disease
b. Malabsorption syndrome
c. Malnutrtion and muscle wasting disease
2. Increased loss
a. Nephrotic syndrome (20-30g/day)- albumin excretion is increased when the
glomerulus no longer
functions to restrict thepassage of proteins from the blood
b. Massive burns
c. Protein-Hosing enteropathy
d. Orthostatic albuminuria
3. Increased Cotabolism
a. Massive burns
b. Widespread malignancy
c. Thyrotoxicosis

Teminologies:
1. Analbuminemia
t i s the hereditary absence
2. Bisalbuminemia
of albumin or inability to synthesize albumin.
i t is the presence of
i t is the presence of
two albumin bands instead of a single band in electrophoresis.
albumin with unusual molecular characteristics in the blood.
I t is associated wth excess amount
of therapeutic
drugs in serum.
Albumin/Giobulin Ratio
I t is determine to
If globulin is
validate if globulin is higher than albumin.
greater than albumin it is known as inverted A/G ratio seen in
myeloma and Waldenström's macroglobulinemia. cirrhosis, multiple
Serum and even urine proteln electrophoresis may
help define the clinical situations.
Reference value: 1.3-3:1

18
I . TEST MEASURING CONJUGATION AND EXCRETION FUNCTION

Bilirubin
tis the end product of hemogtobin metabolism and the principal pigment in bile.
t is also formed from destruction of heme-containing proteins such as myogtobin, catalase
and cytochrome oxidase.
ONJUGTou «ub IN UNE

Bilirubin Metabolism:
Red Blood Cell (120 days)

Hemoglobin

Heme (Iron Porphyrin) + Globin (Protein)

Heme Oxygenase
Biliverdin
Biliverdin Reductase

Bilirubin (Bi)
Attaches to albumin
i
Liver
Uridine Diphosphate Glucoronyl Transferase (UDPGT)

Bilirubin Monoglucoronide
UDPGT
Bilirubin Digucoronide (B:)

Bile
Intestine (normal flora)
dkconjug onon nappen
a URDBILINGEN
Urobilinogen
Reabsorbed Oxidation

Reconjuated Stercobilinogen
Urobilin (urine) Stercobilin (stool)

119
 Comparison Between Conjugated Biltrubin and Unconjugated Blirubin
Billrubin 1 Bilirubin 2
Unconjugated bilirubin Conjugated bilirubin CXcICiCd Fm
Water Insotuble Water Soluble
Non-polar bilirubin Polar biirubin
Indirect reacting Direct reacting
Hemobilirubin Cholebilirubin
Slow reacting One-minute/Prompt bilirubin
Prehepatic bilirubin Post hepatic bilirubin/Hepatic Bilirubin/
Obstructive and Regurgitative bilirubln

Reference Value
Conjugated bilirubin: 0-0.2 mg/dL (0-3umol/L)
Unconjugated bilirubin: 0.2-0.8 mg/dL (3-14umol/L)
Total bilirubin: 0.2-1.0 mg/dL (3-174mol/L)

Delta bilrubin
Dormgirorm It is conjugated bilirubin tightly bound to albumin.
It has a longer half-life than other forms of bilirubin.
t is formed due to prolonged elevation of conjugated bilirubin in biliary obstruction.
t helps in monitoring the declineof serum bilirubin following surgical removal of gailstones.
It reacts with diazo reagent in the direct bilirubin assay.
It is computed by usingthis formula: TB-DB +1B Delta bilirubin
t is not calculated on neonatal patients (s14 days ).
Reference value: <0.2 mg/dL (<3 umol/L)

Notes to Remember
The plasma concentration of bilirubin increases upon birth and reaches its peak on the S day.
The intracellutar conjugation of gucoronic acid onto two sites of the bliubin molecule confers
negative charge to it, making conjugated biliubin solubie in aqueous phase.
Only small amounts of conjugated bilirubin (B-} circulates in blood because of minor leakiness of
the hepatocytes in directions away from the formation and excretion of bile.
f the rate of blirubin formation exceeds the rate of liver clearance (ie., a state of
in serum.
overproduction of bilirubin) there will be a rise in the bilirubin level

120
Jaundice
t is also called Icterus or hyperbilirubinemia.
It is characterized by yellow discoloration of the skin, sclerae and mucus membranes.
It is clinically evldent when bilirubin level exceeds 2 mg/dL

Classtication of Jaundca/tvperbilirubinemla:
1. Pre-hepatic Jaundice
Cause: Too much destruction of RBC.
Bilirubin Assay: Elevated indirect bilirubin

2. Posthepatic Jaundice
Couse: Failure of bile to flow to the intestine/impaired bilirubin excretion.
Bilirubin Assay: Elevated direct bilirubin

3. Hepatocellular Combined Jaundice

Cause: Hepatocyte injury caused by viruses, alcohol and parasites.
Bilirubin Assay: Elevated direct and indirect bilirubin

Derangements of BIllirubin Metabolism:

1.Gibert's Syndrome-Bilirubin Transport Deficit
t is characterized by impaired cellular uptake of bilirubin.
t is diagnosed in young adults ( 20-30 years old).
Affected individuais may have no symptoms but may have mild icterus.
Lab result:elevated Bi (3 mg/dL)

2. Crigler-Najjar Syndrome-Conjugation Deficit

Infants are treated by means of phototheraphy.
Lab result: Elevated B
a. Type I Crigler-Najjar Syndrome
- It is characterized by the deficiency of the enzyme glucorynl transferase (UDPGT).
- Lab result: Total absence of Ba production
- (+) kernicterus; bile is colorless
b. Type Ii Crigler-Najar Syndrome
It is charactertzed by the partial deficiency of UDPGT.
Only small amount of B2 is produced.

3. Dubin-Johnson Syndrome and Rotor Syndrome- Bilirubin Excretion Deficit

Thls is a hereditary defect defect characterized by defective excretion of conjugated bilirubin
into the canalicull, caused by hepatocyte membrane defect.
There is an intense dark pigmentation of the liver ("black liver") due to accumulation of
lipofuscin pigment.
Lab result: elevated 82 and total bilirubin

4. Lucey-Driscoll Syndrome
is a familial form of unconjugated hyperbilirubinemia caused by a circulating inhibitorof bilirubin
conjugation.
Lab result: elevated B

121
Notes to Remember.
A fetus with hemolytic
disease does not develop hyperbilirubinemia (jaundice) because the
placenta normaly removes the bilirubin.
Some of the bilirubin may appear in the amniotic
fluid, and monitoring the concentration by
spectrophotometer at 450nm wavelength (Llley's test) provides a means of determining the
degree and progression of fetal hemolytlc disease.
The blinding capacity of
albumin for unconjugated bilirubin is the basic defense for
preventlon of kernicterus in HDN and infantile jaundice.
Kernicterus, seen in Criggler-Naljar syndrome, is the deposition of bilirubin in the brain,
particularhy affecting the basal ganglia, mainly the lenticular nucleus, causing severe motor
dysfunction and retardation.
Unbound bilirubin (free bilirubin) may cause the blood-brain barrier resulting to kernicterus.
Measurement of B is a sensitive and specific marker for hepatic and post hepatic
jaundice
because lt is not elevated by hemolytic anemia.
In adults, cholelithiasis (presence of gall stones) is the most common cause of
hyperbilirubinemia.
f there is obstruction, conjugated bilirubin will re-enter the circulation and accumulate, and
spontaneously form a covalent bond with albumin, thereby prolonging its haif-life in the
circulation.
Obstruction of the biliary tract also causes elevated total cholesterol due to block in the
normal excretion of cholesterol in bile.
Hepatocyte injury causes loss of conjugation of transported bilirubin, so that indirect
(unconjugated) bilirubin also rises.
In panhepatic cirrhosis, because insufficient viable liver tissue remains, and because fibrosis
destroys the cholangioles, both indirect and direct bilirubin tend to be elevated.

Methods
Serum specimens should be free from hemolysis and lipemia -hemolysis will cause increase
bilirubin while lipemia will cause decrease bilirubin.
Serum should be stored in the dark and measured ASAP or within 2 to 3 hours after collection.
Signtficant decreased of bilirubin occurs after exposure to fluorescent, indirect and direct
sunlight.
Visible icterisia occurs when bilirubin is> 25 mg/L
Because unconjugated bilirubin reacts slowly, accelerants such as caffeine or methanol are used
to measure total bilirubin.
Deletion of accelerants allows determination of direct-reacting or conjugated bilirubin.
Bilirubin standard solution is usually made from unconjugated bilirubin. (B,
I. Blirubin Assay
Principle: Van den Berg Reactionis diazotzation of bilirubin to produce azoblirubin.
a. Evelyn and Malloy Method
Coupling Accelerator: Methanol
Diazo Reagents:
Dlazo A 0.1% Sulfanilic Acid + HCI
Dlazo B 0.5% Sodium Nitrite
Diazo Blank 1.5% HCI
Final reaction: pink to purple azobllirubin

122
 bAendrassik and Grof
It is the most commonly used method.
disract andmaer t is a popular technique for the discreet analyzers.
ph oepcnaca ha t is more sensitive than Evelyn and Malloy method.
Is not affected by hemoglobin up to 750 mg/dL and pH changes.
Main reagent: Diazo reagent
Coupling Accelerator: Caffeine Sodium Benzoate
Buffer: Sodlum Acetate
Ascorbic acid: Terminates the initlal reaction and destroys the excess diazo reaget
Alkaline tartrate solution: Provides an alkaline pH
Final reaction: pink to blue azbilirubin

Notes to Remember.
Conjugated bilirubin produced a color in aqueous solution.
Unconjugated bilirubin is the fraction that produced a color only after the addition of alcohol.
Deta bilirubin is the conjugated bilirubin bound to albumin, elevated in obstructive jaundice.
The purpose of adding a methano! or caffeine solution is to allow indirect bilirubin to react
(solubilize) with the color reagent.
Total bilirubin is measured 15 minutes after adding methanol or caffeine solution.
Caffeine-benzoate is preferred over methanol because the latter promotes protein precipitation
and increases turbidity.
Measurement of total bilirubin involves solubilization of the unconjugated form before chemical
quantitation.
Bilirubin absorbs light maximaly at 450nm and imparts a yellow color to amniotic fluid.

IncreasedB1 Increased82
1. Gilbert's Syndrome 1. Biliary obstruction (gall stones)
2. Criggler-Najar Syndrome 2. Pancreatic (head) cancer
3. Hemolytic anemia 3. Dubin-Johnson Syndrome
4. Hepatocellular disease 4. Alcoholic and viral hepatitis
5. Lucey Driscoll Syndrome 5. Biliary atresia
6. G-6-PD deficiency 6. Hepatocellular disease

. Bromsufonthalein (BSP) Dye Excretion Test

It is a test for hepatocellular function and potency of bile duct; rarely used.

Dose (BSP Dye) Admtntstration Methods:

1. Rosenthal White (Double Collection Method)
Dose 2mg/kg body weight (BW) of the patient
Specimen Collection after 5 minutes and after 30 minutes
Normal Value
After 5 minutes 50% dye retention
After 30 minutes 0% dye retention

123
 2. Mac Donald Method (Single Collection
Method)
Dose Smg/kg body weight (BW) of the patient
Specimen Collection after 45 minutes
Normal Value
After 45 minutes +-5%dye retention
Uroblinogen
I t is a colorless end
product of bilirubin metabolism that is oxidized
brown pigment urobilin.
by intestinal bacteria to the
I t is elther
excreted in urine and feces, or reabsorbed into the portal blood and returned to the
Itver.
Absence of this substance in urine or stool denotes
In the collection of the
complete bilary obstruction.
sample, avoid exposure to direct light.
Specimen: 2-hour freshly collected urine or
freshlycollected stool
Method: Ehrlich's method (p-dimethyl
aminobenzaldehyde reagent)
Reference value: Urine =0.1-1.0 Ehrlich units/2 hour or 0.54 Ehrlichs units/day urine
Stool 75-275 Ehrlich units/100g
of feces

M. TEST FOR DETOXIFICATION

FUNCTION
It involves enzyme and ammonia tests.
MOrKCTS: ALT. 84T
A. Enzyme Tests
t is used to assess the extent
of Iiver damage and to differentlate
from obstructive (mechanical) disease. hepatocellular (functional)
Any injury to the liver that resuts in cytolysis and necrosis
causes the liberaticn of various
enzymes.
Enzyme tests are often the only indication of cell injury in
early or localized liver disease.
Enzymes secreted by the liver:ALP, aminotransferases,
5'nucleotidase, G6T, OCT, LAP and LD
B. Ammonia
t arises from deamination of amino acids, which occurs
and bacterial enzymes (bacterial mainty through the action of digestive
proteases, ureases and amine oxidases)on proteins in the
intestinal tract.
It is also released from metabolic
reactions that occur in skeletal muscles
The liver normally removes most of this NPN via the during exercise.
urea, then eliminated by the kidneys (urine).
portal vein circulation and converts it
to
Reference value: 19-60 Hg/dL (11-35 mmol/l)
Diagnostic Slantficance:
Increased levels: cirrhosis, hepatitis,Reye's syndrome, chronlc renal disease and
acetaminophen poisoning
For the diagnosis of
hepatic failure (hepatic coma) and Reye's syndrome.
In severe liver disorder, it accumulates
and reaches the
converted to glutamine In the brain, thus compromising the systemic
Kreb's
cireuiation, which Is then
cycle
lack of ATP for the brain ammonia
increases CNS pH.
leading to coma due to
Elevated plasma levels of ammonia are
neurotoxie and are often associated with
encephalopathy an important mechanism by which ammonta can cause
toxicity to the CNS is
124
 its ability to lower the concentration of y-aminobutyric acid (GABA), a critically important
neurotransmitter in the CNS, by reacting with glutamic acid to form glutamine via reversal of the
glutaminase-catalyzed reaction.
Plasma levels of ammonia are not dependent on renal function, though an NPN, but on liver
function, thus, are not useful In the study of kidney diseases.

Methods
Smoking is a source of contamination (patient and the phlebotomist) which leads to elevated
concentrations.
Prolonged standing of the specimen rises ammonia level due to enzymatic deamination of labile
amides like glutamine.
Preferred specimen: arterial blood (venous bloodis not recommended, if used, tourniquets
should be used minimally, and fist clenching and relaxing avoided during collection)
Specimen requlrements: Heparin or EDTA plasma (fasting); plasma/serum kept in ice water
immediately; hemolysis should be avoided.
Common Methods: Berthelot and Glutamate Dehydrogenase

1. Digestion (Kjeldahl) Method

Nitrogen ion in a protein-free filtrate (PFF) of the specimen is converted to ammonia
using hot oncentrated sulfuric acid in the presence of catalyst.
Catalyst: copper sulfate, mercury and selenium
HSO
Nitrogen NH3
CuSOs
Hg
Selenium

2. Measurement of Ammonia
a. Nesslerization Reaction
Gum Ghatt
NHa +KHgol NHaHgah
Yellow end color-N low to moderate
Orange brown end color- Na high

b. Berthelot Reoction
Na Nitroprusside
NH+Phenol+ Hypochlorite Indophenol blue

3. Glutamate dehydrogenase
Glutamate dehydrogenase

NHg+a-ketoglutarate+ NADPH Glutamate +NADP

125
 ENZYMES
These are proteins produced by
living cells that hastens chemical reactions in organic matter.
They are measured in terms of their activity and not in terms of thelr absolute
They are large molecules and they are normally confined within values.
permeability allows them to enter the blood. cells unless increased membrane
They frequenty appear in the serum after cellular injury,
Abnormal large amounts of enzymes in serum are used degradation of cells or from storage areas.
Each enzyme catalyzes a single reaction or a limited clinically evidence of organ damage.
as
number of chemical reactions, and It is specific for
a substrate that it converts to a
defined product.

Factors Atfectina Enzmatie Reactions

1. Enzyme Concentration
The higher the enzyme
concentration, the faster is the reaction, because more enzyme is
present to bind with the substrate.

2. Substrate Concentration
With the amount of enzyme
exceeding the amount of substrate, the reaction rate steadily
increases as more substrate is added.
However, when the substrate concentration reaches a maximal value,
substrate no longer result in increased rate of reaction higher concentration of
(saturation kinetics).
3. Cofactors
Nonprotein entities that must bind to particular enzymes before a reaction occurs.
a.
Coenzymes-is an organic compound (second substrates)
increasing its concentration wil increase the velocity of an enzymatic reaction
it is essential to achieve absolute
-

enzymatic activity
example: NAD and NADP
b. Activators are inorganic iorns which alters the spatial configuration of the enzyme for proper
substrate binding
-example: calcium, zinc, chloride, magnesium and potassium
c. Metalloenzymes are inorganic ion attached to a molecule
example: catalase and cytochrome oxldase
-

4. Inhibitors
Enzymatic reactions may not progress if an inhibitor interferes with the reaction.
a. Competitive Inhibitor
ftphysically binds to the active site of an enzyme -
both the substrate and inhibitor
for the same active site of the compete
enzyme.
The inhibrtion is reversible when the substrate concentration is
concentration of the inhibitor.
significantly higher than the
The effect of the inhibitor can be counteracted by
adding excess substrate to bind the
enzyme.
Dilution of serum results to reduction in the concentration of this
the rate of reaction.
inhibitor, thus increasing
It has the ability to ater the apparent Michaelis-Menten constant
(Km).

126
b. Non-Competitive Inhlbltor
t does not compete wth the substrate but look for areas other than the active site.
The substrate and inhibltor (commonly metallic ion) may bind an enzyme
simultaneously.
Because the Inhibitor binds the enzyme independently from the substrate, increasing
o substrate concentration does not reverse the inhibtion.
The presence of the inhibitor when it is bound to the enzyme, slows the rate of the reaction.

c. Uncompetitive Inhibitor
the inhibltor binds to the enzyme-substrate (ES) complex.
increasing the substrate concentration resuts in more ES complexes to which the inhibitor binds
and thereby Increases the inhibition.
Increasing substrate concentration results to increase inhibition.

5. Isoenzymes
These are enzymes (polypeptide chains) having the same catalytic reactions but slightly different
molecular structures-various forms accur because of differences in the amino acid sequence of
enzymes.
The importance of the total enzyme activity is enhanced by fractionating the isoenzymes.

6. Temperature
Enzymes are active at 25C, 30*C, or 37"C.
37C is the optimum temperature for enzymatic activity.
Increasing temperature usually increases the rate of a chemical reaction by increasing the
movement of molecules.
The rate of denaturation increases as the temperature increases, and is usually significant at
40°Cto 50°C.
60-65 C may result to inactivation of enzymes
Temperature Coefficient (Q10means for everý 10"C increase in temperature, there will be a
two-fold increase Inenzyme activity.

7. Hydrogen lon Concentration or pH

Most physiologic reactions occur in the pH range of 7to8
Extreme pH level may denature an enzyme or influence its ionic state resulting in structural
change or change In the charge of amino acld residue In the active site.

8. Storage
Low temperatures (refrigeration/freezing) render enzymes reversibly inactive.
Repeated freezing and thawing tends to denature protelins and should be avolded,
-20 C ideal temperature for preservation ofenzymes (longer period oftime)
2 to 8 ideal storage temperature for substrates and coenzymes
22'C or room temperature ldeal for storage of LDH (LD4 and LD5)

9. Hemolysis mostly increases enzyme concentration

10. Lactescense or milky specimen - decreases enzyme concentration

127
 EnzymeNomencature
enzyme nomenclature, the Enzyme Commission (EC) adepted dassifcation
Tosystem
standardize
in 1961, and revised the standards in 1972 and 1978.
a

Enzymes are dassifed according to their biochemical functions, indicating substrate and cass of
reaction catalyzed, and are designated by fndividual identfication numbers.
The first digit, places the enzyme in its clasifications (six classificatons)
The second andthird digits, represents the subclass to which the enzyme is assigned.
The final andfourth number/s, is a serial number that is specific to each enzyme
in a subclass.
1. Acid Phosphatase E.C. 3.1.3.2
2. Alkaline Phosphatase E.C. 3.1.3.1
3. Amylase E.C. 3.2.1.1
4. Alanine Aminotransferase E.C. 2.6.1.2
5. Aspartate Aminotransferase E.C. 2.6.1.1
6. Aldolase E.C. 4.1.2.13
7. Angiotensin Converting Enzyme E.C. 3.4.15.1
8. Creatine Kinase E.C. 2.7.3.2.
9. True/Acetyl Cholinesterase E.C. 3.1.1.7
Pseudocholinesterase E.C. 3.1.1.8
10. Gamma Glutamyl Transferase E.C. 2.3.2.2
11. G-6-PD EC. 1.1.1.49
12. Lipase E.C. 3.1.1.3
13. Lactic Dehydrogenase E.C. 1.1.1.27

14.5'Nucleotidase E.C. 3.1.3.5

Classificaton of Enzyme
Class Function Example
1. Oxidoreductases Catalyze theremoval.oraddition of CO, LDH, MDH, ICD,G-6-PD
electrons(redox reaction)
2.Transferases Catalyze the transfer of a chemical group other CK, AST, ALT, OCT
than hydrogen from onesubstrate to another.
Catalyze hydrolysis or spliting of a bond by the Esterases
3.Hydroloses
addition of water(hydrolytic reactions). ACP, ALP, CHS, LPS
Peptidases
Trypsin, Pepsin, LAP
Glycosidase
AMS, Galactosidases
4.Lyases Catalyze removal of groups fromsubstrates Glutamate decarboxylase,
without hydrolysis. Theproduct contains double pyruvate decarboxylase,
bonds tryptophan decarboxylase and
aldolase
5.Isomeroses Catalyze the intramolecular arrangement ofthe Glucose phosphate isonerase
and ribose phosphate isomerase
Substratecompound
Catalyze the joining of two substrate Synthase
6.Ligases
molecules, coupled withbreaking of the
PYrophosphate bondin ATP orsimilar compound.

128
Co-Cytochrome Oxidase
MD Malate Dehydrogenase
ICD- Isocitrate Dehydrogenase
LAP-Leudine Aminopeptidase

General Propertiea.ot Enzymes

Each enzyme contains:
substrate interacts with particularcharged
1. Actlve site i s a water-freecavity, where the
structure
amino acid residues; is a 3-dimensional protein
bind regulator molecules
2. Allosterlc site is a cavity otherthan the active site; may

Notes to Remember. amino acid (primary structure), forming

protein, each enzyme is composed of specific
a
As a and resutts In
chalns (secondary structure), which then folds (tertiary structure)
polypeptide
structural cavities.
generally on the integrity of its
structure.
The catalytic actvity of an enzyme molecule depends its contents.
occur until an active site discharges
When all sttes are filled, no further binding can

Teminologies a prosthetic group.

is called
W h e n bound tightly to the enzyme, the coenzyme

Apoenzymelenzyme portion)and coenzyme

forms acomplete and active system known as
holoenzyme (apoenzyme+ prosthetic group =holoenzyme). called a
secreted from the organ of production is
Digestive enzymes in its inactive form originally
proenzyme or zymogen.

Enzyme Theory
1. Emil Fisher's/Lock and Key Theory
must fit into the lock (enzyme).
It is based on the premise that the shape of the key (substrate)

2. Kochland's/Induced Fit Theory

I t is based on the substrate binding to
the active site of the enzyme.

Enzyme Knetics if the free energy or available kinetic energy is

A chemical reaction may ocur spontaneously
higher for the substrate than the product. the activation energy level that the
Enzymes catalyze physiologic reactions by lowering
substrate must reach for the reaction to occur.
substrate and catalyzes only one reaction is known as
An enzyme combines with only one
absolute specificty.
a chemical group is called group specificity.
Enzymes combine with all the subsrates in
bonds is knon as bond specificity.
Enzymes reacting with specific chemical

Enzymatic Reaction
1.Zero-order reaction the reaction rate depends only on enzyme concentration
2. First-order reactdon the reaction rate is directly proportional to substrate concentration

129
Tomeasure theextentofenzymaticreactions,2 general methods may be used:
1. for a
Fixed-time the reactants are combined; the reaction proceeds designated time; the reaction is
stopped and measurement is made
made
2. Continuous monitoring/kinetic assay multiple measurements of changed in absorbanceare
during the reaction; it is preferred than fixed-tine

Units for Egoresing EnzmaticActhdtyi

1. Internatlonal Unlt (U or U) 1 micromole of substrate/minute
2. Katal Unt (KU) -1 mole ofsubstrate/second
Enzymes are quantifed based on their activity rather than absolute values.
The unts used to report enzyme levels are activity units.
The definition for acthity unit must consider change in pH, temperature, substrate, cte

EnzymeAcivity
Enzymes are measured in terms of
1. Change in the substrate concentration.
2. Change in the product concentration.
3. Change in coenzyme concentration.

Causes of Elevated Plasma Enzvme Levels

1. Impaired removaB of enzyme from plasma.
2. Increased permeability of cell membrane.
3. Increased in the number of cells or the production of cells.
4. Increased in the normal cell turnover.
5. Decreased clearance of enzymes from the circulation.
6. Tissue necrosis and degeneration death ofenzyme-containing cells.

Notes toAn
Remember
enzyme accelerates the rate of reaction, reducing the time required to reach equilibrium.
A constant change in absorbance per unit time accurs only when the rate of the reaction is zero
order.
Asubstrate concentration of >99x Km is needed to achieve zero-order reaction.
Itis easler to measure small increases in product than to measure small decreases in a large
amount of substrate.
An enzyme do not alter the free energy or direction of a reaction, but it alters the energy of
acttvation by forming a metastable intermedlate, the ES complex.
Most enzymes are measured by monitoring the rate of absorbance charige (kinetic assay) at
340nm as NADH is redured or consumed, and it allows direct reporting elther by IU or KU.
in first-order reaction, the enzymes ere used as reagents to measure a specific analyte.
in nonkdnetic assay, absorbance is made at 10-second intervals for 100 seconds.
Endpoint measurement determines the concentration of substrate or product at specific time
after addition of the sample (bedside glucose testing using strips).
Enzyme actvity measurements may not be accurate if enzyme inhibitors are present, essential
cofactors are not included in the assay, and improper specimen storage.

130
MAJOR CLINICAL ENZYMES:

LPHOSPHATASES
A. Allkaline Phosphate/Allaline Orthophosphoric Monoester Phosphohydrolase
I t is a nonspecific enzyme capable of reacting with many different substrates.
ester with the
It functions to liberate inorganic phosphate from an organic phosphate
concomitant production of an alcohol.
Major tissue sources: liver, bone, placenta and intestinal
Reference value: 30-90 UL

Malor isoenzymes:
1. Liver ALP
2. Bone ALP
3. Placental ALP
4. Intestinal ALP
derived from Iliver and bone (osteoblasts).
healthy sera, alkaline phosphatase (ALP) levels
are
in
and is normally elevated in children
Bone isoenzyme increases due to osteoblastic activity
during periods of growth and in adults older than age years (geriatric).
50
AB.
Placental ALP is also lower in pregnant women of groups A and
detected between 16-20 weeks of
In normal pregnancy, increased ALP activity can be

pregnancy.
The presence of intestinal ALP isoenzyme in serum depends on the blood group (secretor gene
intestinal ALP after
and H substance) the individual B or O blood group increases
of
consumption of a fatty meal.
in A and AB individuals because of
ALP is also higher in individuals of group B and O than
differences in intestinal ALP levels.
ALP and creatinine levels increase.
During perlod of growth and muscle development, serum

Diagnostic Significance
When total ALP levels are increased, it is the major liver fraction that is most frequently
elevated, especially in obstructive jaundice.
rate of secretion.
ALP is increased in obstructive jaundice due to greater
For bone disorders, highest elevations occur Paget's
in disease(osteitis deformans).
Bone ALP isoform, 81x, was detected in the serum
of dialysis patients.
B1x isoform is used to study low bone mineral disease (BMD) in patients with chronic kidney

disease.
of individuals with BMD of the hip, which is
B1x isofom is also increased in the serum
predominantly made up of trabecular bone.

Carcinoplacental AL cancers; bone ALP co-migrator; most

1. Regan ALP-is found in lung, breast, ovarian gynecological
and
heat stable ALP (65 Cfor 30 minutes); inhibited by phenylalanine reagent
2. Nagoo ALP-found in adenocarcinoma of the pancreas and bile duct, pleural cancer; variant of
Regan ALP; inhibited by L-leucine and phenylalanine.

131
 Methods:
1. Electrophoresis
Liver and bone ALPs are the mast anodal
Use of isoenzymes; intestinal ALP is the least anodal.
neuraminidase and wheat germ lectin improves separation of bone and liver ALPs.
High-resolution electrophoresis using polyacrylamidegel and isoelectric
resotving multlple bands of ALP. focusing are capable of
CICcho:P bL cathoo
2. Heat
Fractionation/Stablity Test
I t is performed at
56°C for 10-15 minutes.
Placental ALP is the most heat stable; bone ALP is the
most heat labile.
Decreasing order of ALP heat stability: placental, intestinal, liver and bone
Ncat: PILB tobic
3. Chemical Inhibltion Test
This method uses different concentrations of
solutions.
phenylalanine, synthetic urea and levamisole
Placental and intestinal ALPs are inhibited
by phenylalanine reagent and 3M urea inhibits bone
ALP.
Levamisole reagent inhibits liver and bone
ALP
4. Bowers and Me Comb
(Szasz modification)
Is considered as the most
specific method; IFCC recommended method.
It is a
continous-monitoring technique which requires a pH environment of 10.15 and should be read
405nm.
ALP
p-nitrophenylphosphate p-nitrophenol+ phosphate ion

Summary of ALP Methods

Methods Substrate EndProducts
1. Bodansky
2. Shinoworo Beta-glyceroPO4
3. Jones InorganicP04 +
4. Reinha Glycerol
5. King and Armstron2g Phenylphosphate Phenol
6. BessyLowry & Brock p-nitro phenyl PO« (PNPP) p-nitrophenolor yellow
7. Bowers and McComb
nitrophenoxide ion
PNPP
p-nitrophenolor yellow
8. Huggins and Talalay nitrophenoxide ion
Phenolphthalein diphosphate Phenolphthalein red
9. Moss Alpha naphthol PO Alpha-naphtol
10. Klein, Babson &Read Buffered phenolphthalein PO4 Free phenolphthalein

Notes to Remember.
Zinc is a component of ALP, and magnesium is the enzyme activator.
ingestion of food leads to release of intestinal ALP into lymphatic fluld, and may transiently
Increase plasma levels of ALP
Hemolysis and diet (fatty meals)-sources of analytical errors; elevated serum ALP.
ALP is sensitive if stored at low temperature (4°C), leads to increased serum level.

132
 buffer
ALP is inhibited by phosphorus - the addition of 2-amino-2-methyl-1-propanol (AMP)
binds phosphorus under Bowers-McComb method.
Decreased ALP Is seen in zinc deficiency.
transfusion or cardiopulmonary bypass.
Transient low serum AuP may occur after blood
Prolonged low levels of ALP Occur in hypophosphatasla.
marker in serum and cerebrospinal fluid
Placental alkaline phosphatase (PLAP) Is a useful tumor
PLAP are of diagnostic value in differentiating
(CSF) for most germ cell tumors- CSF levels of
cell tumor.
whether a tumor in the pineal body is a pinealoma or a germ

Increased AL2
1. Osteltis deformans 5.Osteoblastic bone tumors-osteosarcoma
2. Obstructive jaundice 6. Sprue
3. Osteomalacia 7. Hyperparathyroidism
4. Rickets 8. Hepatitis and cirrhosis (slight increased)

B. Add Phosphatasef Acdd Orthophosphoric Monoester Phosphohydrolase

is active at pH 5.0.
It catalyzes the same reaction made by ALP, except that it
fluid in the sample.
ACP activity > 50 IU/L indicates the presence of seminal
bone
(major source), RBCs, platelets, liver and
TisSue sources: prostate
Reference Value: Male 2.5-11.7 U/L (Total ACP)
=

0-3.5 ng/mL (Prostatic ACP)

Diagnostic Significance:
For detection of prostaticadenocarcinoma.
faster than PSA, and plasma levels
After surgicai treatment of prostate cancer, ACP Jevels falls
are expected to be undetectable following complete
removal of tumor.
It alsc usefu! in forensic clinical chemistry, in the investigation of rape cases vaginal washings
are examined for seminal fluid-ACP activity,
which can persists for up to 4 days.

tract obstruction, acute urinary retention, extensive

Other causes of increased serum ACP; urinary
infarction/ischemia, and prostatic manipulations (needle
prostatic massage, prostatic inflammation,
biopsy and ystoscopy)

Summary of ACP Methods

Method Substrate End Products
1. Gutman and Gutmon Phenyl PO4 Inorganic PO
2. Shinowara PNPP p-nitrophenol
3. Babson, Read &Phillips Alpha naphthyl PO4 Alpha-naphtol
4. Roy ond Hlman M4 PLCekc, iIThymolphthalein Free thymolpthalein
MoroPO

Notes toSerum
Remember.
sample must be free from hemolysis.
Thymolphthalein monophosphate is the specific substrate; substrate of choice for quantitative
endpoint reaction.
a-naphthy! POs is preferred for continuous monitoring methods.
Normal men and women up to about age 55 have the same reference ranges for ACP.

PHOMACKP Torvott 133

 Serum ACP decreases within 1 to 2 hours if left at room
f not assayed immediately, serum should be
temperature.
frozen or acidified to a pH lower than 6.5. With
acidification, ACP is stable for 2 days at room temperature.
Fluid collected from the vagina on a cotton swab will
give a positive test for ACP if semen is
present, provided a stabilizing fluid with an acidic pH is used.
Prostatic ACP is Inhibited by 20mM L-tartrate ions while 1mM
cupric sulfate and 2%
formaldehyde ions inhibit red cel ACP.
Tartrate-resistant acid phosphatase (TRAP) is present in certain chronic leukemias and some
fymphomas, most notablyin hairy cell leukemia.
Elevated serum bilirubin causes falsely lowwalues for TRAP
activity, but not for total ACP.
Increased ACP (with Metastatic Bone Involvement
1. Prostatic carcinoma
2. Breast, Iung and thyrold carcinoma
3. Gaucher's disease
4. Niemann Pick Disease

Notes to Remember.
Increased ACP is observed in
thrombocytopenia.
ACP activity in the bones is associated with the osteoclasts.
Prostatic acid phosphatase (PAP) is used
together with prostate specific antigen (PSA) to
monitor recurrence of prostate cancer.
PSA is more sensitive than PAP in detecting
stages A ar1d B prostatic cancer.

. TRANSFERASES/TRANSAMINASES
A Aspartate Aminotransferase (AST)
t i s involved in the transfer
of an amino group between aspartate and u-keto acids with the
formation of oxaloacetate and glutamate.
It has 2 isoenzyme fractlons,
cytoplasm and mitochondrial ASTs- the cytoplasmic isoenzyme is
the predominant form in serum.
Major tissue source: cardiac tissue, liver and skeletal muscle
Other sources: kidney, pancreas and RBC
Reference value:5-37 U/L

diagnosticIn Slantficance:
the evaluation of myocardial infarction, hepatocellular disorders and skeletal muscle
involvement.
In acute myocardial infarction (AMI), AST levels begin to rise 6-8 hours, peak at 24 hours and
normalize within 5 days.
It is released to a greater degree in chronic disorders of the
liver with progressive
AST is used for monitoring therapy with damage.
potentialy hepatotoxic drugs; a result more than three
times the upper border of normal should signal cessation of
therapy.

134
 Method:
Karmen Method pH 7.5; 340nm
I t uses malate dehydrogenase (MD) and monitors the change in absorbance at 340 nm.
AST
Aspartate+a-ketoglutarate Oxaloacetate Glutamate
MD
Oxaloacetate + NADH H* Malate +NAD+

B. Alanine Aminotransferase (ALT)

t has enzymatic activity similar to AST.
t catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the formation
of glutamate and pyruvate.
The highest concentration is in the liver; more liver-specific than AST.
Major tissue source: liver
Other sources: kidney, pancreas, RBC, heart, skeletal muscles, lungs
Reference value: 6-37 U/L

Diagnostis Sionlfkance:
It is significant in the evaluation of hepatic disorders - markedly increased concentration in

acute inffammatory conditions than AST.

It monitors the course of liver (like hepatitis) treatment and the effects of drug therapy.
ALT levels are used to screen blood donors.
ALT measurement is a more sensitive and specific screening test for posttransfusion hepatitis or
occupational toxic exposure compared to AST.

Method:
Aminotransferases are present in human plasma, bile, CSF and saliva.
Aminotransferases require pyridoxal phosphate (vitamin Bs) as coenzyme (prosthetic group).
Hemolysis should be avoided because it increases AST 10x. 6(t) NUrhcnathy vcno
Heparin may inhibit the activity of AST (but not all methods). GTREPTO COCCI
PyndoxO phtsplatt
Haemophilus ( 5a1euj1iS
Coupled Enzymatic Reaction: using pH 7.5; reading at 340nm
ALT
P y r u v a t e + Glutamate
Alanine+ a-ketoglutarate
LD
Pyruvate+ NADH * H+ Lactate + NAD+

AST/SGOOT ALT/SGPT
Mojor Orgon offected Heat Liver
Substrate Aspartic Aipha Alanine Alpha
Ketoglutarlc Acid Ketoglutaric Acid
End products Glutamic Acid+ Glutamic Acld+
Oxaloacetic Acid Pyruvic Acid
Color developer 2.4 DNPH 2,4DNPH
Color intensifier 0.4N NaOH 0.4N NaOH
Methods Reitman and Frankel Reitman and Frankel

135
 uncreased Transferase
1.Toxic hepatitis 7. Hepatic cancer
sAcute Myocardial Infarction -AST 8. Reye's syndrome
3. Wolff-Parkinson White
4. Trichinosis AST-
Syndrome 9. Viral hepatitis
10. Muscular
5. Chronic alcoholism Dystrophy -AST
11. Acute
NDermatomyositis AST pancreatitis - AST
LMP
Notes to Remember
The highest elevations Vif
Severe viral or toxic
of transferase is seen in
acute,hepatitis.
hepatitis may produce elevations of
the De Ritis ratio (ALT:AST) Is> 1.0. transferase up to 20x the normal limits.
In acute
hepatitis,
Moderate elevation of transferase
in chronic
mononucleosis. hepatitis, hepatic cancer and infectious
Slightly increased in hepatic cirrhosis, alcoholic
ALT is slightly increased in obstructive jaundice hepatitis
but
and obstructive
jaundice.
With most forms of acute markedly increased in necrotic jaundice.
initially because of the higher hepatocellular injury, such as hepatitis, AST will be
higher than ALT
ongoing damage occurs, ALT willactivity
of AST in
hepatocytes.
become higher than
Within 24-48 hours,
particularly f
In end-stage
cirrhosis, the levels of both enzymes AST, based on its longer half-life.
the result of massive
tissue generally are not elevated and may be iow as
destruction.

I. AMYLASE! ALPHA-14
t GLUCAN4-GLUCOHYDROLASE
catalyzes the breakdown of (AMS)
physiologic digestion of starch.starch glycogen a n important enzyme in the
and
It is the smallest
enzyme in size
the renal glomerulus and also (with MW of 50,000
a
to 55,000 daltons)- normally filtered by
appears in the urine.
It is the earliest pancreatic marker.
P3 is the most
predominant pancreatic amylase isoenzyme in acute
Isoenzymes: S-type (ptyalin) and pancreatitis.
Major Tissue source: acinar cells ofP-type.amylgpsin) both present in normal
the pancrëas and the salivary glands healthy sera
Other tissue source: adipose
tissue, fallopian tubes, small intestine and
Normal serum contains both skeletal muscles
salivary and pancreatic AMS.
Reference volue: 60-180 SU/dL (somogyi units)
95-290 U/L

Diagnostic Sianificance:
In acute
pancreatitis (AP), AMS levels rise 2-12
hours after onset of
normalize within 3-5 days. attack, peak at24 hours, and
In AP, increased AMS
blood levels are accompanied by
AMS in urine remains elevated for increased urinary excretion.
up to 7 days.
Salivary gland inflammation (parotitis) due to mumps can also
causing an elevated serum AMS. release AMS into the circulation
In renal failure in the
absence of AP, increased plasma AMS is
AMS. accompanied by decreased urine

136
Methesa:
Heparin may inhiblt the activity of AMS (using some, but not all, methods).
Triglycerides may Inhibltserum AMS actvity.
Samples with high activity of AMS should be diluted wlth NaCl to prevent inacthivation.
Many endogenous inhibtors of AMS, such as wheat germ are present In serum.
The administration of morphine and other oplates for pain relief before blood sampling will lead
to falsely elevated serum AMS levels.

Substrate for all the methods: Starch

1. Saccharogenic
I t is the classic reference method expressed in Somogyi units.
I t measures the amount of reducing sugars produced by the hydrolysisof starch by the usual
giucose methods.

2. Amyloclastic
t measures amylase actlvity by following the decreases in substrateconcentratiori (degradation
of starch).

3.Chromogenic
t measures amylase activity by the increase in color intensity of the soluble dye-substrate
soBution produced in the reaction.

4. Coupled-enzyme
It measures amylase activity by a continuous-monitoring technique.
AMS
Matopentose Maltrotriose maltose

gtucosidase
Maltrotriose maltose 5-glucose
hexokinase
5-glucose +5 ATP 5-glucose-6-phosphate +5 ADP
G-6-PD
5-glucose-6-phosphate +5 NAD 5,6-phosphogBuconolactone +5 NADH

Notes to Remember
>Three-fold amylase increase with normal 24 hours urine amylase repeat serum AMS after
polyethylene glycol precipitation.
Salivary AMS is inhibited by wheat germ lectin.
Enzyme molecules are too large to pass through the healthy glomerulus of the kldneys, thus,
urinary excretion is not a major route for elimination except amylase.
Macroamylasemia AMS+ immunoglobulin (to large to befiltered across the giomerulus)
Normal Amylase/creatnine ratio: 1%-4% (0.01-0.04)
A:C ratio (AP) >4% ( up to 15%)

137
 Formula for A/C ratio:
Urine amylase Serum creatinine x 100
Serum amylase Urine creatinine
Increased Serum Amylaose
1. Acute pancreatitis
2. Ectopic pregnancy
3. Peptic ulcers
4. Alcoholism
5. Mumps

V. LIPASE
Latc Markcr t hucok is an (LPSTRIACYLGLYCEROL
enzyme thathydrolyzes the
ACYLHYDROLASE
ester inkages of fats to
It
catalyzes partial hydrolysis of dietary TAG produce alcohol and fatty acid.
in the intestine to the
intermediate, with the production of long chain 2-monoglyceride
It is the
most fatty acids.
specific
renal disorders.
pancreatic marker secreted exclusively in the
pancreas; not affected by
Plasma concentrations are
normal in conditions of
Major tissue source: Pancreas salivary gland involvement.
Reference value: 0-1.0 U/mL
Diagnostic
In
Sianificance
acute
pancreatitis (AP), LPS levels rise 6 hours after onset
elevated for 7 days, and normalize in
8-14 days.
of attack, peak at 24 hours, remains
In chronic pancreatitis, acinar cell degradation ocurs
production. resulting in loss of
amylase and lipase
Methode:
It uses olive oil as the substrate because other
esterases can
hydrolyze
digycerides. TAG andsynthetic
Addition of colipase (protein secreted
by the pancreas) and blie salts will make
sensitve and specific for AP detection. assay more
Hemoglobin inhibits the activity of LPS leading to
Triolein (more pure form of TAG) is used also as a falsely
low values.
substrate for LPS assay.
1. Chery Crandal (reference method
Principle: Hydrolysis of olive oil after incubation for 24 hours at 37"C
Substrate: S0% olive oil and titration of fatty acids
using NaOH.
End product: Fatty acid
LPS
Triglyceride +2 H0 2-monoglyceride + 2 fatty acids
(olive oil)

2. Tietz and Fiereck

3. Peroxidase coupling most commonly used method; does not use 50% olive ol.
PANCAERTIG 1AKKGA
mylaye
Lipayt
CHTMOTKYPStN 138
CLASTR
 V. LACTATE DEHYDROGENASE (LD)
t is an enzyme that catalyzes the interconversion of lactic and pyruvic acids.
t ls a zinc-containing enzyme that is part of the glycolytic pathway and Is found in virtually all
cells in the body.
It is a hydrogen-transfer enzyme that uses the coenzyme nicotinamide dinucleotide (NAD+).
oMo It is a tetrameric molecule containing four subunits of two possible forms (H and M).
In plasma, the majority of LD comes from breakdown of erythrocytes and platelets, with varying
contributionsfrom other organs.
Tissue sources: heart, RBCS, kidneys (LD-1 & LD-2); lungs, pancreas, spleen (LD-3); skeletal
muscles, liver, intestine (LD-4& LD-5)
Reference value: 100-225 U/L (Forward reaction)
80-280 U/L (Reverse reaction)

Dlagnestic Slanifcance:
Highest serum levels are seen in pernicious anemia and hemolytic disorders.
In AMI, LD levels begin to rise within 12-24 hours, peak levels within 48-72 hours and remains
iaLK pu ricyelevated for 10-14 days.
Hepatic carcinoma and toxic hepatitis will have 10-fold increased.
Viral hepatitis and cirrthosis would give LD slightly increased values (2-3x URL).
LD-1> LD-2 also known as the "fipped pattern" is seen in myocardial infarction and hemolytic
anemia, rCnai nfarction
LD2: LD1 ratio rises to >0.75 and often exceeds 1.0, which occurs only about 36 hours after the
onset of symptoms.
LD-2, LD-3, LD4 LDcancer markers (predominanthly LD-3); acute leukemia, germ cell
tumors, breast and lung cancers
LD-5 is moderately increased in acute viral hepatitis and cirrhosis and markedly increased in
hepatic carcnoma and toxic hepatitis.
t will be clinically significant if separated into isoenzyme fractions.
An elevated total LD is a nonspecific resut because of its presence from several tissues.

LDIsoenzyme as a Peroentage of Toal LD:

LD-1 17-27% Hcort.ked cM Renal orc ASt udol, let sidkie
LD-2 27-37%
LD-3 18-25% :lung. spucn PonCrDS, wec

LD-4 3-8% tivtt. musuk. scum,*in

LD-5 0-5%
RBCs and cardiac tissues contain high levels of LD-1.
LD1 is relatively abundant in cardiac muscle, whereas LDS is more abundant in skeletal muscle.
LD-1 is not found in the steletal muscles and Iiver; LD-2 is never found in the skeletal muscles.
LD-2 is the major isoenzyme in the sera of healthy persons.
LD-2 greater than LD-1 is seen in healthy sera.
LD-S has undetectable level in the heart, RBCs and renal cortex.
LO-6 represents the alcohol dehydrogenase enzyme; 6h band in electrophoresis; elevated in
drug hepatoxicity and obstructive jaundice; it is responsible for the metabolic conversion of
methanol and ethylene giycol to toxic compounds; present In patients with arteriosclerotic
failure.
Another form of LD composed of four C subunits is found in spermatozoa and in semen but has
never been detected in serum, even in individuals with seminoma.

139
 Methods:
Lactate is a more specific
substrate compared to pyruvate.
LD-1 prefers the forward
LD is stable at room reaction, whereas LD-5 prefers the reverse reaction.
temperature for 48 hours.
1. Wacker Method
(forward/direct reaction)- reaction is at pH 8.8
It is the most
LD commonly used method because it produces
affected by product inhibition. a positlve rate (NADH) and not
LD
Lactate+NAD Pyruvate+ NADH @340nm
2. Wrobleuski La Due
LDs
(reverse/indirect
t is about 2x faster reaction- reaction is at pH 7.2
It is the
as the forward reaction.
preferred method for dry slide technology
t uses a less
costly cofactor and it has a smaller specimen volume
LD requirement.
Pyruvate+ NADH Lactate +NAD
3. Wrobleuski Cabaud
4. Berger Broida

Notes to Remember
Red blood cells contain
very high levels of LD.
LD can use other substrate in addition to
lactate sucth as a-hydroxybutyrate.
a-hydroxybutyrate dehydrogenase (a-HBD) represents the LD-1 activity.
a-hydroxybutyrate actlvity is elevated in conditions in which both the LD-1 and LD-2
increased. are
LD activity in pleural fluids is useful
for differentiating transudates (low
Total LD increases temporarily after biood transfusion LD) from exudates (high LD).
but returns to baseline within24 hours.
Decreased values of LD are observed when
samples are frozen (LD-5 is cold-labile), therefore
samples should be processed within 24 hours after collection and stored at 25°C.

Increased LDH
1 Anemias pernicious, hemolytic, megaloblastic
2. Myocardial infarction
3. Leukemia
4. Renal infarction
5. Hepatitis and hepatic cancer
6. Muscular dystrophy
7. Delirium tremens
8. Malignancy
9. Pneumoeystis jerovecii pneumonia

140
VI. CREATINE KINASEJATP-CREATINE-N-PHOSPHOTRANSFERASE (CK)
I t catalyzes the transfer of a phosphate group between creatine phosphate and adenosine
diphosphate.
tis involved in the storage of high-energy creatine P0, in the muscles.
t is a dimeric molecule with small molecular size, composed of a pair of two different
monomers called M and B.
t is found in small amounts throughout the body, but is found in high concentrations only in
muscle and brain, although CK from brain virtually never crosses the blood-brain barrier to reach
plasma.
Major tissue sources: brain tissue, smooth and skeletal muscles and cardiac muscles
Reference voalues: Male = 15-160 U/L
Female 15-130 U/L
CK-MB <6% of total CK

(so in sera.
Isoenzmes: CK-B8 (brain type), CK-MB (hybrid type), CK-MM (muscle type) ajor eniry me

CK-1 Is the most anodal and lable isoenzyme; CK-3 is the least anodal.
CK-88 is the dominant isoenzyme of CK found in brain, intestine, and smooth muscle.
Serum of adults rarely contains CK-BB of brain origin due to its high molecular size; it may be
normaly present in neonatal sera.
Cardiac tissues contain significant amount of CK-MB (20%) - myocardium is the only tissue from

which CK-MB enters the serum in significant quantities.

CK-MB in serum of healthy person is < 5 ug/L
CK-MM is both abundantly present in the cardiac and skeletal muscles.
in the sera of healthy persons, CK-MM is the major isoenzyme (94-100%).
Physicalily well-trained Individuals tend to have elevated baseline levels of total Ck.
Direct muscle trauma, as seen in contact sports, surgery, strenuous exercise, and intramuscular
injection, are common causes of mild elevations of serum CK (up to about five to six times
reference limits).
Intramuscularinjections are known to increase CK(<5x URL).
Bedridden patients may have decreased CK activity.

Diagnostic Signficance
I t is a very sensitive indicator of acute myocardial infarction (AMI) and Duchenne disorder.
Highest elevation of total CK is seen in Duchenne's muscular dystrophy (50x URL).
CK-MB is found mainly in myocardial tissue- it is used as a serodiagnostic test for AM
Demonstration of elevated levels of CK-MB, 2 6% of the tatal CK, is considered the most specific
indicator of myocardial damage, particularly AMI.
Following AMI, the Ck-MB levels begin to rise within 4-8 hours, peak at 12-24 hours and
normalize within 48-72 hours.
CK-MB is not elevated In angina.
Injury to both cardiac and skeletal muscle accounts for the majority of CK-MM elevations.
Total CK is markedly elevated after trauma to skeletal muscle from crush injury, convulsions,
tetany, surgical incision or intramuscular injections.
DuCHTNNE MUSU M 0YSTKOPy
CK-MM 1 1tnyne skectul 4ker)

141
 Methods:
1.
Tanzer-Glbarg AssayCPK(forward/direct method)- pH 9.0; 340nm
Creatine+ATP Creatine P04 +ADP
PK
ADP+phosphoenolpyruvate
LD Pyruvate +ATP
Pyruvate+NADH Lactate+ NAD
2. Olver-Rosalki ( reverse/lndirect
method)- most commonly used method; faster reaction; pH
CPK 6.8; 340nm
Creatine PO4+ ADP Creatine + ATP
HK
ATP+glucose ADP+glucose-6-PO
G-6-PO
Glucose-6-POa+ NADP 6-phosphogluconate+ NADPH
HK- hexokinsse

Notes to Remember.
Adenylate kinase (AK) released after
red cell iysis interferes with CK assay
hemolysis of > 320 mg/L. *
kd ds A ontan & hoycver
particularly with
Liver cells and RBC do not contain
AK
nierrcres
CK.
To increase both the
sensitivity and the
specificity CK-MB in the diagnosis of acute
of
been found necessary to
perform seria! determinations of MB fraction (at 3- to 4-hourAMI, has
it
over a 12-to 16-hour intervals
period) that show a progressive rise that reachesa
to low levels. peak, followed by a fall
Adenosine monophosphate (AMP) is added to the reverse method to
present in the serum from hemolysis-AK hydrolyzes ADP. inhibit AK which may be
N-acetyicysteine is added to CK reagent to activate the enzyme (aside from
reversed the inhibition of oxidized Mg*) and partialy
sulfhydryl groups.
Imidazole serves as a buffer; urate and cystine are potent CK inhibitors.
CK is ight and pH
sensitive; it is also lost with excessive storage.
deland's reagent and
CK mass units
glutathione-partially restore lost activity of CK.
assay are more sensitive than electrophoresis, but
reference method for CK. electrophoresis is still the

CK relative index (CKI)

I t is an expression of the
percentage of the total CK that is attributed to CK-MB. This is
computed to know possible release of CK-MB from noncardiac tissues when total
high. CK is very
CKI
CK-MB ug/or lL x 100
Total CK IU/L

increased
1.
Creatine kinase
Duchenne's muscular dystrophy 7. Cerebral vascular accident
2. Myocardial Infarction (occasional)
3. Hypothyroidism
8. Rocky Mountain Spotted Fever -CK-MS
9. Carbon monoxide
4. Pulmonary infarction poisoning
5. Reye's syndrome
6. Strenous exercise and intramuscular injections

142
VII. ALDOLASEFRUCTOSE 1,6-DIPHOSPHATE ALDOLASE
It s a gycolytic enzyme that splits fructose-1,6-diphosphate Into two triose phosphate
molecules in the metabolism of glucose.
Increased: skeletal muscle disease, leukemia, hemolytic anemia and hepatic cancer
Iscenzymes CK A1A.AS. Ads ke A. y 44t
Aldolase A Skeletal muscles
Aldolase B WBC, Iver, kidney
Aldolase C Brain Tissue

BKAINALdcl9e
QTHER CLINICALLY STIGNIEICANT ENZYMES;
1.5' Nucleotidase (5'N)
it is a phosphoric monoester hydrolase; predominantly secreted from the liver.
It is a marker for hepatobiliary diseas and infiltrative lesions of the liver.
Method: Dixon & Purdon, Campbell, Belfield & Goldbeng
Reference value: 0-1.6 units
Fo hepoti junch¢e
2. Gamma Glutamyl Transamine Peptidase/Transferase (GGT)
I t catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at
Oneys
gammy carboxyl group.
t is located in the canaliculi of the hepatic cells and particularly in the epithelial cells lining the
sm biliary ductules; also in the kidney, prostate and pancreas.
I t affects the cell membrane and microsomal fractions- elevated among individuals undergoing
warfarin, phenobarbital and phenytoin therapies.
Substrate: Y-glutamyl-p-nitroanilide
Method: Szass, Rosalki& Tarrow, Orlowski
Reference value: 5-30 U/L(F)/6-45 U/L (M)

Diagnostict isSignificance:
useful in differentiating the source of an increased ALP level.
It is elevated in all hepatobiliary disorders -biliarytract obstructions.
it is a sensitive indicator of aicoholism (occult alcoholism) most sensitive marker of acute
alcoholic hepattis.
t is useful in monitoring the effects of abstention from alcohol.
I t is also increased in pancreatitis and prostatic disorders
3. Pseudochotnesterase (PChE)
It is secreted by the liver-it reflects synthetic function rather than hepatocyte injury.
It catalyzes the removal of benzyl group from cocaine-it acts as a "antixenobiotic enzyme."
ts a marker for insecticide/pesticide poisoning (organophosphate poisoning)- low serum PChE.
Is used to monitor the effect of muscle relaxants (succinylcholine) after surgery.
t is involvedinthe metabolism of anticholinergic drugs.
PChE reflects acutetoxicity while AChE (true cholinesterase or choline esterase 1) in the red
blood cells better reflect chronic exposure.
Tissue source: lver, myocardium and pancreas
Decreased:acute hepatitis, cirrhosis, carcinoma metastaticto liver and malnutrition
Method: Eiman technique and potentiometric
Reference value: 0.5-1.3 pH units (plas1ma)

143
 4. Angiotensin-Converting Enzyme (ACE)
It is also known as
peptidyldipeptidase A or kininase ll; a hydrolase enzyme.
t converts angiotensin I to
angiotensin Il within the lungs.
t is a possible indicator of neuronal
dysfunction (Alzheimer's disease- CSF).
Is a critical target for
inhibitory drugs designedto lower blood pressure.
Tissue source: lungs, testes,
macrophages and
epitheloid cells
Diagnostic slgnificance:for the diagnosis and monitoring of sarcoidosis
Increased: sarcoidosis, multiple sclerosis, addison's disease, acute andchronic bronchitis, HIV
infection and leprosy
5. Ceruloplasmin CNLY enmone ntuty
I t is a copper-carrying protein and also an enzyme.
tis a marker for
Wilson's
disease (hepatolenticular disease).
6 Ornithine Carbamoyl Transferase (OCT)
It is a marker for hepatobiliary diseases.
7. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
I t functions to maintain NADPH in the reduced form in the
erythrocytes.
Itis an newborn screening marker
tis found in adrenal cortex, spleen, RBC and lymph nodes.
Deficiency of this enzyme can lead to drug-induced hemolytic anemia after taking primaquine,
an antimalarial drug.
Increased: myocardial infarction and megaloblastic anemia
Specimen: red cell hemolysate and serum
Reference value: 10-15 U/g hemoglobin or 1200-2000 mU/mL packed RBC

144
 ELECTROLYTES

These are ions capable of carrying an electric charge.

Fluid always contains equal numbers of cations and anions this balance of charges is referred
to as electroneutrality.
Dissociation of solutes into charged particles (ions) depends on the chemical composition
compound and on the concentration of other charged particles in the medium.
of the

Distibution
40-75% is the average water content of the human body (advanced age and obesity - decreased
values)
Extracellular fluid (ECF) is one third of the total body water (16 liters).
Intracellular fluid (ICF) Is two thirds of total body water (24 liters).
60% of the body's water is inside cells, and the rest is in the bloodstream or tissue fluids.
About 30 liters offluld
passes fram the blood to the tissue spaces daily.
Normal plasma is composed of 93% water and 7% solutes ( glucose, lipids, proteins, NPN, amino
acids, and ions).
Water content of plasma is 12% higher than that of whole blood.
Retentio of 3 liters of fluid in the tissues will result to edema.
Deficiency of vasopressin caused 10 to 20 liters of water excreted daily.
Sweat contains about 50 mmol/L of sodium and 5 mmol/l of potassium.
Salt content of the body is the maindeterminant of the extracellular volume.

Functiensgf Electobrtes
1. For volume and osmotic regulation.
2. For myocardial rhythm and contractility.
3. important cofactors in enzyme activation.
4. For the regulation of adenosine triphosphatase (ATPase) ion pumps.
5. For neuromascular excitability.
6. For the production and use of ATP from glucose.
7. Maintenance of acid-base balance.
8. Replication of DNA and the translation of mRNA.

I.SODIUM MATOR OSMOTIC PnCLE!

tisalso kaown as "natrium."

it is the major extracellular cation, hence the major contributor of osmolality (ECF is % of the
total body water).
It is the principal osmotic particle outside the cell.
Its plasma concentratlon depends greatly on the intake and excretion of water.
All confirmed serum sodium abnormalities must be followed up with urinalysis (including urine
sodium and urine osmoBality) on the patient, who should be fluid restricted.
Reference value: 135-145 mmol/t
Threshold critical vahue: 160 mmol/L hypernatremia)
120 mmol/L (hyponatremia)
CSF sodium: 136-150 mmol/L

145
 Homones AffectingSodium Levels:
1.Aldosterone balanM¢ Sodium. eccmo requahng normo
i t promotes absorption of sodium in the distal tubule.
It
promotes.sodium retentlon.and potassium excretion.
2. Atrial Natriuretic Factor (ANF)
t is an endogenous
antihypertensive agent secreted from the cardiac atria.
I t blocks aldosterone and renin
secretion, and inhibits the action of angiotensin I1 and
vasopressin.
It causes natriuresis. uriary i0S sodium

Hypenatremia;
1. Excess water loss
3. Increased water intake or retention
a. Diabetes insipidus
a. Hyperaldosteronism (Conn's disease)
b.Renal tubular disorder b. Sodium bicarbonate infusion
c. Prolonged diarrhea c. Increased oral or IV intake of NaCl
d. Profuse sweating d. Ingestion of sea water
e. Severe burns
f. Vomiting
8. Fever
DN Hp.0ta
h. Hyperventilation ICoge ghuGse
2. Decreased water Intake

Hyponatremia:
1. Increased sodlum loss
a. Diuretic use
b. Saline infusion
2. Increased water retention
a. Renal failure
b. Nephrotic syndrome
c. Aldosterone deficiency
d. Cancer
e. Syndrome of Inappropriate ADH Secretion (SIADH)
f. Hepatic cirrhosis
8.Primary polydipsia
h. CNS abnormaities- meningitis,
encephalitis, multiple sclerosis
i. Myxedema

Notes to Remember.
1. Hypernatremia
10Hypernatremia is defined as an increased sodium concetration in plasma
water, and is
generally diagnosed at serum sodium levels >145 mmol/L
Hypernatremia is caused by loss of water, gain of sodiurn, or both.
tusualy resutts from excessive water loss.
Perspiration and breathing would result to one liter water loss/day in adults.
A water deficity of 1-2% leads to a severe thirst.

146
 Serum levels of 150-160 mEq/L is indicative of moderate deficit of water;> 165 mEq/L is severe
water deflcit.
concentration was about 4 mmol/L higher than that
With rapid intake of water, venous sodium
of arterial blood.
reduced arterial serum sodium by about 8 mmol/L and
Drinking 20 ml/kg of water in 15 minutes
amount given by slow sipping did not produce
resulted in rapid water diuresis, but the same
water diuresis.
of hypothalamic disease.
Chronic hypernatremila in an alert patient is indicative
and hypernatremia.
Thirst is the major defense against hyperosmolality

2. Hyponatremia sodium
is defined as reduced plasma
Hyponatremia, the most common electrolyte disorder,
concentration to a value less than 135 mmol/L.
is less than 130 mmol/L
Generally, clinical concern arises when the concentration
concentrate the urine, resulting to
f r e n a l failure occurs, the kidneys ultimately fail to

hyponatremia. of sodium and water.

If urine sodium is than 20 mmol/day, there is ongoing renal loss
more
sodium decreases by 1.6 mmol/L-glucose
For every 100mg/dl increase in blood glucose, serum
to the ECF,diluting its electrolytes.
is osmotically active and induces flow of water from the cells
the ECF is a well-known cause of hyponatremia because,
HpooFumulation of glucose or mannitol in of water from the cells to the ECF, thus
glucose is osmotically active and induces diffusion
diluting its electrolytes.
In diabetes mellitus, sodium loss occurs with ketonuria.
Decreased serum sodium in SIADH is due to excess retention
of water in the collecting ducts.
inverse relatlonship of the two
Potassium deficiency also causes loss of sodium because of the
the tubules will conserve
ions in the renal tubules when serum potassium levels are low,
of the monovalent cation.
potassium and excrete the sodium in exchange for the loss
I n Barterr's syndrome, hyponatremia is notcorrected with fluid restriction.
Translocationalhyponatremiaoccurs with the absorption of glycine during transurethralprostate
resection, as well as in gynecologic and orthopedic procedures.
Symptoms of hyponatremia occurs if the serum level is 125-130 mmol/L.
Serum sodium level of< 125 mmol/L may result to severe neuropsychiatric symptoms.
the causes of hyponatremia and
B y examining the urinary sodium, potassium, and osmolarity,
hypernatremia can be readily determined.

3. Pseudohyponatremia
I t is the reduction in serum sodium concentration caused by a systematic error in measurement.
T h e most common, yet not widely known, cause of pseudohyponatremia is in vitro hemolysis, a
well known cause of pseudohyperkalemia.
Marked hemolysis may cause decreased of sodium levels due to dilutional effect.
as in
Hemoglobin released from the red cells cause additional reduction in serum Na+,
hyperproteinemia; this error occurs because hemoglobin is mostly a protein, and has the
it same

effect as hyperproteinemia in displacing plasma water.

Artifactual hyponatremia is also associated with hyperlipidemia or hyperproteinemia - decrease

sodium is due to excess retention of water in the collecting ducts.

147
 Hyponatromia wth Normal Renal Functlon
Cause Serum Na Urine Na 24-hour UNa Urine Serum
osmolallty
1. Overhydration Low Low Low Low Normal or low

2.Diuretics Low Low High Low Low

3. SIADH Low High High High Nornal or low

4. Adrenal failure Mildly elevated Normal

High High
5. Bartter's Low Low High Low Low
Syndrome
6. Diabetic Low Normal Normal Normal High
Hyperosmolarity

Notes to Remember.
Fractional excretion (FE) is the quantity of a substance excreted in the urine
fraction of the filtered load of the same substance.
expressed as a
FE of sodium is often used to distinguish between acute tubular
necrosis (acute kidney injury)
and prerenal azotemia FE of sodium of <0.01 (less than
1%) suggests prerenal azotemia, and
-

a>0.01 suggests acute tubular necrosis (acute kidney injury).

Methods:
1. Emission Flame Photometry
2. lon Selective Electrode (Glass aluminum
silicate)- most commonly used method
3. Atomic Absorption
Spectrophotometry
4. Colorimetry {Albanese Lein)

I POTASSIUM
I t is otherwise known as "kalium."
I t is the major intracellular cation only 2% of the body' s total potassium circulates in plasma.
I t is the single most important analyte in terms of an abnormality being immediately life
threatenin8.
I t s concentration in the red blood cells is 105 mmol/L {23x its
concentration in plasma).
I t is filtered at the glomerull and is mostly
(709%-80%)
reabsorbed by active and passive
mechanisms in the proximal tubule.
In the
ascending limb of Henle's loop, it is reabsorbed together with Na and Ct by the sodium
potassium chloride cotransporter.
Functions: heart contraction, neuromuscular excitability, ICF volume regulation and
hydrogenion concentration
Reference value: 3.5-.2 mmol/L
Threshold critical vatue: 6.5 mmol/L (hyperkalemia)
2.5 nmol/L (hypokalemia)

148
Specimen Consideratons:
a. Hemolysis of 0.5% RBC can Increase levels by 0.5 mmol/L (30% increase in gross hemolysis).
b. Plasma levels are lower (0.1-0.7mmol/L) compare to serum levels because of the releasedof platelets
into serum on clot formation.
Muscular actlvity like exerclse and prolonged standing 10 to 20% increase
-

c.
- 0.3-1.2 mmol/L = mild to moderate exercise
- 2-3 mmol/L vigorous exercise; fist clenching
d. Prolonged contact of serum and red cell.
e. Prolonged application of tourniquet.

Hyperkalenmla: Hypokalemia
1. Gastrointestinal loss
1. Decreased renal excretion
a. Acute or chronic renal failure a. Gastric suction and laxative abuse
b. Intestinal tumor and malabsorption
b. Severe dehydration
c. Cancer and radio therapy
c. Addison's disease
d.Vomitting and diarrhea

2. Extracullur shift
a. Acidosis 2. Renal loss
a. Diuretics use (thiazides)
b. Muscle/ellular injury
b. Hyperaldosteronism
c.Chemotherapy
d. Vigorous exercise c. Cushing syndrome
d. Leukemia
e. Digitalis intoxication
3. Increased intake- oral or IV infusion e. Bartter's syndrome
f. Gitelman's syndrome
4. Use of immunosuppressive drugs
- tacroiimus and cyciosporine &. Liddle's syndrome
i. Malignant hypertension
overdose
3. Intracellular shift-alkalosis and insulin

Notes to Remember.
1. Hyperkalemia
renai excretion.
Hyperkalemia is almost aiways due to inmpaired
potassium excretion: reduced aldosterone
or
diminished renal
Three (3) major mechanisms of
reduced distal delivery of sodium
aldosterone responsiveness, renal failure, and
serum K' can directly stimulate
the adrenal cortex to release aldosterone.
Elevations in

Reduced aldosterone
excretion of potassium
chronic hyperkalemia due to impaired renal
> The most common.cause of chronic renal insufficiency of primarily
is hyporeninemic hypoaldosteronism, which s caused by
tubulointerstitial disease.
the most common cause of all aidosterone deficiency
Hyporeninemic hypoaldosteronism is of chronic hyperkalemia among nondialysis
cause
states, and is by far the most
common

patients.

Renal Failure
accumulation of potassium (including
Reduced GFR and decreased tubular secretion
causes

magnesium and phosphorus) in plasma.

149
 Effects of Hyperkolemia to Cardiac Muscle
Hyperkalemia decreases the resting membrane potential (RMP) of the cell.
Severe hyperkalemla can utimately cause a lack of muscle excitability ( 8mmol/L).
Plasma K levels of 6-7mmol/ may alter ECG.
Plasma K' levels of 10mmoi/L is fatal (cardiac arrest).

pHImbalance (Acidosis) and Drugs

In acidosis, plasma K' increases by 0.2-1.7 mmo/L for each unit reduction of pH = H* enter RBC
and as an exchange, K'move out of the oells.
Low insulin levels cause high serum potassium.
Therapeutic K administration is themost common cause of hyperkalemla among hospitalized
individuals.
Hyperkalemic drugs captopril, spironolactone (K sparing-diuretic), digoxin, cyclosporine and
heparin therapy
Digitalis inhibits the Na*-K*-ATPase pump.
2. Pseudohyperkalemia
Causes: sample hemolysis, thrombocytosis, prolonged tourniquet application, fist
clenching,
blood stored in ice, IV fluid and high blast counts in acute or accelerated
phase leukemias (blast
may lyse during standard phlebotomy releasing K)
>Thrombocytosis and severe leukocytosis cause potassium release from the platelets and white
blood cells, respectively, during blood clotting.
I n psaudohyperkalemia due to
thrombocytosis, both serum and plasma K* should be obtained
simultaneously.
>Recentrihugation of SST has been associated with pseudohyperkalemia after centrifugation, a
serum layer will develop not
only above but also under the gel within the cellular layer. During
storage, potassBum escapes from the cell Into the serum layer under the gel. When the tube is
recentrifuged, the serum layer under the gel will move above and cause a pseudohyperkalemia.
Electrocardiographic (ECG) abnormalities of hyperkalemia are absent in pseudohyperkalemia,
but the absence of ECG changes does not rule out true
hyperkalemia because ECG changes are
rare in chronic hyperkalemia.
3. Hypokalemia
Plasma K levels of 3.0-3.4 mmol/Lis mild hypokalemia.
Hypomagnesemia teads to hypokalemia by promoting urinary loss of potassium.
f plasma K* levels are low, it wil be
retained, and NH4 ions will be secreted if
are needed. balancing cations

Impaired renal function/Renal loss

I t is the most common cause of
hypokalemia increased renal wasting of potassium can be
attributed to increased activity of aldosterone or other
secondary hyperaldosteronism are other causes. mineralocorticoids; primary and

Extra renal loss

Diarrhea (low urine anion gap) is the mast common cause of extra renal of
Diarrhea causes direct K loss in the stool, but in vomiting, hypokalemia isloss potassium.
mainly the resuit of K
lossin the urine because vomiting causes metabolic alkalosis, and the
excretion of bicarbonate leads to renal K wasting. subsequent renal

150
Effects of Hypokalemia to Cardiac Muscle
Hypokalemia decreases cell excitability by increasing RMP, resulting in arrhythmia and paralysis.
The heart may experience cessation of contraction in either hyperkalemia or hypokalemia.
Heart fallure does not lead to hypokalemia despite secondary hyperaldosteronism, unless distal
delivery of Na* is increased by diuretic therapy.

pH imbolance (atkaiosis) and Hormones

In alkalosis, pBasma K* decreases by 0.4mmol/L/0.1 pH unit rise - alkalemia promotes

intracellular loss of H to neutralize the rise of pH, so K (and even sodium) move into cells to

maintain eiectroneutrallty.
pH balance due to increased concentrationof bicarbonate and pCO2, leads to movement of K*
acumulation of bicarbonate in the cell must be accompanied by
tendsto move into the cells
Na and K
to be an additional
Direct stimulation of aldosterone secretion by metabolic acidosis appears
mechanism that contributes to hypokalemia.
In chronic metabolic acidosis, hypokalemia develops, probably because reduced proximal
reabsorption of NaCl allows increased delivery of NaCl to the distal nephron.
Insulin and cathecolamines influence the distribution of K between cells and ECF by promoting
the entry of K into the cell as glucose is transported into cells - whenever glucose is transported

into the cel, it is accompanied by potassium.

4. Pseudohypokalemia
decrease potassium levels- because K* is taken up by WBC, like
Leukocytosis can cause falsely
active leukemic cells, if sample is left at room temperature.

Methods
Heparinized plasma is preferred oer serum due to potassium released during blood clotting.
Platelets contain K that is released into serum on clot formation.
Diet rich in fats may lead to elevated serum potassium.
1. Emission Flame Photometry
2. lon Selective Electrode (Valinomycin gel)
3. Atomic Absorption Spectrophotometry
4. Colorimetry (Lockhead and Purcell)

Differential
The
Diaancsis
first step in the differential diagnosis of hyperkalemia should be to rule out
pseudohyperkatemia.
Once pseudohyperkalemía is ruled out, the next step is to dfferentiate among the three major
causes of hyperkalemia: Increased K* intake, shft of K* from the celi, and Impaired renal
excretion. Measurement of a 24 hour urine K wll distinguish increased intake from the other
two causes.
For differential diagnosis of chronic hyperkalemia of renal causes, the first step is to measure
piasma renin activity, plasma aldosterone, and urinary excretion of Na' and K"
The first step in the differential diagnosis of hypokalemia is to measure urinary excretionof K. If
urinary K excretion is low (<20 mEq/day or <0.01 mEq/mg ofcreatinine), the cause of
hypokalemia is low intake, extrarenal loss of K, or intracellularshift.

151
 f urinary excretion is normal or
increased (urine K'>30 mEq/day or 0.02 mEq/mg of
the cause of hypokalemia is renal loss. Once a creatinine)
renal cause is suspected, the next step should be
the measurement of plasma renin
actlvity and plasma aldosterone.

I . CHLORIDE
Itis the major extracellular anion chief counter ion of sodium in ECF.
-

t promotes maintenance of water balance and osmotic pressure in

t i s the only anion to serve as an conjunction with sodium.
enzyme activator.
t is excreted in the urine and
sweat
Disorders of chloride are the same as sodium since
Functions: maintains osmolality, blood volume and they
are both extracelular cations.
electric neutrallty
Reference value: 98-107 mmol/L

Spedimen
1. Marked
Conslderations:
hemolysis may caused decreased levels of chloride due to dilutional effect.
2. Slightly lower values are observed in post prandial
3.Low serum values are observed in specimen.
conditions with high HCO3 levels.
Methods:
Interferences: bromide, cyanide and cysteine (chemicai test and
Methods for chloride also measure bromide. coulometry)
1. Mercurimetrie Titration (Schales and Schales)
Diphenyicarbazone indicator
HgCh (blue violet) end product
2. Spectrophotometric Methods
a. Mercuric Thiocyanate (Whitehorn Titration
b. Ferric Perchlorate Method) =
reddish complex
3. Coulometric colored complex
Amperometric
4. lon Selective Electrode Titration Cotlove Chloridometer
using ion exchange membrane
decanol); most commonly used(tri-n-octylpropylamnonium
method chloride

Hvperchloremia Hvpochloremia
1. Renal tubular acidosis
1. Prolonged vomitting
2.Diabetes insipidus
3. Salicylate intoxication
2. Aldosterone deficiency
3. Metabolic alkalosis
4.Primary hyperparathyroidism
5. Metabolic acidosis
4. Salt-losing nephritis

6. Prolonged dlarrhea

152
M.CALCIUM
t i s present almost exclusvely In the plasma.
it is invoved In blood coagulation, enzyme activity, excitability of skeletal and cardiac muscle, and
maintenance of blood pressure.
I t is maxtmally absorbed In the duodenum - the absorption is favored at an acidic pH.
99% is part of the bones and 1% is in the blood and ECF.
Referencee values: 8.6-10 mg/di (adult)/ 8.8-10.8 mg/dL (child) total calcium
-

4.6-5.3 mg/dL (adut}/ 4.8-.5 mg/dt (child) ionized calcium

Eorms ot Caldum
1. lonized(active) Calcium S0%
2. Protein-bound Calclum 40%
3. Complexed with anions 10%
lonized calcium is a sensitive and specific marker for calcium disorders.
total Ca*2
F o r every 1 g/dL serum albumin decrease, there is 0.8 mg/dl decrease in
-

hypocalcemia can be a consequence of reduced plasma albumin.

Factors AftiectingPlasma CaldumLevels:

1.1,25 Dihydroxycholecaiclferol [1,25-40H)-D]
tincreases intestinal absorption ofcalcium.
I t increases reabsorption in the kidneys.
I t increases mobilization ofcaicium from bones.

2. Parathyroid Hormone (PTH)

i t conserves calcium by increasing reabsorption in the kidneys.
I t increases the levelby moblizing bone caBlcium.
It activates the process of bone resorption.
t suppresses urinary loss of calcium.
t stimulates the conversion of inactive vitamin D to active vitamin Da in the kidneys.

3. Calcitonin
l s a thyroid hormone, secreted by the parafollcular Ccells ofthe thyroid gland
I t inhibits PTH andvitamin D- hypocalcemic hormone.
it inhibits bone resorption.
It promotes urinary excretion of calcium.

Practical Consicierations:
1. Serum is the specimen of choice for analysis.
2. A decrease in plasma protein concentration will result in decrease total calcium.
3. Avoid prolonged contact of serum with cell clot-Jca (ionized Cais pH dependent).
4. Recumbent posture LCa
5.Venousocclusion 1Ca
6. Decrease pH of the reagent facidification) results to liberation of calcium from albumin.
7. Urinary excretion is the major net loss of calclum.

153
 Methods:
1. Precipitation and Redox Ttration
a. Clark Colip Precipitation end product: Oxalic acid (purple color)
-

b. Ferro Ham Chloranilic Acid Precipitation end product: Chloranilic acid (purple color)
2. Ortho-Cresolpthalein Complexorne Dyes (Colorimetric Method)
Dye: Arzeno ll
Mg' inhibitor: 8-hydroxyquinoline (chelator)
3. EDTA THtration Method (Bachra, Dawer and Sobel)
4. lonSelective Electrode(Liquid-membrane)
5. Atomic Absorption
Spectrophotometry reference method
6. Emission Flame Photometry
Hypercalcemia Hypocalcemig
1. Primary hyperparathyroidism -main cause 1. Alkalosis
2. Cancer (Lung & Mammary) 2. Vitamin D deficiency
3. Acidosis 3. Primary hypoparathyroidism
4. Increased vitamin D 4. Acute pancreatitis
5. Multiple myeloma 5. Hypomagnesemia
6. Sarcoidosis 6. Malabsorption Syndrome
7. Hyperthyroidism 7. Renal tubular failure
8. Milk-alkali syndrome

Causes of Hypercalcemia: CHIMPS (Cancer,

Hyperthyroidism, latrogenic causes,
Multiple myeloma, Hyperparathyroidism, and Sarcoidosis)
Causes of Hypocalcemia: CHARD (Calcitonin, Hypoparathyroidism, Alkalosis, Renal failure,vitaminD
deficit)

Notes to Remember
lonized calcium is decreased in secondary
hyperparathyroidism.
Acidosis promotes leaching of calcium from bones and elevation of
hydrogen ions which
displace calcium from proteins, while alkalosis promotes deposition of caicium in bones.
Alkalosis aiso causes plasma proteins to have a greater negative
charge that in turn binds more
ionized calcium,
resulting to hypocalcemia.
Parathyrolk hormone-related protein (PTHRP) is increased in hypercalcemia associated with
malignancy
Dehydration or hemoconcentration may elevate serum albumin, resulting in a falsely elevated
total serum calcium.
Rate of fall in ionized calcium initiates tetany.
Estrogen deficlency reduces formation of active vitamin D.
Primary hypocalcemia (low PTH) is due to parathyroid gland disease, and
secondary
hypocalcemia (high PTH) is due to renal faillure.
Symptoms of severe hypocalcemia occurs with total calcium levels <7.5 mg/dL (1.88 mmo/l).
True hypocalcemia frequently develops as a serious medical
emergency in pancreatitis due to
sequestration of ionized calcium in the damaged tissue and surrounding fluld.

154
V. INORGANIC PHOSPHORUS
I t is omnipresent in its distribution; 85% in the bones and 15% in the ECF in the form of inorganic
phosphate.
I t is Inversely related to calcium.
It is maximally absorbed in the jejunum.
Phosphate is essentlal for the insulin-mediated entry of glucose into cells by a process involving
phosphorylation of the glucose and the co-entry of K.
Most phosphate in serum is in inorganic form.
Reference values: 2.7-4.5 mg/dL (adult}/ 4.5-5. mg/dL (child)

Inorsanic.phosphorus.edst a
- principal anion within cells
1.Organic phosphate
2. Inorganic phosphate part of the blood buffer

Formsof Phosphoru
1. Free or unbound form-55%
2. Complexed with ions 35%
3. Protein-bound 10%

Factors Aliecting Phosohate Concentratlon:

1. PTH decreases phosphate by renal excretion
2. Caicitonin inhibitsboneresorption
3. Growth hormone increases phosphate renal reabsorption

Pracicai Coisiderations: decrease levels.

1. Fasting requlred-a high CH0 diet can resut to
is
2. Separate the serum from the red cells immediately
after clotting is completed.

Methods: measured in clinical

Phosphorus is measured in terms of POs -

only inorganic phosphate is

laboratory.
Laboratory results cannot be expressed in mEq/L P0sgroups
because different normally present
at physiologic pH have different valences.
levels in late morning and low levels in
Blood collection is affected by circadian rhythm high
the evening
the parathyroid gland.
Fasting senum phosphate is controlled by

Fiske Subbarow Method (Ammonium molybdate method)

Is the most commonly used method to measure
serum inorganic phosphate.
Amino Naphthol Sulfonic Acid)
Most common reducing agent: pictol(amino phenol), ascorbic acid and
Other reducing agents: elon (methyl
senidine (N-pheny-p-phenylene diamine hydrochloride)
Endproduct: ammonlum-molybdate complex (unstabie)
The unreduced complex at 340nim is the most accurate measurement of inorganic phosphorus
in serum. result in
The pH must be maintained in the acid range because higher range (alkaline) can

reduction of the complex

155
 The reduced form of the endproduct yields a blue color and determined between 600nm to
700nm.

Hvperphosphotemia Hvpophosphotemia
1. Hypoparathyroidism 1. Alcohol abuse- most common cause
2. Renal failure (tubular failure) 2. Primary hyperparathyroidism
3. Lymphoblastic leukemia 3. Avitaminosis D
4. Hypervitaminosis D 4. Myxedema

Notes to Remember
Phosphate (PO«) deficiency can lead to ATP depletion.
Transcellular shift is a major cause of hypophosphatemia - an increase shift of phosphate into
cells can deplete phosphate in the blood and once it is taken up by cells it remains there to be
used in the synthesis of phosphorylated compounds.
The
equilibrium between serum phosphatases and intraceliular phosphate stores is determined
largely by carbohydrate metabolism and blood pH.
I n renal disease, where
tubular failure occurs, phosphate excretion is inhibited by the
nonresponsiveness of the tubules to parathyroid hormone phosphate levels rise, while calcium
levels fall.
>increased serum POs causes serum calcium to diminish.
Hyperphosphatemia and hypocalcemia, in the face of elevated BUN and creatinine, is indicative
of renal disease, strongly suggest tubular failure.
Severe hypophosphatemia will resut to plasma concentration of < 1.0
g/dL or 0.3 mmol/L.

VI. MAGNESIUM
I t is an intracellular cation second in abundance to
potassium.
I t is the fourth most abundant cation in the
body; an enzyme activator.
It is stored in the bones and muscles -53% in bone; 46% in muscles and soft
tissues; 1% in serum
and RBC.
It is a vasodilator and cause decrease uterine hyperactivity in eclampsic states and increase
uterine blood flow.
Life threatening symptoms occur if theserum level reach 5 mmol/L
Magnesium loss leads to decreased intracellular K levels.
Functions: Important in maintaining the structures of DNA, RNA and
ribosomes; synthesis of
CHO, CHONS and lipids; neuromuscular transmission; cofactor, regulates movement
of potassium across myocardium
Reference value: 1.2-2.1 mEq/L

Form
1. Free Mg /lonized form 55 (physiologicallyactive)
2. Protein-bound Mg2 - 30%
3. Complexed w/ ions 15%

156
 Factors Affecting Plasnma Magnesium Level:
1. Parathyrold Hormone
I t increases renal reabsorption of magnesium.
I t increases intestinal absorption of magnesium.
2. Aldosterone and Thyroxine
I t increases renal excretion of magnesium.

Hypermagnesemia Hypomagnesemia
1. Diabetic coma 1. Acute renal failure
2. Addison's disease 2. Malnutrition
3. Chronic renal failure 3. Malabsorption syndrome (sprue)
4. Increased intake of 4. Chronic alcoholism
antacids, enemas and cathartics 5. Severe diarrhea

Methods:
1. Colorimetric Methods
a. Calmagite Method (+) reddish-violet complex
b. Formazen Dye Method (+) colored complex
c. Magnesium Thymol Blue Method - (+) colored complex
2. Atomic Absorption Spectrophotometry- reference method
3. Dye-Lake Method-Titan Yellow Dye (Clayton Yelow or Thiazole Yellow)

VIL BICARBONATE
i t is the second most abundant anion in the ECF.
It accounts for 90% of the total Co2 at physiologic pH.
t is composed of undissociated NaHCO3, carbonate and carbamate.
t buffers excess hydrogen ion by combining with acid.
Maintenance of high plasma bicarbonate concentration occurs in advanced renal faiure, or
when the renal threshold for bicarbonate is increased.
Function: major component of the buffering system in the blood.
Specimen: blood anaerobically collected
Method: lon selective electrode (using the pCO,electrode) and
Enzymatic (phosphoenolpyruvate carboxylase and dehydrogenase)
Reference value: 21-28 mEq/L {venous blood-plasma orserum)

pHImbalance
It diffuses out of the cell in exchange for chloride to maintain ionic charge neutrality
within the cell (chloride shift) - the buffering capacity of blood is malntained by a reversible
exchange process between bicarbonate and chloride.

157
OTHER SIGNIEICANT INFORMAIION:
1. Anion Gap (AG)
I t is the difference between the unmeasured cations (sodium and potassium) and unineasured
anions (chloride and bicarbonate).
Itis a form ofqualitycontrol for the analyzer used to measure these electrolytes.
I t i s used to monitor recovery from diabetic ketoacidois.
Abnormal anion gaps in sera from healthy person indicate an instrument problen.

Formulae: AG Na-(Cr + HCO) RV = 8-16 mmol/

AG (Na +K) - (Ct +HCO) RV =10-20 mmol/l
increased: Uremia/renal failure (phosphate and sulfate retention);ketoacldosis (starvation or
diabetes); polsoning by methanol, ethanol, ethylene glycol or salleylate; lactic acidosis,
hypernatremia and instrument error

Decreased: Hypoalbuminemia ( decreased unmeasured anions), hypercalcemia (elevated unmeasured

cations); hyperlipidemia; elevated myeloma proteins

Notes to Remember
The total concentration of unmeasured anions (i.e, all anions other than chloride and
bicarbonate) is about 23 mmol/L, and the total concentration of unmeasured cations lie.,
all cations other than sodium) is about 11 mmol/L
A change in serum Na' usually does not cause a change in AG because serum C usualiy
changes in the same direction.
Increased AG is most often due to accumulation of anions of acids, such as suifate, lactate,
and ketone anions, while decreased AG is most commonly due to reduction in serum
albumin concentration.
The presence of a widened anion gap signifies the presence of a metabolic acidosis due to a
non-chloride-containing acid.
Bromide Intoxication is accompanied by low serum anion gap because bromide causes a
false increase in serum C
Low anion gaps, typically in the range of 1-3 mEa/L, signify the presence of high leveis of
basic protein, often a monoclonal paraprotein as occurs in plasma cll dyscrasias.
The measurement of urine AG is useful in determining the severity of diarrhea.
Normal urine anion gap is about 40 mmol/24 hours.

2.CysticFibrosis(Mucoviscidosis)
t is usualy recognized in infancy (miconeum ileus) or early childhood.
I s an inherited disorder of the exocrne glands, causing those glands to produce abnormally
thick secretions of mucus, elevation of sweat electrolytes, increased organic and enzymatic
constituents of saliva, and overactivity of the autonomic nervous system.
Increased In electrolytes is due to defective gene and protein known as the cystic fitbrosis
transmembranous conductance regulator, located on chromosome 7.
Signs: chroic cough, frequent foul-smelling stool and persistent URT infection
Affected organs and glands: pancreas, respiratory system and sweat glands
Test requirement: without rashes, cuts or skininflan mation, for neonate, at least 48 hour-oid
Sweat Inducer: Pilocarpine
Diagnostic Test: Sweat Test-Coulometry (1sodium and chioride)

158
 Reference Method:Gibson and Cooke Pilocarpine lontophoresis
5 0 mg of sweat sample collected within 30 minutes
(+) Resut: >65 mmol/L of sweat electrolytes (R.v. = 5-40 mmol/L)

3. Iron
tis a common metalic element important for the synthesis of hemoglobin.
t is a prooxidant, contributing to lipid peroxidation, atherosclerosis, DNA damage and
carcinogenesis.
I t is stored as ferritin and hemosiderin primarily in the spleen, bone marrow and liver.
Of the total 3 to 5 g of iron in the body, 2 to 2.5 is in hemogiobin; 130 mg is in myoglobin; 8 mg
is in tissue; 3 to 5 mg is in plasma (albumin and free hemoglobin).
Serum ferritin mustbe depleted first, before iron level declines a low serum ferritin level is
diagnostic of iron deficiency.
Reference volues: Male=50-160 ug/dL
Female 45-150 Hg/dL
Increased: Iron poisoning(overdose), hemochromatosis, viral hepatitis, noniron deficiency
anemias (thalassemia, aplastic, megaloblastic, sideroblastic, hemolytic and
pernicious anemias)
Decreased: Iron deficiency anemia (1DA), malnutrition, malignancy, chronic infection and
nephrotic syndrome
Method:
1. Colorimetry (HCl and ferrozine- (+) blue color
2. Anodic Stripping Voltammetry
The first step in the quantitation of serum iron is separation from transferrin by acidification.
Serum iron levels are falsely elevated by hemolysis and affected by diurnal variation.
Diurnal variation means highest concentration in the morning and lowest at night.

Terminologies:
1. Total Iron Binding Capacity (TIBC)
It refers to the amount of iron that could be bound by saturating transferrin and other minor
iron binding proteins present in the serum or plasma sample.
Is a direct measureofthe total number offunctional ferrous ion-binding sites in transferrin.
All TIBC methods require addition of excess iron to saturate transferrin.
Excess iron is then removed by adding magnesium carbonate to measure the bound iron.
IDA has high TIBC; noniron deficiency (non-1DA) anemias have low TIBC.
Anemla of chronlc infection has low or normal TIBC.
Reference volues: 245-425 ug/dL (adult male and female)
10-250 ug/dL (40years old)
100-200 48/dL (newborn and child)
Formulae: TIBC = UIBC Serum Iron

TIBC(ug/d serum transferrin (mg/dL) x 1.25"

Increased: DA, hepatitis,iron-supplemented pregnancy
Decreased: non-IDA and nephrosis

2. Unsaturated Iron Binding Capacity (UIBC)

Is a measure of the reserve iron binding capacity of transferrin.
Formula: TIBC Serum Iron

159
3. Percent Saturation
It is also known as the transferrin
saturation; an index of iron storage.
It is the ratio
of serum iron to TIBC the ratio is around 1:3 for normal individuals, and in IDA it
is significantly reducedto values of around 1:5 or lower.
Increased: Iron overdose, hemochromatosis,
sideroblastic anemia
Decreased: IDA (lowest % saturation), malignancy, chronic infectlon, anemia of chronic disease
References value: 20-50%

%Saturation =Totaliron lug/dL x100

TIBC (ug/dL)

4. Serum transferrin
Transferrin is the principal iron transport protein.
Formula: TIBC (ug/dL) x 0.70 mg/dl

160
 BLOOD GASES and pH MEASUREMENTS

Begulatlonof Add-Basa Balanca: Lungsand Kldnevs

Most of the CO combines with H0 to form carbonic acid (HCO), which dissociates
immediately into H' and bicarbonate (HCO3) - the reaction is accelerated by carbonic anhydrase.
The dissociation of H;CO, Increased HC0 in RBC causing it to diffuse into the plasma.
HCO and HCOs are renewable evenbefore renal mechanisms restore the constituents, the
lung alters the ratio of numerator (HCO,) to denominator (HCOs) by blowing off CO,.

Lungs
Respiratory control of CO% excretion allows rapid and very sensitive adjustments in blood pH.
As the lungs eliminate excess CO to resist accumulating H', the proportion between HCO, and HCO
readjusts to 20:1, although the absolute concentrations ofeach can fall below normal.
By regulating the rate of Co excretion, the lungs can maintain the ratio at or about 20:1, thereby
minimizing pH changes.
The CO, diffuses into the alveoli and is eliminated through ventilation.
Slow or non removal of CO% bythe lungs results to increase inH ion concentration respiatory
acidosis.
Rapid orfast elimination of CO2 results to decrease H ion concentration -respiratoryalkalosis.

Kidneys
The most important function ofthe kidneys in acid-base homeostasis is excretion of acid, which
is equivalent to generation of alkali or reabsorption of HCO from the glomerular filtrate
(proximal tubules of the kidneys) and add it to the blood.
Acid is excreted in the form of NH and titrable acid.
Hydrogen ions are also excreted by the kidney, both by direct excretion and through indirect
disposal in,the form of ammonium lon.
HCO concentration is under renal control, in that the kidneys regulate both the generation of
HCO, ions and thelr rate of urinanry excretion.
Fifty to one hundred percent i50-100) mmol/L of ucid must be excreted daily by the kidneys
resulting to urine pH of 4.5.

Plasma and Urine Bicarbonate:

t HCO's: V infusion of lactate, acetate and HCO
HCO : use of diuretics, reduced reabsorption and chronic nephritis
If HCO: Is below 25 mmol/L or if plasma COrises above
normal, the tubule can reabsorb all
theHCO,in the glomerular filtrate, leaving none for the excretion in urine.
A smali amount of bicarbonate is
normally excreted in the urine (about 10 mEq/day).
The kidneys excrete considerable amounts of acid and base for
acid-base regulation.
Urinary excretion of HCO%: when plasma reached 26-30 mmol/L

161
 Blood Buffers:
1. Bicarbonate and carbonic acid (HCO%: H2Coa) -

major extracellular blocd buffer

2. Plasma proteins
3. Hemoglobin
3. Inorganic phosphate
H2CO weak acid because it does not completely dissociate into H' and
is a
HCO3
When acid is added to the bicarbonate-carbonic acid
an
system, the HCO; wili combine with the
H from the acid to fom
H2CO
Plasma proteins have buffering capacity through charges on their surfaces.
Hemoglobin is an effective buffer because it can off-load its oxygen and combine with CO2 that
has diffuse gradients.
across
1 gram of hemoglobin carries
1.39 ml of oxygen; each mole of
oxygen more than 95% of the hemoglobin binds oxygen.
hemoglobin binds 1 mole of
All the above-mentioned blood buffers
also contribute to buffer base.

Henderson-Hasselbalch Eguation
it expresses acid-base
relationship and relates the pH ofa solution to the dissociation properties
of the weak acid.
It indicates that pH
depends on the ratio of HCOa/pco2.
When the kidneys and the lungs are
functioning properly, a 20:1 ratio of HC0'3 to H;CO3 will be
maintained, and it is expressed by the Henderson-Hasselbalch equation.

pH= pKa +log coniugate base

where:
weak acid
pKa is 6.1; combine hydration and dissociation constants for CO2 in blood
conjugate base bicarbonate
weak acid carbonic acid
Parameters in the Assessment of Acdd-Base Balance:
1pH
2. pCO2
3. HCO
4. pO2

1. Evaluate the pH
Normal pH: 7.35-7.45
7.35 Acidosis
745 Alkalosis
pH 7.40 is the optimum level for arterial blood.
To preserve pH within the narrow
physiologic range, short-term buffering capacity rnust neutralize
acids as they are generated, and
long-term corrective measures must eliminate the acid
permanently, but ona continuous basis.
The reference range for arterial biood pH
(7.35-7.45) is only 0.03 pH unit lower for venous blood
owing to the buffering effects of hemoglobin known as
The pH decreases by 0.015 each Celsius above 37"C. chloride-isohydric shíft.

162
 3 major causes of extrarenal acidosis: organic acidosis, diarrheal loss of bicarbonate, and acidosis
due to exogenous toxins.

2. Evaluate the ventilation (Lungs)

Normal pC02: 35-45 mmHg
<35 mmHg Respiratory Alkalosis
> 45 mmH4g Respiratory Acidosis
pCO isan index or efficiency of gas exchange and not a measure of COa concentration in blood.
The lungs regulate pH through retention or elimination of C02
An increasing ratio of heparin to blood can cause marked artifactual rise on measured pCO2 (12-
15%) and the parameters calculated from it.
Whole blood total Coais equal to dissolved CO+ HCO +HcO.
Increased pCOx use of elicit drugs like barbiturates and morphine, and aicoholism

3. Evaluate the metabolic process (Kldneys)

Normal HCOs: 21-28 mEq/
21 mEq/L Metabolic Acidosis
28 mEa/L Metabolic Alkalosis
The kidneys regulate pH by excreting acid {NH4 ions) and reabsorption of HCO from the
glomerular filtrate.

4. Evaluate the degree of oxygenation

Normal pO: 81-100 mmHg (adequate oxygenation)
3 leveis of hvpoxemia:
Mild 61-80 mmHg8
Moderate 41-60 mmHg
Severe 40 mmHg cr less
Hypoxemia is low p02.
pOxreflects the availability of the gas in blood but not its content.
po changes more rapidly than pCO or pH.
pO is 60-70% lower in venous blood after oxygen is released in the capillary tissues.
The degree of association or dissociation of oxygen with hemoglobin is determined by po and
the affinity of hemoglobin for Oz
Low pOz is seen in myocardial infarction, interstitial pneumonia and severe congestive heart
failure.
Healthy persons living at higher altitudes will show lower ranges of arterial po, because of the
naturally lower partial pressure of oxygen in the atmosphere and so in the inspired air.

Four BasicAbnomalSateg
1. Metabotic Acidosis
t is caused by bicarbonate deficiency.
i t is seen in diabetic ketoacidosis (DKA),
Jactic acidosis (alcoholism), renal failure and diarrhea.
In DKA, the chlorlde remains normal (normochloremic acidosis) or low with elevated anion gap.
Diarrhea result in metabolic acidosis via bicarbonate loss.
Metabollc acidosis causes greater potassium efflux than
respiratory acidosis.acid) causes greater
Metabolic acidosis due to inorganic acids (e.g., sulfuric acid, hydrochloric
potassium effux than that due to organic acids (e.g., lactic acid, keto acids) because organic

163
 anions accumulate $ubstantialy in the
cell, as well as in the ECF, whereas inorganic anions
accumulate mainly in the ECF.
Compensation: Breathing rate increases to lower pCO-
After compensation: low HCO+low hyperventilation
The maximal compensation is pCO+pH < 7.4 (pCO% 10-15 mmHg)
completed within 12 to 24 hours.
pCO% drops 1 to 1.3 mmHg per mEq/L fall in HCO
Electrolyte imbalonce: hyperkalemia and hyperchloremia
2. Metabolic Alkalosts
I t is caused
by
I t is seen in
blcarbonate excess.
vomiting (with the loss of chloride from the stomach).
Compensation: breathing rate decreases to increase pCO2 (hypoventilation)
After compensation:high HCO +high pCO +pH> 7.4
The maximal compensation is
completed within 12 to 24 hours.
Among the four types of acid-base disorders, compensation is least
alkalosis probably because hypoxemia, an inevitable consequenceeffective in metabolic
of hypoventilation,
stimulates ventilation.
For every 10 mEa/L rise in
HCO3, the pCO2 rises by 6
mmHg.
Electrolyte imbalance: hypokalemia and hypochloridemia
3. Respiratory Acidosis
t is due to excessive carbon
dioxide accumulation.
tis seen in chronic obstructive
the accessory muscles
pulmonic disease (COPD), myasthenia gravis (partial paralysis of
of breathing), CNS disease, drug overdose (morphine, barbiturates &
opiates), botulism, stroke, myxedema and pneumonia.
Compensation: kidneys retain HC0, because of increased
After compensation: high pCO2 +high HCOat pH < 7.4 pCO2
Maximal compensation requires 5
days but is 90% complete in 3 days.
Excretion of acid is another way of
compensating the rise of pCO
Restriction of NaCl intake during the recovery
phase of chronic respiratory acidosis results in the
maintenance of a high serum HCOO.
HCO rises1 mEq/L for each 10 mmHg rise in pCO2
4. Respiratory Alkalosts
I t is due
to excessive carbon dioxide loss (because of rapid breathing).
It is observed during anxiety, pain, aspirin overdosage, hepatic cirrhosis,
severe
sepsis, salicylate and progesterone drugs. gram-negative
Blood pH tends to be extremely
high when
stimulation of the resplratory center, because therespiratory alkalosis is caused by
psychogenic
condition is usually superacute, and
there is no time for
compensation. therefore
High progesterone levels are responsible for chronic
respiratory alkalosis of pregnancy.
Compensatlon: decreased reabsorption of HCO
After compensotion: low pCO2 +low HCO,+ pH> 7.4
Compensatlon is completed within 2 to 3 days.
Among the four types of acid-base disorders, compensation is
most effective in
alkalosis-pH after compensation sometimes returns to normal levels. respiratory
When complete
compensation does occur, one should look for evidence of
metabolic acidosis. complicating

164
 HCO fals 2 mEa/L for each 10 mmHg fall in pCO2.
Electrolyte imbalance: hypokalemia

Mixed Acid-Base Disorders

t refers to a ctinical condition in which two or more primary acid-base disorders coexist.
They generaly present with one obvious disturbance with what appears to be an inappropriate
(excessive or inadequate) compensation.
The "inappropriateness" of the compensatory process is probably the result of a separate
primary disorder.
When two disorders infuence the blood pH in opposite directions, the blood pH wll be
determined by the dominant disorder.
f pCO and HCO: have changed in opposite directions (e.g, pCO2 is high and HCO3 is low, or
pCO2 is low and HCO3 is high), the presence of a mixed acid-base disorder is certain.

Notes to Remember
The body' s cellular and metabolic activities pH dependent, thus, during compensation, the
are

body
tries to return the toward normal whenever imbalance
pH an occur.
After full compensation, ph would returned to its normal range.
After partial compensation, the pH would be near normal.
T h e lungs can compensate immediately but the response is short term and incomplete; the
kidneys are slow in its response but long term and complete.
> Base excess is increased in metabolic and respiratory alkalosis and decreased in metabolic and

respiratory acidosis (reference values: -2 to +3 in adult; -4 to +2 among children).

Specimen Coliection
Specimen: Arterial blood
Anticoagulant: 0 05mL heparin/mL of blood
The best method for blood gas collection in the newborn is by indwelling umbilical artery
catheter.
Syringe and needle for arterial blcod collection must be preheparinized by drawing up heparin
into the syringe to wet its interior; excess heparin should be expelled.
The use of butterfiy infusion sets is not recommended.
The liquid form of heparin i not recommended bécause excessive amounts can dilute the
sample and possibly contaminate the sample if equilibrated with room air.
Any air trapped in the syringe during blood collection should be immediately expelled at the
completion of the draw.
Arterial and venous blood differ in ph, pCO2 and pO2

of
Common errors in specimen collecton and handling: form and concentration of heparin, speed
syringe filling, maintenance of anaerobiosis, miing of samples, collection device, transport and storage

time before analysis

Common analytcalerrorS temperature error and protein coating of electrodes

165
 Specimen
1. On
Considerations:
standing, pH and poz (decreased) and pcO
2. Blood samples should be
chilled to
(increased) are affected.
prevent oxygen consumption by the RBC and release of acidic
metabolites, thereby altering the pH.
3. Glycolysis results to
decrease blood pH.
4. Excess heparin causes downward shifting
of ty ,
5. Lower
temperatures cause increased oxygenblood pH-most common preanalytic error
solubility
oxyhemoglobin curve resulting in more oxygen combiningblood
in and a left-shift in the
with hemoglobin.
Quality Control
The minimum
requirement for blood gas quality control is one
levels (acidosis, normal,
alkalosis) of control every 24 hours. sample every 8 hours and three
A single-point calibration is performed between each
instrument drift. gas sample to correct electrode and

Methods:
I.Gasometer . Electrodes
A. Van Slyke
A. pH (potenticmetry)
B. Notelson
1. Silver-siver chloride
1. electrode- reference electrode
mercury to produce vacuum 2. Calomel electrode
2. caprylic alcohol anti-foam
-
(Hg-Cl2)- reference electrode
reagent 3. Glass electrode mostcommonly used for pH
3. lactic acid
8. pO2 Clark electrode (polarography-armperometry)
4. NaOH and NaHSOs
Modern blood gas analyzers routinely
C pCO-Severinghaus electrode (potentiometry)
contain three
electrodes that give very rapid and accurate
results for direct
measurement of pH, pCO2 and pO2.
Continuous monitoring for pO
This is done by using transcutaneous
(TC) electrodesplaced directly on the skin of
ft is commonly used for neonates and infants; a noninvasive procedure. the patient.

Factors affecting blood gases and pH measurement:

1.Temperature (37 Ct 0.1)-most important factor
It is essential that the electrode sample chamber be
maintained at constant temperature for all
measurements.
F o r each degree of fever in the patient, p02 will fall 7% and
2. Elevated
pCO2 will rise 3%.
plasma protein concentrations.
POtest is affected by build up of proteins on the surface of the membrane.
3. Bacterial contamination within the measuring chamber, if present, will consume
low value of pO2. oxygen and cause
4. improper transport of the blood specimen.
When blood samples are not transported on ice (during transport to other laboratory), the p02
changes rapidly than pH and pCO2
Samples should be kept at room temperature andanalyzed immediately ( in less than 30
minutes) after blood collection.

166
Notes toBlood
Remember
gas resuts are affected by the gas mixture the patient is breathing and by the patient's
body temperature
When serum Is used to measure total CO, the dissolved CO is insignificant because all COa gas
has escaped into the air.
The total Co in arterial blood (plasma or serum) is equal to HCO% in arterial blood.
Calculations of base excess uses pH and pCO, values.
Blood gas results should be back to the physician preferably within 10 minutes after draw to
obtain maximum benefit from them.
Total COa 19-24 mmol/L(arterial whole blood)
22-26 mmol/L (venous whole blood)

167
 VITAMINS, TRACE ELEMENTS AND TUMOR MARKERS
Vitamins
These are essential organic
synthesize.
substances that the body cannot synthesize, or does not sufficiently
These are present in almost all
foods, but no single food group is the source of all vitamins.
Functions: antloxidants, enzyme
cofactors, hormones and important in blood cell
maturation, bone formation and active in energy metabolism

|Fat Soluble Vitamins VITAMINS and its CLINICAL SIGNIFICANCE

Chemical Name
1. VitaminA Retinol
Functionn Deficiency
Maintenance of good vision, Night blindness, growth
resistance to infection retardation, abnormal taste
2. Vitamin E
Tocopherols (a,B,,6) response, dermatitis
Antioxidant; for cellular Mild hemolytic anemia
respiration (newborn), RBC fragility,
3. Vitamin Da ataxia
Ergocalciferol, Rickets (young),
Cholecalciferol 0steornalacla (adult).
4. VitaminD Activated sterol-1,25 Absorption of dietary Ca Hypocalcemia
5. Vitamin K
dihydroxycholecalciferol
Phytomenadione Cofactor of procoagulants Bleeding disorder,
like prothrombin hemorrhage
Water Soluble Vitamins
1. Vitamin B1 Thiamine Enzyme cofactor Infants: Dyspnea, cyanosis,
diarrhea and vomiting
Adult: Reri-beri, Wernicke
Korsakoff syndrome
(apathy, ataxia and visual
2. Vitamin Bz problems
Riboflavin Enzyme cofactor Angular stomatitis,
3. Vitamin Bs Pantothenic acid
dermatitis, photophobia
Enzyme cofactor Depressed immune systen1,
4. Vitamin Bs muscle weakness
Pyridoxine, Pyridoxal Enzyme cofactor Irfants: irritability, seizures,
anemia

5. Vitamin Bi2 Adult: facial seborrheaa

Cyanocobalamin Synthesis of DNA andfolate Megaloblastic anemia,
6. Vitamin C Ascorbic acid
neurologic abnormalities
Hydroxylation of collagen; Scurvy
participates in redox
reactions
7. BBotin Enzyme cofactor Dermatitis, hair loss,
8. Carnitine
depression
Fat catabolism Muscle wakness, fatigue
9. Folic acid Pteroylglutamic acid Synthesis of amino aclds Megaloblastic anemia
and DNA
10. Niacin/Niacinamide Nicotinic Enzyme cofactor Pellagra (dermatitis,
acid/nicotinamide disorientation, weight loss)

168
Trace Eleme
These are consist of metals, except selenium, the halogens, fluoride, andiodine
Essential trace elements are important for maintenance of normal health, and tissue and organ
functions.
Trace elements have specific in vivo metabolic functions that cannot be effectively performed by
other similarelements.
Concentration in tissue<1 ue/g of wet tissue and <0.01% of dry body weight

TRACEELEMENTS and its CLINICAL SIGNIFICANCE

Essential Element Function Deficiency Toxicity
Chromium Enhances insulin action; insulin resistance, Skin ulcers, renal and
for glucose and lipid impaired glucose hepatic necrosis
metabolism tolerance (type 2 DM),
hyperlipidemia
Cobalt Hemoglobin synthtesis; | Anemia, growth Heart failure,
component of depression Hypothyroidism
vitamin B12
Copper Cellular respiration; | Menkes' kinky hair Interferes with
collagen synthesis syndrome' muscle absorption of iron and
weakness zinc
Fluorine Prevents dentalcaries Dental carries
lodine Thyroid hormone Goiter, cretinism, Thyrotoxicosis
synthesis myxedema
Iron Oxygen transport, Anemia Hemachromatosis
component of
hernogiobirn
Manganese Bone and connective Skeletal defects Psychiatric disorders,
tissue functions Parkinson's disease
Molybdenum DNA metabolism Growth depression, Anemiz, thyrotoxicois
cretinism, goiter
Selenium Prevents oxidative Keshan disease Hair and nail loss, liver
damage oflipid fall
Zinc Protein synthesis Acrodermatitis Gastrointestinal
enteropathica, growth irritation
retardation, immune
deficiency, infertility,
delayed wound healing,
osteoporosis

169
Tumor markers
These are substances in the body that is associated with the presence of a cancer.
It may be enzymes, hormones, receptors, oncofetal (glycoproteins) antigens or oncogenes.
Theyare expressed at very low levels in the blood.
Measurements of these substances is mostly essential to test recurrence of cancer.

Tumor Markers Associnted Cancer

AFP Hepatic and testicular cancers
ALP (placental-ALP) Lungcancer
Amylase Pancreatic cancer
BRCA-1 Breast or ovarian cancer
CA-125 Ovarian cancer (recurrence and treatment)
CA 15-3 Breast cancer (treatment and recurrence)
CA-19.9 Gastric, pancreaticand colorectal cancers
CA-50 Gastric and pancreatic cancers
recurrence and treatment)
CA 27.29 Breast cancer (treatment and recurrence)
Calcitonin Medullany thyroid cancer
Cathepsin-D Breast cancer
CEA Breast, lung colorectal and ach cancer
(recurrence and treatment)
CK-1 Smallcelllung cancer; prostate cancer
Estrogen receptor(ER) Breast cancer
GGT Hepatoma
HER-2/neu Breast cancer (efficiency of trastuzumab or
herceptin therapy)
Nuclear matrix protein (NMP) Urinary bladder cancer

170
 LABORATORY MATHEMATICS

PERCENT SOLUTION
It is equal to parts per 100 or the amount of solute per 100 total units of solution.
it is determined in the same manner regardless of whether it is w/w, v/v or w/v units are used.

1. Wetghtolume (w) % soluttons

t is the most common type of solution prepared in clinical laboratory.
It refers to the number of grams of solute per 100 ml of solution.

Grams of solute %solutiondesired ix total volumedesired

100

2. VolumeNolume (v/N) % solutions

t is used when both solute and sovent are liquid.
It refers to the amount of solute in ml in 100mL of solvent.

mi of solute =%solution desired x total volume desired

100

3. Weightwetght (w/w) % solutions

t refers to the number of grams of solute per 100 gms of solution.

Grams of solute =%solutiondesired xgramsofthe totalsolution

100

add ocid to water

Note: When preparing concentrated acid solutions, alwoys

MOLARITY
It is the number of moles of solute per liter of solution.
One mole of substance equals its gram molecuBlar weight (gmw).
Gram Moleculor Weight (GMW) is obtained by adding the atomic weights
- of the
component elements

Molorty ofsohution (M} Grams of solute

GMWXvolume of solution (1)

Moles weigtt fems)

GMW

171
 To prepare a molar solution:
Grams of solute Molarity x GMW of the solute x Volume (liter) desired
Toconvert %w to Molarity (M:
M % w/v x 10Q
GMW

NORMALTY
t is the number of
equivalent
It has often been used in
weight of solute per liter of solution.
acid-base calculations.

Normolity (N= Grams of solute

EWx volume (L)

Equivolent weight (EW) = MW

Valence

ToPrepare a Normal Solution of Solids:

Grams of solute EW
=
x
Normality x Volume (Liter)
Toconvert %wfv to Normality (N:
N %w/y x 10
EW

Relationship between Molarity and Normality

Normality Molarity x valence
Molarity Normality
valence

MOLALITY
It is the amount of solute
I t is expressed in terms
per 1 kilogram of solvent.
of
Molecular Weight (MW) isweight/weight
or moles per
kilogram (mol/kg).
obtained by adding the atomic weights
of the given compound.

Molality (m) Grams of solute

MWx kg of sovent

172
 MILLIEQUTVALENT
I t is commonly used for reporting electrolytes.

mEq/l mg/dL x 10 xvalence

MW

MILLIMOLES
t is the molecular weight in millimoles (mmol/L).

mmol/l mg/dlx 10
MW

RATIO AND DILUTION

Ratio Volume of solute
Volume of solvent

Dilution Volume ofsolute_

Volume of solution

Problem Solving:
1. What is the normality of a 200ml solution containing 5grams of HS04?
(MW =H-1; 5-32:0-16)
2. A glucose quality control chart has the following data:
2SD 6.0
nean = 98 mg/dL
Compute the coefficient of variation.
3. A mean value of 100 mmol/L and a standard deviation of 1.4 were obtained froma setof chloride
determination usinga control solution. What would be the 95% confidence interval in mmol/L?
4. What would be the average if the total glucose value in a group of 25 samples is 1740 mg/dL?
5. How much NaCl must be dissolved to prepare 500mL of 5molar solution?
(MW Na 23; C- 35.5)
6. How many milliliter of 8N H2S04 will neutralize 45mL of 6N KOH?
7. Solve for the equivalent weight of HCO3. (MW = H-1; C-12; 0-16)
8. Compute the molarity:
1. 10N H2S04
2. 8N HC
9. Compute the normality:
10M H2S04
b. 8M HCI
10. Convert 9.8 mg/dL of calcium to mmol/L.
odesiatsseye

173
 for EMR havinga frequency of 1.58* 1015 Hz?
11. What is the wavelength in nm

a (nm) 300x 10 m/s

1.58 x 1015 Hz
A (nm)= 190

Answers
1. 0.51 N 8. a. 5MM
2. CV 3.06% b. 8M
3.97.2-102.8 9. a. 20N
4. 69.6 b. 8N
5. 146.25 gms 10. 2.45 mmol/L
6. 33.75 ml 11. 190 nm
7. EW 31

174
 CONVERSION FACTORS FROM CONVENTIONAL UNITS TO SI UNITS

Analytes Conventional units Conversion

toSIunits Factor
1. Albumin 8/dLto g/L 10
2. Ammonia H8/dL to umol/L 0.587
| 3.Bicarbonate mEq/L tommol/L 1.0
4. Bilirubin mg/dt to umol/L 17.1
5. BUN mg/dL to mmol/L 0.357
6. Calcium mg/dL tommol/L 0.25
1.0
ZChloride nEq/L tommol/l
8. Cholestero mg/dL to mmol/L 0.026
mg/dL to umol/L 88.4
9. Creatinine
10. Glucose mg/dL to nmol/L 0.0555
mg/dL to umol/L 0.179
11. Iron
mEq/L to umol/L 1.0
12.Lithium
mEq/L to mmol/L 0.5
13. Magnesium
0.01
14. Phospholipid g/dLtog/l
mg/dL to mmol/L 0.323
15. Phosphorus
1.0
16. Potassium nEq/L to mmol/L
Meq/L to mmol/L 1.0
17. Sodium
18. 1hyroxine HE/dL to nmol/L 12.9
19. Total Protein g/dL to g/L 10
mg/dL to mmol/L 0.0113
20. Triglyceride
21. Uricacid mg/dL to mmol/L 0.0595
22. pCO2 mm/Hg to kPa 0.133
23. pO2 mm/Hg to kPa 0.133

TemperatureConversions:
Centigrade (C) to Kelvin ("K) C+273
Centigrade ('C) to Fahrenheit ('F) Cx 1.8)+32
Fahrenheit (°F) to Centigrade ('C} (°F 32) x 0.5565

175
 Chemicals Used for Reagent Preparation:
1Analytical Reagent Grade (AR)
I t is important for qualitative and quantitative
Specifications were established by the Americananalyses;
Chemical
essential for accuracy.
Labels these reagents either state the
on Society (ACS).
actual impurities for each chemical lot
maximum allowable impurities or list the
Use: trace metal
( percentage of impurities).
analysis and preparation standard solutions
of

2.Utrapure reagents
These types of reagents have been
Exomple: Spectrograde, nanograde andthrough
put additional purification steps.
Use: HPLE pure
chromatography, atomic absorption and immunoassays
3. Chemicaly Pure
(CP) or Pure Grade
The impurity limitations of this os
this type of chemicalare
the tolerance limits of usually not stated- it fails to reveal
impurities.
Preparation of these chemicals is not uniform.
Purity is
usually delivered by measurement of melting
It is notrecommended for research and point or boiling point.
reagent blank is included. analytical chemistry unless further puritication or a

4 Technical or Commerclal Grade

t i s used
primarily in manufacturing.
It should never be used in clinical laboratory testing.
5. United States
Pharmacopoela (USP)
I t is approved for human
and National Formulary (NF)
for laboratory analysis.
consumption ( not injurious to individuals) but may not be applicable
Use: for drug
manufacturing
Three
1.
Grades of Reagent Water:
Type Reagent Water
l
i t is used for test methods
requiring minimum
I t is used for procedures that interference.
require maximum water
It should be used immediately (storage is discouraged) purity
after
for accuracy and
precision.
I t is production.
used for utramicrochemical analyses, measurements of
concentrations, tissue or cell methods (microscopy) and preparationnanogram
of
or
subnanogram
Use: flame Photometry, AAS, blood standard soButions.
gases and pH, enzyme studies,
trace metal and iron studies electrolyte testing, HPLC,
2. Type lI Reagent Water
t is utilized for hematology, microbiology, immunolog8y and chemistry.
t i s acceptable for
preparation of reagents and quality control materials.
3. Type HI Reagent Water
tis utilized for preparation of reagents for urinatysis, parasitology and
It is also
used for washing glassware. histology

176
Notes to Remember:
Water should be classified in terms of type instead of the method of preparation, according to
Clinical and Laboratory Standards Institute (CLSI).
Filtration is the first step before the processes are performed in reagent grade water
preparation.
Processes involved in the prepuration of reagent grade water: distillation, ion exchange, reverse
osmosis and UV oxidation
CLSI and CAP have defined three grades of water purity.

Distilled water
t the condensate collected from steam, and created when water is boiled and vaporized.
It has been purified to remove almost all organic materials.

Deionized water
I t is prepared by using deionizer (anion or cation) and it is free from mineral salts; removed by ion
exchange processes.
or all ions removed, but organic material may still be
t has some present.
is purified from previously treated water such as prefiltered or distilled water.

Notes to Remembe
Testsfor water purity: microbioiogical cotent, pH, resistivity, chemical oxygen demand (COD),
ammonia, ions and metals
Occupational Safety and Health Act (0SHA) requires manufacturers to clearty indicate the lot
number, physicai or biological health hazard of the chemicai reagents, and precautions for safe
use and storage.
The College of American Pathologists (CAP) recommends that a laboratory document cuture
growth, pH and specific water resistance on reagent grade water.
Water may be distilled more than once and each distillation cycle removes more impurities.
Water can also be purified by ultrafiltration, UV light, sterilization or ozone treatment.
Detergent-contaminated water will have an alkaline pH.
Hard water contains calcium, iron and other dissolved elements.
The most commonly found organisms in water after the purification process is complete are
Gram-negative bacteria.

Irres ofSolutions Usedin the Clinical Laboratorv

1. Dilute Solution -it has the presence of relatively ittle solute
2. Concentrated Solution-it has a large quantity of solute in solution
3. Saturated Solution -a solution in which there is an excess of undissolved solute particles
4. Super Saturated Solution-it has a greater concentration of undissolved solute particies than
does a saturated solution of the same substance

177
 Reference Materials:
Calibration materials should meet the identity,
labeling and performance reguirement of CLSI
(formerty NcCLS).
1. Primary Standard
t is a highly purified chemical that can be measured directly te produce a substance of exact
known concentration
the International Union of Pure and
Applied Chemistry (IUPAC) has established the criteria for
primary standard.

2. Secondary Standard
it is a substance of lower purity whose
concentration is determined by comparison with a
primary standard

LABORATORY SAFETY
Class of Type of Hazard Type of Extinguisher
Fire
1. Type A Ordinary Combustibles Water, dry chemical and
cloth, paper,rubbish, plastics loaded steam

2. Type B
and wood
Flammable liquids Dry chemical, carbon dioxide
3. Type C
grease, gasoline,paints and oil halon foam
Electrical equipment and Carbon dioxide, dry chemical

4. Type D
motor switches and halon
Flammable metals Metal X
mercury, magnesium, sodium -should be fought by fire
and lithium fighters only
5. Type E Detonation (arsenal fire) Usually allowed to burn out
and nearby materials
protected
Source: National Fire Protecdon Association (

Halogenated hydrocarbon extinguishers are recommended particularly forcomputer

equipment.
Do not use water on burning liquids or electrical equipment.

Standard Hazards-Identification SYstem (diamond-shaped color coded symbol):

1. Blue Quadrant Health hazard
2. Red Quadrant Flammable hazard
3. Yelow Quadrant Reactivity/Stability Hazard
4. White Quadrant Other Special Information

178
Notes to Remenber
1. Personal Protective Equlpment (PPE)
A l l contaminated personal protective equipment must be removed and properly disposed off
before leaving the laboratory and must not be taken home or outside the laboratory.

2. Disinfection
Common decontamination agents: heat (250°C for 15 minutes), ethylene oxide, 2%
glutaraidehyde, 10% HO2 10% formalin, 5.25% hypochlorite (10% bleach), formaldehyde,
detergents, phenols, UV radiation, ionizing radiation and photo-oxidation.
Bleach should be in contact with the area for at least 20 minutes.
hepatits B virus in 10 minutes and HIV in
10% solution of common household bleach inactivates
2 minutes.

3. Chemical Precaution
fa chemical spill occurs, the first step should be to assist/evacuate personnel.
the quantities of chemicals needed
Arrangements for the storage of chemicals will depend on
and the nature or type of chemicals.
Poisonousvapors: chloroform, methanol, carbon tetrachloride, bromide, ammonia,
formaldehyde and mercury
benzene,
Flammable and combustible solvents:acetone, ethanol, toluene, methanol, xylene,
isopropanol and heptane
cabinets is 5
The maximurn working volume of fiammable solvents allowed outside storage
gallons/room.
Aflamnable liquid has a flash point below 37.8°C and combustible liquids at or above 37.8°C
never be added to
Strong acids bases should be neutralized before disposal water should
or
concentrated acid.
Some chemicals deteriorate over time and become hazardous ether forms explosive

peroxides.
with volumes
Safety carriers for transport should be provided for bottles of acids and alkalis
greater than 500mL
>Benzidine is a known carcinogen.

4. Fume Hoods and Safety Shower

Fume hoods must be examined periodically as to ventilation (velocity of 100-120 ft/min),
direction of airflow and smoke testing (to locate dead or turbulent areas in the work place).
I t i s recommended that safety showers deliver 30-50 gal/minute of water at 20-50 psi.

5. Other significant information

T h e parts of the body most frequently subject to injury in clinical laboratory are the eyes,
offer eye protection).
mouth, respiratory and digestive tracts (contact lenses do not
Specimens should remain "capped" during centrifugation
because biologic specimens produce
infection.
finely dispersed aerosols that are a high-risk source of
T h e speed of a centrifuge should be checked once every 3 months by a tachometer.
rmust wash their hands after removal of gloves, after any contact
with blood or body
Employees
fluids, and between patients.

179
Gloves should not be washed and reused
because
difficult to remove (Doebbeling et al, 1988 cited by Mcmicroorganisms
Pherson
that adhere to gloves are

Masks, protective eyewear, or face shields must be worn to and Pincus 2017).
the mouth, eyes, or nose. prevent exposure from splashes to
A protective
equipment that has the potential for
including laboratory coats, must be removed beforecoming into contact with infectious material,
be taken home or outside the leaving the laboratory area and must never
laboratory (such as during lunch or personal breaks).

180
 ENDOCRINOLOGY

Endocrine system
t is a network of ductless glands that secrete hormones directly into the blood.
It is considered to be the regulatory system of the body.
It is regulated by means of control of hormone synthesis rather than by degradation.

Hormones
into the blood stream and
These are chemical signals produced by specialized cells, secreted
carried to a target tissue.
of an individual.
They play an important role in the growth and development
feedback mechanism.
They are regulated by the metabolic activity either positive or negative
of extracellular and
Major function: To maintain the constancy of chemical composition
intracellular fluids, and control metabolism, growth, fertility, and responses to stress.
There are hormones influenced by physiologic factors such as age the elderly secrete less
cortisol.
triiodothyronine, parathyroid hormone, aldosterone, and

Feedback Mechanis1ns:
1. Positive feedback system
t is a system in which an increased in the product results to elevation of the activity of the
system and the production rate (example: gonadal, thyroidal and adrenocortical hormones).
2. Negative feedback system
t is a system in which an increased in the product results to decreased activity of the system
and the production rate (example: leutenizing hormone)
Typesof Hormone Acions
1. Endocrine
I t is secreted in one location and release into blood circulation; binds to specific receptor to elicit
physiological response.
2. Paracrine
i t is secreted in endocrine celis and released into interstitiai space; binds to specific receptor in
adjacent cell and affects its function.
3. Autocrine
I t is secreted in endocrine cels and sometimes released into interstitial space;binds to specific
receptor on celi of origin resulting to self-regulation of ts function.
4. Juxtacrne
t is secreted in endocrine cells and remains in relation to plasma membrane;acts on immediately
adjacent cell by direct cel-to-cell contact.
5. intracrine
i t is secreted in endocrine cells and remained as well as function inside the synthesis of origin.
6. Exocrine
I t is secreted in endocrine cells and released into lumen ofgut; it affects theirfunction.
7. Neurocrine
I t is secreted in neurons and released into extraceliular space; binds to receptor in nearby cell and
affects its furction.
8. Neuroendocrine
I t is secreted in neurons and released from nerve endings; interacts with receptors of cells at
distant ste.
181
Control of Homone Secretion:
The majority of endocrine functions are regulated through
pituitary gland, which in turn is
the
controlled by secretions from the
hypothalamus
Classification of
A. Peptides and Hormones
Proteins According to Composition or Structure:
These are synthesized and stored within the
cel! in the form of secretory
cleaved as needed. granules and are
They cannot cross the cell membrane due to their large molecular size and
effects on the outer surface of the cell. thus; produce their
They are water soluble and not bound to carrier
protein.
1. Glycoprotein
FSH, HCG, TSH, erythropoetin

2.Potypeptides
ACTH, ADH, GH, angiotensin, calcitonin, cholecystokinin,
gastrin, giucagon, insulin, melanocyte-
stimulating hormone (MSH), oxytocin, PTH, prolactin, somatostatin
B.Steroids
These are lipid molecules that have
cholesterol as a common
precursor.
They are produced by adrenal glands, ovaries, testes and
placenta.
They are water insoluble (hydrophcbic) and circulate bound to a carrier
Examples: aldosterone, cortisol, estradiol, progesterone, testosterone andprotein.
activated vitamin Da.
C. Amines
They are derived from an amino acid and they
hormones.
are intermediary between steroid and protein

Examples: epinephrine, norepinephrine, triiodothyronine and thyroxine

Hypothalamus
t is the portion of the brain located in the
walls and floor of the third ventricle.
It is above the pituitary gland, and is
connected to the posterior pituitary by the
(pituitary stalk). infundibulum
t is the link between the nervous
system and the endocrine system.
The supraoptic and paraventricular nuclei
produce vasopressin and oxytocin.
The neurons in the anterior portion
release the following hormones (hypophyseal
hormoanes)
thyrotropin-releasing
hormone (TRH); gonadotropin-releasing
hormone(Gn-RH); somatostatin
also known as growth hormone inhibiting hormone
(GH-IH); 8rowth hormone releasing
hormone (GH-RH); and prolactin-inhibiting factor (PIF).

182
ENDOCRINE GLANDs
PINEAL GLAND
t is attached to the midbrain.
it secretes melatonin that decreases the pigmentation of the skin.
Secretions are controlied by nerve stimuli.

PITUITARY GLAND (Hypophysis)

t is known as the "Master Gland".
It is located in a small cavity in the sphenoid bone of the skull called the sella turcica or Turkish saddle.
t is connected by the infundibular stalkto the median eminence of the hypothalamus.
All pituitary hormones have circadian rhythms.

A. Anterior Pituitary (Adenohypophysis)

t is the "true endocrine gland".
I t regulates the released and production of hormones such as prolactin, growth hormone,
gonadotropins (FSH and LH), TSH and ACTH.
The hormones secreted by this anterior lobe are either peptides or glycoproteins.

STYnes of Celts byImmunochemical Test:

1. Somatotrophs -secrete growth hormone
2. Lactotrophs or mammotrophs -secrete prolactin
3. Thyrotrophs - secrete TSH

4. Gonadotrophs secrete LH and FSH

5. Corticotrophs secretes proopiomelanocortin (POMC is cleaved within the pituitary to produce
ACTH, B-endorphin, and B-lipotropin)

Notes to Remember.
The hypothalamus coordinates with the anterior pituitary by synthesizing its own trophic
hormones that are specific for each of the cell population. These trophic hormones pass through
the infundibular stalk to the adenohypophysis.

Hormones Secretedby the Anterior Pituitary Gland:

Growth Hormone (GH)/Somatotropin
it is the most abundant of all pituitary hormones.
t is controlled by GH+-RH (the amount released) and somatostatin (governs the frequency and
duration of secretory pulse).
It is structurally similar to prolactin and human placental lactogen.
Its secretion is erratic and occurs in short burst.
its overall metabolic effect is to metabolize fat stores while conserving glucose.
Major stimulus: deep sleep (markedly increased GH)
Major inhibitor: somatostatin
Physiologic stimuli fincreased GH); stress, fasting and high protein diet
Pharmacologic stimuli (increased GH)sex steroids, apomorphine and levodopa
GH suppressors: glucocorticoids and elevated fatty acid
Increased: acromegally, chronlc maBnutrition, renal disease, cirrhosis and sepsis
Decreased: hyperglycemia, obesity and hypothyroidism
Method.chemiluminescent irnmunoassay
Reference volue (fosting: <7 ng/mt.

183
 Disorders
1. GH deficiency (GHD)
a. Idiopathic growth hormone deficiency
I t is the most common cause
In children with
of GH deficiency in children.
pituitary dwarfism, normal proportions
are retained and show no inteliectual
abnormalities.
b. Pituitary adenoma
I t is the most common
etiology in adult-onset GH deficiency.
2. Acromegaly
t is due to
overproduction of GH (> 50 ng/ml or 2210 pmol/l).
Dagnosti
Potient
Tests;
Preparation:complete rest 30 minutes before blood collection
Specimen requirement: preferably fasting serum
1. For GH deficiency
a.
Screening test: Physical activity test (Exercise test)
Result of the test: elevated serum GH
If GH fails to increase, confirmation must
be made.

b. Confirmatory test
Insulin Tolerance Test-gold standard
Arginine stimulation test-2d confirmatory test
Procedure:24-hour or nighttime monitoring of GH
Interpretation:Failure of GH to rise >5ng/mL (adults) and> 10 ng/mL (child) in ali the tests is
confirmed GH deficiency.
GHD in childhood is defined by failure of serum GH to reach defined levels when at least two
different pharmacologic stimuli are used.

2. For acromegaly
a.
Screening test: Somatomedin C or insulin-like growth factor 1 (IGF-1)
IGF-1 is produced in the liver.
IGF-1 is increased in patients with acromegaly.
IGF-1 is low in GHD.

b. Confirmatory test: Glucose Suppression Test -OGTT (75g glucose)

Blood is collected every after 30 minutes for 2 hours;
fasting sample is required.
Interpretatonof resut for acromegaly:
A normal response for this test isa suppression of GH less than 1ng/ml.
I f GH fails to dectine less than 1 ng/ml, it is
Failure of GH to be suppressed below 0.3 Hg/L,
acromegaly
of acromegaly.
accompanied by an elevated IGF-, is diagnostic
Suppression of GH below 0.3 g/L with normal lGF-I excludes acromegaly.
Suppression of GH but increased IGF-1, requires follow-up and monitoring.

184
Gonadotropins Follicte Stimulating Hormone (FSH) and Leutinizing Hormone (LH)
I t is an important markers in diagnosing fertility and menstrual cycle disorders.
It is present in the blood of both male and female at all ages.
FSH aids in spermatogenesis (male).
LH helps Leydig cells to produce testosterone (male), and for female, it is necessary for
ovulation and the final follicular growth.
LH acts on thecal cells to cause the synthesis of androgens, estrogens (estradiol and estrone)

and progesterone.
Elevation of FSH is a clue in the diagnosis of premature menopause.
Increased of FSH and LH after menopause is due to lack of estrogen.

Thyroid Stimulating Hormone (TSH)

it is aiso known as thyrotropin.
It is the main stimulus for the uptake of iodide by the thyroid gland.
t acts to increase the number and size of folicular cells; it stimulates thyroid hormone

synthesis.
t is composed of 2 mono-covaiently linked to a and B subunits; a subunit has the same amino

acid sequences of LH, FSH and HCG

The 3-subunit carries the specific information to the binding receptors for expression of

hormonai activities.
Blood levels may contribute in the evaluation of infertility.

Adrenocorticotrophic Hormone (ACTH)

I t is a single-chain peptide without disulfide bonds.
i t is produced in response to low serum cortisol; regulator of adrenal androgen synthesis.
will lead to atrophy of the zona glomerulosa and zona reticularis (layers of the
deficiency of ACTH
adrenal cortex).
Highest level is between 6:00am to 8:00am; lowest level is between 6:00pm to 11:00pm.
increased: Addison's disease, ectopic tumors, after protein-rich meals

Specimen requirement:
Blood should be collected into prechilled polysterene (plastic) EDTA tubes to prevent
degradation of ACTH.
Specimen for testing should not be allowed to have contact with glass because ACTH adheres to
glass surface resuting to decreased levels
Best time for collecting specimen: 8:00am to 10:00am

Prolactin (PRL)
t isa pituitary lactogenic hormone; a stress hormone; a direct effector hormone.
its amino acid structure is similar to GH.
It fanctions in the initiation and maintenance of lactation.
I t also acts in conjunction with estrogen and progesterone to promote breast tissue
development.
Major inhibitor: dopamine (secreted by the hypothalamus)
Consequence of protactin excess: hypogonadism
Increased: pituitary adenoma, infertility, amenorrhea, galactorrhea,acromegaly, renal failure,
polycystic ovary syndrome, cirrhosis, and primary and secondary hypothyroidism

185
 Prolactin serum level >200mg/dL:
pituitary tumor (prolactinoma can result in anovulation)
Specimen requirement: blood should be collected 3 to 4 hours after the individual has
awakened; fasting sample
Highest serum level (during sleep): 4:00am and 8:00am, 8:00pm and
10:00pm
Method:immunometric assay
Physiologic
stimuli (increased): exercise, sleep, stress, postprandially, pain, coitus,
pregnancy, nipple stimulation or nursing
Pharmacologic (increased): intake of verapamil, phenothiazines, olanzapine, prozac, cimetidine
and opiate
Reference value: Male: 1-20 ng/ml (1-20
ug/L)
Female: 1-25 ng/ml (1-25 ug/L)

Notes to Remember.
I t is essential to obtain TSH and free T4
(or total thyroxine and Ts resin uptake) to eliminate
primary hypothyroidism as a cause for the elevated prolactin.
Thyroid hormone replacementtherapy will usually return the PRL to normal piasma level.
Three specimens should be obtained at20-to 30-minute intervals
because of physiologic stimuli
these samples can be measured
separately andtheir results averaged, or, an equal aliquot from
each samplecan be pooled into one final
sample that is then analyzed.
Elevations inPRL due to physiologic and
pharmacologic stimuli rareiy exceed200 ng/ml.
B. Posterior Pituitary (Neurohypophysis)
It is capable of releasing the hormones'
oxytoxin and vasopressin but not capable of producing
it.
The hormones released by
neurohypophysis are synthesized in the magnicellular neurons of the
supraoptic (ADH) and paraventricular nuclei (oxytocin) of the hypothalamus, and stored in the
nerve terminals that end in the
posterior pituitary.
The release of the hormones occurs in
response to serum osmolality or by suckiing.
Hormones produced by the neurohypophysis are controlled
by the central nervous system
(CNS).
1. Oxytoxin
I t is a nonapeptide, very similar in composition to ADH.
It is secreted in association with a carrier protein.
It stimulates contraction of the gravid uterus at
Itis released in response to neural stimulation of
term-"Fergusson reflex."
receptors in the birth canal and uterus, and of
touch receptors in the breast.
Itplays a role in hemostasis at thee placental site followingdeivery.
It stimulates muscle contraction during delivery and lactation with bursts of oxytocin secretion
occurring with anticipation of nursing or on hearing a baby cry.
Synthetic preparations: to increase weak uterine contractions during labor and to aid
in lactation

186
2. Anti-Diuretic Hormone (ADH)/Arginine Vasopressin (AVP)
t is a nonapeptide that acts on the distal convotuted and colecting tubules of the kidneys.
Major function:to maintain osmotichomeostasis by regulating water balance
It decreases the production of urine by promoting reabsorption of water by the renal tubules
maintains water homeostasis.
It increases blood pressure a decrease in blood volume or blood pressure will likewise
stimulate ADH release.
Vi factor release from
It is potent pressor agent and affects blood clotting by promoting
a
Willebrands factor) release from the endothelium.
hepatocytes and factor Vll (von
Physiologic stimuli to ADH secretion: nausea,cytokine, hypoglycemia, hypercarbia and nicotine
and emotional stress due to major
Physiologic stimuli to ADH release: dehydration, physical
surgery
Potent physiologic stimuli to ADH release: emetic stimuius
lithium and demeclocycline
Inhibitors of ADH release: ethanol, cortisol,
Reference value: 0.5-2 pg/ul

InRelation to Osmolality osmolality

Principal regulator of ADH secretion: increased plasma and suppressed
stimulated at a serum osmolality of > 295 mOsm/kg
ADH secretion is maximally
when the osmolality is less than 284 mOsm/kg.
stimulate
osmoreceptor cells, which then
A rise in effective osmolality shrinks the hypothalamic
ADH production in the supraoptic and
the thirst center in the cerebral cortex and stimulate
paraventricular nuclei.
Conversely, a decline in effective osmoiality causes swelling of the osmoreceptor cells, resulting
in inhibition of ADH production.

Clinical Disorder: results in polyuria (23L of urine/day).

Diabetes Insipidus is deficiency of ADH; severe

Clinical picture includes:

1. Normoghycemia
2. Polyuria with low specific gravity
3. Polydipsia (secondary polydipsia)
4.Polyphagia occassional

Major Types of Diabetes Inisipidus:

(Hypothalamic/Neurogenic/Cranial/CentralDiabetes Insipidus)
a. True Diabetes Insipidus
it is deficiency of ADH with normail ADH receptor.
ADH.
it is due to failure of the pituitary gland to secrete
there is a large volume of urine excreted (3-201/day).

b. Nephrogenic Diabetes Insipidus

ADH but abnormal ADH receptor renal resistance to ADH
it is characterized by having normal
action.
failure of the kidneys to respond to normal or elevated ADH levels.
it is due to

nephrogenic Di is either congenital or acauired.

187
 Diagnostic Test for Diabetes Insipidus: Overnight Water Deprivation Test (Concentration Test)
wCungenlt Fasting: 10:00 pm onwards (8 to 12 hours)
After8 to 12 hours without fluid intake, urine osmolality does not rise above 300 mOsm/kg.
I n neurogenic Di, ADH fevels are low and the kidneyrapidly acts to conserve water in response to

Nephiognio I nexogenous ADH administration.

nephrogenie DI, ADH levels are either normal or increase, and administration of additional
TADH
ADH has littte or no effecton renal water reabsorption.

Notes to Remember
Without ADH, urine osmolality is abet50-mesm/kg, while serum
osmolality-increases.
T o establish diagnosis, the patient's ability to produce concentrated urine must be tested.
f anything occurs to disrupt the
thirst mechanism or the
prevent patient from drinking water,
rapid development of hypernatremla will occur.
yroneirapproprtate-artidturetit hofmone secPHOn SADA) refers to the sustained
production of ADH in the absence of a known stimuli. It is characterized by decreased urine
AD volume, lew-plasme-esmolatity and normal or elevated urine sodium levels (low serum
Na Rtl uietectrolytes).

THYROID GLAND
I t is also known as thte butterfly-sheped gland.
Itis consist of twetebes (one on either side of the trachea) located in the lower part of the neck,
just below the voice box (larynx).
The lobes are connected by a narrow band called the
isthmus.
By 11 weeks of gestation, the gland begins to produce measurable amounts of hormon.
Follicleis the fundamental structural unit of the thyroid gland.
2 types of celsfollicular cells (Tand Ta
parafollicular or Ccells (Calcitonin)
Thyrogiobutin is a glycoporotein; it acts as a preformed matrix containing tyrosy! groups; it is stored in
the follicular colloid of the thyroid gland.

Bleenthesis of Tnvreid-HoEmones
ledine is the most important element in the biosynthesis of thyroid hormones.
lodination of tyrosine residues in thyroglobulin results in formation of
monoicdotyrosine (MIT)
anddiiodotyrosine (DIT)
TSHstimulates synthesis of thyroid-hermones.
Protein beund hormones are metabolically inactiye free hormones (FT and FTa) are the
physiologically active portions of the thyroid hormones.
Protein-bound thyroid hormones do not enter cells and are consldered to be
and function as storage stes for circulating thyroid hormones.
biologicaly inert
The minute levels of free hormone fractions readily enter cells by
specific membrane transport
mechanism to exert their biological effects; the effects are mediated by Ta receptors located in
the nucleus of the cell.
When lodidesources are diminished, MIT is produced in greater quantities, leading to increased
Tformation and release.
4 Reverse Ta is produced by removal of one iodine from the inner ring of T metabolicaly inactive;
product of Ta metabolism.

188
 The hypothalamic-pituitary-thyroid axis (HPTA) is the neuroendocrine system that regulates the
production and secretion of thyroid hormones.
lodine intake below 50ug/day is an indication of the deficiency of hormone secretion.
Thyroid hormones affect synthesis, degradation, and intermediate metabolism of adipose tissue
and circulatinglipids

Eunctions of Thyrold Hormones

1. For tissue growth.
2. For mental development.
3. For development of the central nervous system.
4. Elevated heat production.
S. Control of oxygen consumption.
6. It influence carbohydrate and protein metabolism.
7. For energy conservation.

Maior Thyroid Hormones

1. Triodothyronine (T. 3,5,3" triiodothyronine.
I t has the most active thyroid hormonal activity.
Almost 75-80% is produced from the tissue deiodination of Ta- conversion of Ta to T takes place
in many tissues, particularly the liver and the kidneys.
The principal application of this hormone is in diagnosing Ts thyrotoxicosis.
It is a better indicator of recovery from hyperthyroidism as well as the recognition of recurrence
of hyperthyroidism - it is helpful in confirming the diagnosis of hyperthyroidism, especially in
patients withno or minimally elevated Ta.
An increase in the plasma level of Ta is the first abnormality seen in cases of hyperthyroidism.
Reference value: Adult= 60-160 ug/dL or 0.9-2.46 nmol/L
Children 1-14 years old = 10S-245 ng/dL or 1.8-3.8 nmol/L

2. Tetraiodothyronine (Ta/3,5,3*'5' tetraiodothyronine

It is the principal secretory product.
t has the major fraction of organic iodine in the circulation.
It is a prohormone for Ia production.
Al! circulating Ta originates in the thyroid gland-it is secreted 100% in the thyroid gland.
The amount of serum Ta is a good indicator of the thyroid secretory rate.
Elevated thyroxine causes inhibition of TSH secretion, and vice versa.
Reference value: S.5-12.5ug/dL or 71-161 nmol/L (adult)
11.8-22.6 ug/dL or 152-292 nmol/L (neonate)

Thyroid Hormone Binding Proteins:

1. Thyroxine-Binding Globulin fTBG)
i t transports majority of Ta (affinity for T is lower than Ta).
it transports 70-75% of total Ta.
2. Thyroxine-Binding Prealbumin (transthyrctin}
it transports 15-20% of total Ta.
T, has no affinity for prealbumin.
3. Thyroxine-8inding Albumin
i t transports Ta and 10% of Ta.

189
 Thyroid autoantibodies responsible for autoimnmune thyroid disorders
1. Thyroperoxidase
(TPO)-is involved in the tissue destructive process (Hashimoto's disease)
2. Thyroglobulin (Tg)-
3. TSH receptor (TR)-is involved in Grave's disease

Clinical Dleorder
Screening of thyroid disorders is recommended when a person reaches 35 years old and every 5
years thereafter.

1. Hyperthyroldism
It refers to an
excess of circulating thyroid hormone.
Signs and symptoms: tachycardia, tremors, weight loss, heat intolerance, emotionallability and
menstrual changes
Primary hyperthyroidism - elevated Ta and T, decreased TSH
Secondary hyperthyroidism increased FTa and TSH (due to primary lesion in the pituitary gland)
a. Thyrotoxicosis
i s applied to a group of syndromes caused by high levels of free thyroid hormones in the
circulation.
Ta thyrotoxicosis or Plummer's disease: FTa increased but FTa normal with low TSH
Tathyrotoxicosis: Tanormal or low but Taincreased with low TSH

b.Graves't disease (dijuse toxiegoter)

is the most common cause of thyrotoxicosis.
Itis an autoimmune disease in which antibodies are produced that activate the TSH receptor.
adendt occurs 6x more commonly in women than in men.
Features: exophthalmos (bulging eyes) and pritibial myxedema
Diagnostic test: TSH receptor antibody test

. Riedel's thyroiditis
t h e thyroid turns into a woody or stony-hard mass.

d. Subclinical hyperthyroidism
shows no dinical symptoms but TSH level is low, and FTs and FTa normal.

e. Subacute granulomatous/Subacute nonsuppurative thyroiditis/De Quervain' thyroiditis (painful thyroiditis)

in
itis associated with neck pain, low-grade fever and swings thyroid
function tests.
the thyroidal peroxldase (TPO) antibodies are absent; FSR and thyroglobulin levels are elevated.

2. Hypothyroidism
hormone are available to tissues.
I t develops whenever insufficient amounts thyroid (levethyroxine).
of

t is treeted with thyroid hormone replacement therapy

coarsened skin, cold intolerance and mental
signs and symptoms: bradycardla, weight gain,
duliness

190
 a. Primary hypothyrodism
i t is primarily dueto deficiency of elemental iodine (Ts and Ta TSH).
t is also caused by destruction or ablation of the thyroid gland.
Other causes: surgical removal of the gland; used of radioacthve lodine for hyperthyroidism
treatment; radlation exposure; drugs such as lithium

Hashimoto's disease (chronic autoimmune thyroiditis)

i s the most common cause of primary hypothyroldism.
t is characterlzed by a thyrold replaced by a nest of lymphold tissue sensitized T
ymphocytes/autoantibodies bind to cell membrane causing cell tysis and inflammatory reaction.

t is associated with enlergement ofthe thyrotdgandtgoter).

lab result: high TSH and positive-TPeantibody

Myxedema
i t describes the peculiar.nonpittingswelling of the skin.
the skin becomes infitrated by mueopohsacchartdes.
clinical features: puffy"face, weight gain, slow speech, eyebrows thinned, dry and yellow skin,
andanemia
yxedema coma is the severe form of primary hypothyroidism

b. Secondary hypothyroidism
it is due to pituitary destructrion or pituitary adenoma.
l a b resuit: Ta and Ta low levels, TSH is also decreased.

c. Tertiary hypothyrodism
it is due to hypothalamic disease
lab resut: Ts and Ta low levels, TSH is also decreased.

d. Congenital hypothyroidism/cretinism
it is a derect in the development or function of the gland.
of the child retarded
symptoms: physical and mentai deveiopment
are

screening test: T ldecreased)

confirmatory test: TSH (increased)
interpretation: TSH value <10 miU/L( no further test)
TSH value 10-20-miU/L( repeat test in 2-6 weeks)
TSH 20 mlU/L- for endocrinologic evaluation to diagnose hypothyroidism

e. Subclinical hypothyroidism
increased
lab result: Ts and Ta normal but TSH is slightly

Thyroid Function Tests

1. TRH Stimutatton Test (Thyrotropin Releasing Hormane)
it measures the relationship between the TRH'and TSHsecretions. TSH
who both had undetectable
tis used to differentiate euthyroid and hyperthyroid patients
levels.
resistance syndromes.
it may also be helpful in the detection of thyroid hormone
itis used to confirm borderline cases and euthyroid Grave's disease
dose needed: 500 4g TRH by V

191
 increased: primary hypothyroidism
decreased: hyperthyroidisn
2. TSH Test
t i s the most
important
thyroid dysfunction. thyroid
function test the best method for
detecting clinicalily significant
tis the most
clinicaly sensitive assay for the detection of
it helps in the
early detection of primary thyroid disorders
it is used to
differentiate hypothyroidism.
it is used to monitor and primary hypothyroidism from secondary hypothyroidism.
the sensitlvity of the adjust thyroid hormone replacement therapy.
third-generation
subclinical disease-or a mild
TSH assays has ied to the
ablity to detect what is termed
the large reciprocal degree of thyroid
dysfunction (due to
change in TSH levels seen for even small changes in free
reference vatues: 0.5-5 uU/ml Ta).

Increased TSH
1. Primary hypothyroidkism Decreased TSH
1. Primary hyperthyroidism
2. Hashimoto's thyroiditis
3. 2. Secondary and
Thyrotoxicosis
due to pituitary tumor Tertiary hypothyroidism
4. TSH antibodies 3. Treated Grave's disease
5. Thyroid hormone 4. Euthyroid sick disease
resistance 5. Over replacement of thyroid hormone in
3. Radioactlve lodine Uptake (RAIU) hypothyroidism
it is used to measure the
it is
ability of the thyroid gland to trap iodine.
helpful in establishing the cause of
hyperthyroidism.
high uptake indicates metabolically active
gland (active hormone production).
high uptake +TSH deficiency autonomous
thyroid activity
4. Thyroglobulin
(Tg) assay
i t is normaly used as a
it is used in
postoperative marker of thyroid cancer
monitorlng the course of metastatic or
differentiated tumors typically display a 10-foid increase inrecurrence of thyroid cancer( a well-
when Tg in response to a high
measuring thyroglobulin as a tumor marker for TSH).
simultaneous sample for thyroglobulin antibodies. thyroid cancer, always check a
it differentiates subacute
thyraiditis ( Te) from thyrotoxicosis factitia (
increased: untreated and metastathc differentiated Tg).
thyroid cancer, nodular golter and
hyperthyroldism
decreased: Infants with goitorous hypothyroidism and
reference value: Adult= 3-42 ng/mLor g/ml thyrotoxicoxis factitia
Infant 38-48 ng/mi or ug/ml
methods for testing: double-antibody RIA, ELISA, IRMA
immunochemiluminescent assay (ICMA
5.Reverse Ta (r Ts
t i s formed by the removal
i t is an
of one iodine from the inner ring of Ta.
endproduct of Te metabolism; the 3 major circulating thyroid hormone.
it identifles patients
t is used to assessed
with euthyroid sick syndrome (elevated ra.
borderline or conflicting laboratory results.

192
 reference value: 38-44 ng/dt

6. Free Thyroxine Index (FTI or T)

it indirectly assesses the level of free Ta in blood.
it is based on the equilibrium relationship of bound T, and FT
it is important in correcting euthyroid individuals.
it is elevated in hyperthyroidism and decreased in hypothyroidism.
reference value: 5.4-9.7
FTI I A x TU(%) or TT x THBR
100

7. Total Ta (TTs), Free T, (FT) and FreeT (FT)

FTa test is used to differentiate drug induced TSH elevation and
hypothyroldism.
the value of TTy or FT, is in confirming hyperthyroidism.
direct/reference method: Equilibrium dialysis (FTa)

8. Ta Uptake most
available binding sites of the thyroxine-binding proteins,
i t measures the number of
notably TBG; a test for TBG.
serum level of TBG.
it does not measure the level of Ta in serum but it reflects the
results to elevated TBG result, and vice
versa.
is inversely related to TSG decreasedTa uptake
chronic liver disease
increased: hyperthyroidism, euthyroid patients,
acute hepatitis
decreased: hypothyroidism, oral contraceptives, pregnancy,
reference vaBue: 25-35%

9. Thyroxine binding globulin (TBG)

and FTa, or abnormalities in the relationship of the total
it is used to confirm results of FT
thyroxine (TTa) and THBR test.
it is useful in distinguishing between hyperthyroidism (
Ta +
N TBG) and euthyroidism (Ta
and TBG). the
elevated Ta and Ta levels but no correlation with
it helps in the diagnosis of patients having
with clinical findings.
other thyroid function tests, or not compatible
levels T and Ta, but the unbound or free form of these
TBG excess leads to increased serum
hormones in the blood remain unchanged.
hormonal effect: estrogen increases
TBG while androgens depresses TBG
increased: euthyroidism, pregnancy
and estrogen surge
decreased: anabolic steroids and nephrosis
nmol/L)
reference alue: 13-39 ug/dL (150-360
nodules
the most accurate tool in the evaluation of thyroid
10. Fine-needle aspiration- is

TSH
11. Recombinant Human of residual or recurrent disease.
i t is used to test patients with thyroid cancers for the presence

12. Tanned Erythrooyte

Hemagglutination
i t is a measure of antithyroglobulin antibodies.

193
 13. Serum Calcitonin
it is a tumor marker for detecting residual thyroid metastasis in medullay thyroid carcinoma (MTC)
it shouldbe measured before and 6 months after surgery.

14. Pentagastrin (PE) Stimulation Test

i t is used for the diagnosis of MTC.
procedure: an intravenous Pg (0.5 ug/kg body weight) is given within 5 seconds; blood samples
are collected at baseline and 1, 2, 5, and 10 minutes after the start ot the infusion

Notes to Remember.
Abnormal values of total Ta or Ta must be evaluated with TBG measurement.
Free-Frand-TSH are the bestindicatorsofthyroid.status.
Eree T and Ta are more specific indicators of thyroid function than the measurements of total
hormone because the values are not affected by the TBG amount.
Patients with increased Ta-binding protein have an elevated Ts or Ta but not free TaorTSH.
Euthyroid sick syndrome is acutely ill but without thyroid disease low Ta and Ta, and normalor
increased TSH, but elevated rT
Euthyroid with elevated T-binding protein is due to increase in TBG.
Patients with nonthyroidal illness (NTI) have low or low-normal TSH andnormai or low-normal Ta
but very low T during their acute illness.
In severe hypothyroidism, totalCK and LDHvaluesrise moderately.
Calcitonin is measured by two-site immunometric assays using monoclonal antibodies, and it is
also elevated in autoimmune thyroid disorder, hypercalcemia and ali neuroendocrine tumors.
Cutoff value for calcitonin is 4OngL (adults).

sUMMARY OF THRYoID DISORDERS AND LABORATORY TESTs

Disorders Ts TSH FT A T TBG_

1. Grave's disease
2.Primary hypothyroidism N/ N/
3. Hashimoto thyroiditis P N/ NI N/ N/
4. Nonthyroidalilness N/ VMA N
N/T
5. Thyroid hormone resistance
6. Neonatal hypothyroidism N/
N-Normal; V-variable

PARATHYoID GLAND
sometimes within the
It is located on or near the thyroid capsuBe (region of the thyroid gland);
thyroid gland. between the hyoid bone in the neck
normal anatomic site
It may also be found outside their
and mediastinum.

194
Role ofPTH:
prime role: to prevent hypocalcemia (regulates blood calcium)
i t preserves calcium and phosphatewithin normal range.
it promotes bone resorption - release calcium into the blood stream.

it increases renal reabsorption of calcium.

it stimulates conversion of inactive Vitamin D to activated vitamin D.
indirectly stimulates intestinal absorption of calcium.
as calcium levels increase, PTH secretion is suppressed allowing urinary loss of calcium and
calcium to remain in bone.
if calcium levels decrease, PTH is reieased.
Clinical Disrders:
1. Hyperparathyrotdism
a. Primary hyperparathyroidism (physiologic defect lies with the PT gland)
i t is the mest commoncause of hypercaicemia.
is due to the presence ofa ftunctioning parathyroid adenoma.
it is accompanied with phosphaturia.
if it goes undetected, severe demineralization may occur (osteitis fibrosa cystica).
lab results: PTH orhigh normal range, ionized calciun1, hypercalciuria,
hypophosphatemia (fasting state)

b. Secondary hyperparathyroidism
i t develops in response to deerease serum catcium.
4glands.
there is diffuse hyperplasia of all
the patient develops severe bone disease.
causes: vitamin D deficiency and chronic renal failure
lab results: PTH, Vionized calcium

c. Tertiary hyperparathyroidism
i t occurs with secondary hyperparathyroidism.
t h e phosphate levels are normal to high; calcium phosphates precipitate in soft tissues.

2. Hypoparathyroidism
it is due to accidental injury to the parathyroid glands (neck} during surgery-postsurgical cause
other cause: autoimmune parathyroid destruction
individuals are unable to maintain calcium concentration in blood without calcium
supplementation

Notes toRemember.
I n hyperparathyroidism, the distal convoluted tubule reabsorbes bicarbonate as well as
phosphate resuting in acidosis.
Parathyroid hormone normally interferes with bicarbonate reabsorption in the proximal tubule;
therefore, the renal tubular bicarbonate threshold tends to be increased in hypoparathyroidism.
Low PTH Causes elevated bicarbonate
reabsorption alkalosis.
Caicium level « 6 mg/dL (1.5 mnol/) laryngeal stridor and tonic-clonic
Calciurn level 8
-

seizures
mg/dL (2.0 mmol/L) leads to tetany and altered neuromuscular activity
(chvostek's sign and treusseau's sign)

195
 The test method for PTH
measurement involves the use of antibodies that detect both the
amino-terminal fragment and intact PTH.

APRENALGLAND
I t has
pyrarnid-like shape (adult gland) located above the
composed of distinct but conjoined glands, the kidneys.
t is
adrenal medulfla (dark outer adrenal cortex (yellow) and inner
clo &Ouio mahogany).
OC: Cutr corttx yehow
o Adrenal Cortex
I t is the outer region of the
adrenal gland secreting the steroid hormone.
It is the major site of
steroid- heFmone production.
Structure and Synthesis of Cortical Homones:
the cortical hormones
are
composed of a basic structure known
cyclopentanoperhydrophenantrene (CPPP) ring., a 17-carbon skeleton derived from cholesterol.
as

cholestero is the
parent cell of
alt-steroidhormones only G cells convert cholesterol to
-

pregnenolone.
cortical hormones are
it is taken up by LDL synthesizedfrom LDL - this lipoprotein is delivered to theadrenals where
receptors.
the secretion of adrenal
control of the glucocorticolids and androgens is regulated by ACTH, which is under
hypothalamic the
mineralocorticoid secretion iscorticotropin-releasing
controlled by the
hormone (CRH).
renin-angiotensin system.
gm
3Layers of the Adrenal Cortex:
a. zona glomerulosa
(outermost layer) 10%
principal source of aneraleerticoid
Fg b. zona fasiculata
CDRA Alsite of
(middlemost layer) 75%
aC. zona reticularis
glucocorticoid syn hesis; also synthesize unsulfated
DHEA
(innermost layer) 10%
produces androstenedione and
dehydroepiandrosterone (weak androgens).
A. Cortiso
itis the principal glucocorticoid t/ asuituaTO
its synthesis is
regulated by ACTH; It is mostly bound to
in the liver resultirng in glycoprotein, transcortin.
i t stimulates
gluconeogenesis
it is the only adrenal hvperglycemla (anti-insulin effect).
hormone that inhibits the secretion
cortisol is elevated). of ACTH
(when plasma level of
it has anti-inflammatory and
immunosuppressive actions à valuable
rheumatoid arthritis, SLE and multiple
its
secretion is diurnal and is sclerosis. therapeutic agent for
associated with a person's sleep-wake
high levels in the morning (8:00am to 10:00am) and cycle.
specimens: serum (red top), urine; blood lowest at night
urine free cortisol sample should be drawn at(10:00pm
8:00an
to
12:00mn).
levels are sensitive indicators of
corticolism)-24 hour urine collection. adrenal hyperfunction (endogenous
reference alue: 5-25 ug/dL (140-690
nmol/L) at 8 am to 10 am

196
Urinary metabolites:
The liver degrades all the glucocorticoids to metabolites excreted in urine.
1. 17-hydroxyeorticosterdid
method:Porter-Slber Method (yellow color)
reagent: Pheny! hydrazine in HSO4 t aicohol
2. 17-Ketogenic steroids
method: ZitmmermanmReattion (reddish purple)
reagent:Meta-dinitrobenzene
oxidation procedure: Norymberski (Na"bismuthate)

Clinical Disorders:
1.Hypercortisolism (Cushing's syndrome)
it is a group of clinical and metabolic disorders characterized by adrenocortical hyperfunction.
it is caused primarily by excessive production of cortisol and ACTH but decreased aldosterone
and renin.
it is also caused by overuse of corticosteroids.
signs & symptoms: obesity but with thin extremities ("'buffalo hump"), hirsutism,
hyperglycemia, thinning of the skin, poor wound healing,
hypertension, hypercholesterolemia, low WBC count (lymphocytes)
screening tests: 24-hour urinary free cortisol test (four-fold increased)
overnight dexamethasone suppression test (blood level not suppressed)
midnight salivary cortisol test (high saliva cortisol)
confirmotory tests: lowdose dewemethasonesuppression-test
midaight plasma-cortisol (>7.5 ug/dL serum cortisol is confirmatory)
corticotrophin-releasing hormone (CR) stimulation test

Patient Preparation:
For midnight saliva cortisol test, it is recommendedto have the patient avoid smoking on the day
of specimencollection.
For midnight plasma cortisol, hospitai admission is required for at least48 hours, insertion of a
line for V access before 10 pm, and patient should be sleeping at the time of the blood
collection.

Procedurefor
One
Overnight/Rapid Dexamethasone Suppression
of dexarmethasone is
Test:
milligram orally
taken between 11:00pm to 12midnight.
Blood is coliected the following day 8am to 9am, and urine may be tested for 17-0HCS.
Normal patient without Cushing's syndrome has a cortisol value « 5.0 Hg/dL and 17-0HCS of
4mg/g creatinine after the test.
()result: cortisol level notsuppressed (> 5.0 g/dL)
17-OHCS=> 4mg/g creatinine
Bma a uhrs/2 days
Procedure for Lon-dose Dexarnethasone.Suppression Test:
0 . 5 mg oral dexamethasone is given every 6 hours for 2 days.
Cortisol is measured 15 minutes after the oral dose.
24-hour urine and serum samples are also collected
as specimens.
)result: elevated serum cortisol level

197
 Notes to Remember
HPLG-MS the current reference method for measuring urinary free cortisol.
test for excess cortisol
24-hour urine free cortisol is the most sensitive and specific screening
cortisol is affected by diurnal variation.
production using HPLC-MS or GC-MS because plasma
-

unbound circulating cortisol that is freely

The 24-hour urinary free cortisol is a reftection of the
filtered by theglomerulus.
that passes through glomerular filtration.
Urinary free cortisol is the only portion of cortisol
Preservatives for urine sample such as 10 grams of boric
acid may be used for measurement of
cortisol and aldosterone.
aliva) specimen is analyzed by immunoassayto or by liquidchromatography-mass spectrometry
normative values varyaccording the reference laboratory.
(LC-MS/MS);

2. Hypocortisolism
a. Primary hypocorticolism (Primary adrenal insufficiency) destruction aldosterone
It is due to decreased cortisol production
90% of the adrenal cortex;
deficiency; excess ACTH reiease. weight loss,
Disorder: Addison's disease ( hypotension, hyponatremia, hyperkalemia,
hyperpigmentation)
tuberculosis, hemorrhage, HIV-AIDS
Addison's disease is due to autoimmune adrenalitis,
infection.
cortisol, aldosterone and renin; high ACTH)
Screening test: ACTH Stimulation Test (low
Insulin Tolerance Test
Confirmatory test:

adrenal insufficiency)
b. Secondary hypocorticolism (Secondary
with loss of ACTH.
I t is due to hypothalamic-pituitary insufficiency
secretion; absence of hyperpigmentation.
No problem with mineralocorticoid cortisol and ACTH)
Test (delayed response-low
Screening test: ACTH Stimulation
Confirmatorytest: Insulin Tolerance Test

ACTH Stimulationknown
Test Gorsyntropin stimulation-test
I t is also as
adrenal insufficiency (decreased
or no ACTH response)
from tertiany
it differentiates secondary
ACTH responsej.
adrenal insufficiency (increased
IV or IM
Dose:250 Hg of Corsyntropin
Blood collection:anytime of
the day (random)
than 18-20 ug/dl (500-550 nmol/1)
Normal response: cortisol level greater (associated
differentiate primary adrenal insufficiency
baseline may help
Serum ACTH level at tertiary forms
than 50-100 pg/ml) from secondary
or

with an elevated ACTH usualiy greater

leve! <10 pg/mL).
(which would have a low ACTH over baseline suggests primary
adrenal
atdosterone
Failure of to increase by more than (4 ngdL
dysfunction.
and aldosteronestimulator.
Corsyntropin is a syntheticcortisol

198
Insulin Tolerance Test (ITD:
I t is the gold standard for secondary ar1d tertiary hypocorticolism.
I t confims borderline response to ACTH stimulation test.
- with signs and symptoms of
Requirement:adequate hypoglycemia must be attained
hypoglycemia (serum glucose <40 mg/dL)
Oral dose: 0.05 U/kg of insulin
Blood collection:baseline, and then at 15, 30, 45, 60, 90,and 120 minutes following insulin

administration.
Measurement: serum cortisol and glucose
Normal response:increased plasma cortisol 2 18 ug/L or 20 ug/L at anytime duringthe test

OvernightIt Metyrapone
measures theTest
abillty of the pituitary gland to respond to declining levels of circulating cortisol,
thereby secrete ACTH.
It is used as an alternative diagnostic or confirmatory test for secondary or tertiary adrenal

insufficiency.
It is performed only if the ACTH stimulation test gives normal results.
Precaution: it may aggrevate an addisonian crisis in those with central hypocortisolism
Oral dose: 30 mg of metyrapone is given at midnight
Blood collection:blood is collected the following morning at 8 am
Normai response: elevated plasma 11-deoxycortisol >7 ug/dL (200 nmol/L)
(+} Resut: serum 11-deoxycortisol less than 7 4g/dL and cortisol less than 5 ug/dL
Metyrapone is an inhibitor of11 B-hydroxylase (blocks cortisol formation).

Notes to Remember
Andoes8amnottorequire
9am plasma cortisol of3 ug/dL (83 nmol/L) is indicative of adrenal insufficiency and
additionai testing.
>A random cortisol of <10 ug/dL is strongty suggestive of adrenal insuficiency.
Serum ACTH differentiates Cushing's disease (high ACTH) from Cushing's syndrome (zero value
of ACTH).
Impaired response to ACTH stimulation is a definitive diagnosis of primary adrenal insufficiency.
Hyperpigmentation (Addison's disease) occurs if the pituitary responds normally to declining
glucocorticoid levels because secretion of MSH & ACTH are closely linked.
Both 17-OHCS and 17-KS are increased when ACTH stimutation is excessive.

3. Congenttal Adrenal Hyperplasia (CAH)

I t resuts from deficiency of enzymes such as 21-hydroxylase, 11 B-hydroxylase and 3-
hydroxysteroid dehydrogenase-isomerase, necessary for the synthesis of cortisol.
This wil result to decreased plasma cortisol, and increased ACTH and andogen levels.
For the diagnosis of hirsutism and virilization, measurement of serum total testosterone and
DHEA-S wouid be of great help.
The use of 24-hour urine free cortisol test is not consistent with CAH.
Definitive tests: 17-0HP measurement in amniotic fluid
Genotyping cells fron1 chorionic villous sampling- most preferred

199
 Ivpes of GAH:
a. 21-hydroxylase deficiency
it is the most
common form of CAH.
it leads to
hirsutism in women and other
virilization; infertilty and amenorrhea. symptoms caused by excess androgen levels such as.
diagnosis is made
by measuring the level of 17-OHP in amniotic fluid
cells (by Southern (by PCR) or by
blotting) obtained by chorionic villous genotyping
definitive method: genotyping sampling
common
ab
method for detection: ACTH stimulation and
17a-hydroxyprogesterone tests
findings: elevated plasma levels of
increased urinary 17a-hydroxyprogesterone and pregnanetriol
defective gene: CYP21 pregnanetriol and 17-KS
b. 11
8-hydroxylase deficiency
it is the 2d most
common form of CAH.
it is associated with
ab
virilization and hypertension.
findings: increased serum deoxycortisol,
defective gene: CYP1181 and urinary 17-OHCS and17-KS

c.
38-hydroxysteroid
it results to dehydrogenase-isomerase deficiency
elevated ratio of
increased ratio of DHEA to 17a-hydroxypregnenolone to
17a-hydroxyprogesterone and
t is
characterized by
androstenedione.
in male infants. pseudohermaphroditism in femmale infants, and incomplete masculinization
lob findings:elevated serum 17-hydroxypregnenolone,
increased urinary 17-KS pregnenolone and DHEA
defective gene: HSD382
d. 17-hydroxylase deficiency
it is characterized by the inability to convert
hydroxyprogesterone to androstenedione. 17-hydroxypregnenolone to DHEA and
17a
it wil result to
decrease androgen, cortisol and
synthesis. estrogen synthesis; decrease progesterone
indicators: amenorrhea, defective ovarian
hypertension and hypokalemic alkalosis maturation, pseudohermaphroditism in males
lab findings:increased serum
defective gene: CYP17 deoxycortisol

B. Aldosterone (Aldo)
Itis the most potent mineralocorticoid (electro-regulating hormone).
I t is a steroid hormone
that helps regulate water and
potassium} and blood pressure sodium levels in serum electrolytes (sodium, chloride and
depend almost completely on the
interplay between aldosterone and ADH.
tis the main determinant
of renal excretion of
t acts on renal tubular potassium
epithelium to increase retention of Na'and d, and excretion of K* and H.*
t promotes the 1:1 exchange of sodium for
The synthesis of this potassium or hydrogen ions.
hormone is primarily controlled by the renin-angiotensin system.

200
 18-hyroxysterold dehydrogenase is an enzyme needed for the synthesis of aldosterone.
method: RIA and chromatography

Clnical
1.
Deorder:
Primary hyperaldosteronism (Conn's disease)
I t i s caused by aldosterone-secreting adrenal adenoma.
I t i s assoclated with elevated plasma aldosterone and low plasma renin.
I n this conditlon, distal delivery of Na' is increased because increased NaCl reabsorption in the
cortical collecting duct by the acton of aldosterene inhibitssat reabsorptiom in the proximal
elbxasle as the result of velkame-axpansion.
Symptoms: hypertension, hypokalemia, mild hypernatremia and metabollcalkalosis
Partient preparation: patient should be upright for at least 2 hours prior to blood collection
Measurement: (PAC/PRA) ratio, serum aldosterone and cortisol
Screening test: plasma aldo concentration/plasma renin activity ratio (PAC/PRA)
(+) result: >30 ratio- suggestive ofprimary hyperaldosteronism
50 ratio-diagnostlc of primary hyperaldosteronism
Confirmatory test: saline suppression test
oral sodium loading test
fludrocortisone suppression test (0.1 mg fludrocortisone oral intake)
captopril chalenge test (25-50 mg of captopril oral intake)

Saline Suppresslon Test:

t invoves infusing 2 liters of 0.9% saline over 4 hours, or by administering 10-12 mg NaCi
tablets daily for 3 days.
()result:>10ng/dL PAC
2. Secondary hyperaldosteronism
I t occurs as a resut of excassive-production
of renin,
t is accompanied by
elevated plasma levels of aldosterone and renin.
I n this condition, hypokalemia occurs only in conditions that are
accompanied by increased
distal Na+ delivery.
Secondary hyperaldosteronism that resut in hypokalemia: renal artery stenosis, diuretic
therapy, malignant hypertension, and congenital defects in renal salt transport such as
Bartter's syndrome and Gitelman's syndrome

Assaciated
a. Liddle's
Disorders:
syndrome (pseudohyperaldosteronism)
t is a congenital
disorder that is characterized by increased ENaC (epithelial sodium channel)
activity in the collecting duct in the absence of lIncreased aldosterone.
It resembles primary aldosteronism
clinically, but aldosterone and renin levels are low, with
absence of hypertension.

2. Bartter's syndrome (bumetanide-sensitive chloride channel

mutation)
I t is a rare potassium-losing autosomal recessive
disorder, is caused by defective NaCl
reabsorpton in the thick ascending imb of Henie.
Is accompanied by elevated concentrations of
plasma aldosterone and renin.

201
 3. Gitelman's syndrome (thiazide-sensitive transporter mutation)
I t is associated with the defect in NaCl reabsorption that occurs in the distal convoluted tubule
I t is accompanied by
elevated
aldosterone.
3. Hypoaldosteronism
I t is due to destruction of the adrenal
glands and deficiency of glucocorticoid.
I t is also associated with
21-hydroxylase deficiency.
Symptoms: hyperkalemia and metabolic acidosis.
Tests: a. Furosemide stimulation test or upright posture-(4) result: low aldo levels
b. Saline suppression test
(+) result: high aldo leveis
-

Patient Preparation and Sample Collection:

Blood samples shoutd be drawn in the
morning before the patient has gotten out of bed
plasma level is influenced by postural changes.
In case the patient has
gotten out of bed, patient should be upright for at least 2 hcurs prior
to
blood collection to avoid markedly increased results.
Blood sample for renin
should be drawn into an iced EDTA tube, which inactivates the
enzymes (angiotensinases)

Notes to Remember.
>Postural stimulation test is an aldosterone test.
Aldosterone levels are lower at night.
Aside from posture, dietary intake of
sodium and potassium is controlled
collection. prior to blood
PAC/PRA ratio is measured after having the patient remain
Plasma samples for aldosterone upright for at least 2 hours.
measurement is treated with extraction
aldosterone from plasma proteins. reagent to remove
Urine samples for aldosterone are
assayed using acid hydrolysis and extraction.
Florinef is a synthetic mineralocorticoid.
Mineralocorticoid deficiency can lead to hyponatremia caused
The effect of the by increased renal sodium loss.
renin-angiotensin system is to increase blood pressure.
C.Weakandrogens/Adrenal
They serve as
androgens
for the
preeursers production of more potent androgens and estrogens in tissues.
They are produce as by-products of cortisol
synthesis that are regulated by
ACTH
They circulate bound to steroid hormone binding
Precursors: pregnenolone and 17-0H globulin (SHBG).
pregnenolone.
Examples: dehydroepiandrosterone (DHEA) and androstenedione.
DHEA, the principal adrenal androgen, is converted to
estrone.
Excessive production of androgens results in
virilization (pseudohermaphroditism).
Excessive levels androgens can be confirmed
by measuring total and free testosterone
dehyroepiandrosterone sulfate (DHEAS). and

202
 Adrenal Medulla
It is composed primarily of chromaffin cells that secrete catecholamines.
L-tyrosine is the precursor of the catecholamines.

Synthesis of Medullary Hormones:

Norepinephrine and epinephrine are metabolized by monoamine oxidase (MAO) and catechol-
O-methyttransferase (COMT) to form metanephrines and VMA.
COMT converts epinephrine to metanephrine, norepinephrine to normetanephrine and
dopamine to methoxytyramine, all of which in turn can be oxidized to vanilylmandelic acid
VMA) by MAO.
The ratio of norepinephrine to epinephrine in serum is 9:1
The hormones are 50% protein bound.

A. Norepinephrine (prkmaryamine)
tis produced by the sympatheticganglia.
*The highest concentration is found in the brain (CNS).
It acts as a reurotransmitter in bothCNSand symphathetic nervous system (SNS).
MHPG is the major metabolite in CNS.
Maior metabolites: 3-methoxy-4-hydroxyphenylglycoi (MHPG) CSF and urine
venmtytmamdetic acttf )
B. Epinephrine (adrenalíne/secondary amine) VM
I t i s most abundant medullary hormone.
I t is produced from norepinephrine and comes only from the adrenal.
I t is called the "fnght or figh-aoFmone", because it is released in response to physiologic
(injuries) or psychologlcal (stress,anxiety)threats
I t increases glucose.concentrationlglycogenolusis).
Any fomm of stress that increases cortisol levels stimulates its production.
It is best collected from an indwelling catheter, since venipuncture may cause levels of
cathecholamines to rise.
Major metabolite: vanillylmandelic acid(VMA)
Minor metabolites: metanephrines, normetanephrines homovanillic acid
VMA Is the major (60%) catecholamine metabolite in urine derived largely from norepinephrine.

C. Dopamine (primary amine)

I t is a catecholamine produced in the body by the decarboxylation of 3,4
dihydroxyphenylalanine (DOPA).
itis present in highest concentration in the regions of thebrain.
Dopamineis the major inteet-cateshelamines present in urine
Maior metabolite: homovanillic acid (HVA)

Clinical Disordersi
1. Pheochromocytoma
i t is tumor ofthe adrenal medulla or sympathetic ganglia.
it is due to overproductlon of catecholamines.
it is commonly seen in 3d to 5th decades of life.
clussic "spels": hypertension, tachycardia, headache, tightness of chest and sweating
screening test: plasma metanephrines and normetanephrines by HPLC (four-fold increased)

203
 diagnostic test: 24-hour urinanry excretion of metanephrines and
normetanephrines (increased levels)
patient preparation:avoid caffeine, nicotine, alcohol and acetaminophen, monoamine
oxidase inhibitors, and tricyclic antidepressants for at least 5 days
before testing

Pharmacologic Tests:
a. Clonidine Test
it differentiates pheochromocytoma (not
suppressed) to
decreased in catecholamine levels) or those individuals with neurogenic hypertension (50%
test.
borderline elevations in urinary
the clonidine (0.3 mg oral)
suppressiontest is used only if the plasma catecholamines are greater
than1000 pg/mL (5.9 nmol/L).
b. Glucagon Stimulation Test
i t is used if it is highly suggestive of
it is only used for individuals with pheochromocytoma.
nermat btood pressure and when catecholamines are only
modestly elevated (three-fold increased).

2. Neuroblastoma
it is a fatal
malignant condition in children resulting to excessive production of
norepinephrine.
(+) high urinary excretion of HVA or VMA or both, and dopamine.

Methods:
Specimens: 24-hour urine and blood (plasma)
Patient Preparation:
The patient should undergo
overnight fasting.
Avoid smoking or drinking coffee at least 4 hours prior to blood collection these will increase
plasma catecholamines.
The patient Is placed in a reclining
position in a quiet envircnment and a heparin iock is inserted
intravenously. After 20-30 minutes blood is collected in a prechilled EDTA tube.
Plasma concentrations of catecholamines are affected by body
be collected after 30 minutes in
positioning, and samples must
a stable position decreased values when
supine.
1. Chromatography- HPLC or GC-MS (VMA and metanephrines)
2. Radioimmunoassay sensitive screening test for total plasma catecholamines
2000pg/ml of plasma catecholamines diagnostic for pheochromocytoma
SpecimenConsiderations:
1. Catheterization-is the preferred method of blood collection to eliminate
2. Urine Preservation: anxiety ofvenipuncture
urine samples with 10 ml of 6N HC catecholamines and metabolites are rapidly
oxidized at higher pH> pH 2)
3.24 hour-urine creatlnine test- to assess the quality of urine collection (0.8 g/day of urine creatinine
is needed to validate the
completeness of the urine collection)
4. To prevent catecholamine
oxidation, blood samples must be transported on ice.

204
REPRODUCTIVE HORMONES

Synthesis and Transport of Hormones:

The testesand ovaries produce sex steroids such as androgens and astrogens from chelesterol.
The overyenvertetestosterone-toestradiol, and androstenedionetoestrone
Peripheral tlssues (ncluding those targeted by androgens) reduce testosterone to
dihydrotestosterone (DHT), hydroxylate estradloi to estriol, convert adrènal androgens to
testosterone and androgens to estrone and estradiol.
Major transport proteins: sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CB6)
and albumin
SHBG transports androgens and estogens
CHG delivers progesterone and glucocorticoids.
About 1% to 2%ofthe sex steroids areffree(unbour the remaining are beund.to proteins
aHbemin, SHB&and ¬HG).
Only the free fraction of the hormone is biologically active, because only the free form can
diffuse into the vascular system and interact with target cells.

réstosterone
t i s the principal andregenhermone in the blood -most petentmale androgen.
Itis synthesized by theeydigels of the testis-ofthe made; also derived from pregesterone.
i t is controlled primarily by FSHand LH.
Functions: growth and developmentof the reproductive system, prostate and
external genitalia
Physiologic factors:
o Levels demonstrate a cirecadian pattern and peak at the time of awakening (8am); fall to
their lowest level at 8pm.
There is a gradual reduction in testosterone after age 30, with an average decline of
about 110 ng/dL every decade.
o Obesity may cause decrease plasma testosterone concentration.
o After age 50, men experience a decrease in secretion rate and concentration of
testosterone, and women have an increase in pituitary gonadotropins, especiallyfollicle
stimulating hormone (Young, 2007 cited by Mc Pherson and Pincus, 2017)
Tests for male infertiity: semen analysis, testosterone, FSH and LH
Reference values: 3.9-7.9 ng/mL (serum)

Transport Proteins:
1.Albumin-50%
2. Sex hormone binding-globulin (SHBG) 45%
The concentration of birnding protein determines the ievel of total testosterone but not the free
testosterone levels during laboratory estimation.

205
 Types of Testicular Infertility (HDogonadism:
a. Pretesticular infertillty (Secondary hypodonadism)
It is due to hypothalamic or pituitary lesions.
F,LH Normal or decreased levels: testosterone, FSH and LH levels

b. Testicularinfertilty (Pelmary.hypogonadism)
It may be congenital
D (cryptorchidism, Klinefelter's syndrome and 5-alpha-reductasedeficiency)
or acquired (varicocele, tumor, orchitis).
t is characterized by having decreased testosterone levels and increased FSH and iH levels.

c. Post-testicular infertilty
I t is due to
disordersof sperm transhort and function.
Normal blood level: testosterone, FSH and LH levels

Other Disorders of Sexual Development:

a. Testicular Feminization Syndrome
t is the mest-seere-ferm
of androgen-Fesistance-syndreme, resulting in leek-of-testosterone
aetienintheterget tissue.
The physical development pursues the female phenotype, with fully developed breast and
female distribution of fat and hair.
There is no utility or response to administration of
exogenous testosterone.
Lab tests: normal levels of testosterone with elevated
FSH and LH !evels
b. Sertoli Cel-Only Syndrome
Its1t4 Itis characterized by
a of lack germ cells.
Men present with small testes, high FSH levels, azoespermie, and nemai-testosterone-levels.
Testicular biopsy is the onty procedure to confirm this diagnosis.

c. Kollmann'sSyndrome
It is a result of an inherited, X-linked recessive trait that manifests
puberty.
as
hypogcnadism during
Associated defects: anosmia (inability to smell) and midiine defects
(cleft palate and lip)
2. Dehydroepiandrosterone (DHEA)
I t is the principal androgen formed
by adrenal cortex; weak androgen.
This androgen is primarily derived from the adrenai
gland.
It is valuable in the assessment of adrenal cortical
function.
3. Estrogen
t is carbon-18 steroid hormones that have a
phenol A ring.
i t arises through
structural alteration of the testosterone molecule.
tis not produced by
the ovaries after
menopause
Functions: promotion of breast development, maturation of the external
body fat and termination of linear growth (secondary sexual characteristicgenitalia, deposition of
in the female)
in conjunction with
progesterone, they function in uterine growth and regulation of
cycle, and maintenance of pregnancy. menstrual
Deficlency: irregular and incomptete development of the endometrium
Precursor: acetate, cholesterol, progesterone and
testosterone

206
 3 Forms: estrone, estradlol and estriol
and extraglandular conversion.
Estrone and estriol are metabolites of intraovarian

a. Estrone (Es)
abundant estrogen in post menopausal
women.
t i s the most

b. Estrodiol (E)
It is the most potent estrogen secreted by the ovary; major estrogen.
low levels in the menopausal stage.
t i s the most abundant estrogen in pre menopausal women;
diffuses out of the thecai cells of the ovaries in the
I t is synthesized from the testosterone, then
female.
It is the precursor of both Es and Es
it is used to assess ovarian function; serves as negative feedback for FSH
albumin (60%) and SHBG (38%)
Transport proteins:
The free form of E, is approximately 2%.

c. Estrio (E)
It is a metabolite of estradiol.
it is the estrogen found in maternal urine.
pregnancy formation in pregnant
a
during
t is the major estrogen secreted by the placenta
function.
women is dependent on fetal and placental
it is used to assess the fetoplacental unit (fetoplacental viability), postdate gestations and
intrauterine retardation.
t promotes uteroplacental blood flow aspotently as other estrogens.
AFP and HCG).
It is used as a marker for Down syndrome (together with
Preferred specimen: plasma

4. Progesterone
It is a carbor-21 compound In the sterold family.
luteum in the female.
it is produced mainly by the granulose (lutein) cells of the corpus
s the prime secretory product of the ovary.
females.
It is a dominant hormone responsible for the luteal phase cycle among
occurred.
It is the single best hormone to determine whether ovulation has
It is used primarily for the evaluation of fertility in femaie.
It serves to prepare the uterus for pregnancy and the lobules of the breast for lactation.
androstenedione.
It is intermediate in the synthesis of adrenal steroids and
Deficiency: fallure of implantation af embryo
Metabolites: pregnanediols (most easily measured metabolite), pregnanediones, pregnanalones

Tests for menstrual cycle dysfunction and anovulation: estrogen, progesterone, FSH, LH
Tests for female infertility: HCG, PRL, FT, TSH, FSH, LH, estradiol and progesterone

207
 Notes to Remember
The CPPP structure is shared
by cholesterol, cortisol, estradiol,
All estrogens have a progesterone and testosterone.
hydroxyl group at C3 E has a 2nd hydroxyl group at C17, arnd E has a 3rd
hydroxyl group at C16.
Ovaries also produce
and dihydrotestosterone.
androgens
like androstenedione, dehydroandrostenedione, testosterone
Excess production of ovarian androgens among females leads to hirsutism, defeminization
virilization. or
SHBG levels are increased with decreased testosterone, increased
and lver disease, whereas levels are decreased with
estrogen, hyperthyroidism,
increased testosterone,
hypothyroidism,and acromegaly.
Neural tube defects (NTDs) and Down
syndrome 4DS) are determined during the early secornd
trimester by screening of maternal serum for levels
of AFP, HCG,
inhibin A unconjugated estriol (uE3) and
When one of the four markers of Down's
syndrome is abnormal, amniocentesis should be
performed.
> Karyotyping or FISH typing is a test for Down syndrome using amniotic fluid as specimen.

PANCREAS
It is a digestive gland in the gastrointestinal system.
Funcions:
1. As Exocrine Gland
It is responsible for the synthesis of digestive enzymes.
Functional secretory unit: Acinus
1. As Endocrine Gland
His responsible for the synthesis of hormones.
alpha celis (20-30%)-glucagon
beta cells (60-70%) insulin
delta cells (2-8 %)-somatostatin

MISCELLANEOUS HORMONES
1. Human Chorionic
Gonadotropin (HCG)
I t is produced by the trophoblast cells of the
placenta during pregnancy.
It is a dimeric molecule, consisting of one alpha subunit and one beta subunit that confers
antigenic individuality.
It has the same a-subunit as LH, FSH, and
TSH.
it has a beta subunit similar to
LH-an "LH-Iike"
hormone.
it serves to maintain progesterone production by the
corpus luteum in the earty pregnancy.
The intact HCG[a+ B) is the predominant form throughout
pregnancy.
It can be detected 2 to 3 days after
ovulation.
For serum assay, an antibody is directed separately to Bsubunit and intact
HCG.
For urine assay, an antibody is directed separately to a and B subunit of HCG.
Qualitative test for urine samples has detection limits of about 50 miU/mL.
Serum HCG Increases above the reference interval (4-6 miU/mL}
by implantation.
Indicative of
pregnancy/trophoblastic disease: >S miu/mt
208
 Serum level during the first trimester
of
pregnancy:>100,000 miU/ml
Method: immunometric (sandwich) method serum and urlne samples
2. Human Placental Lactogen (HPL)
I t is functionally, structurally and immunologically similar to GH and PRL.
It is produced by the pBecenta and can be measured in urine, serum and amniotic fluid.
I t stimulates development of
mammary gland.
It increases maternal plasma
glucose levels (HPL inhibits the action of insulin)) and promotes
positive nitrogen balance.
t is important in the diagnoses of intrauterine growth retardation.
3. Gastrin
I t is a peptide secreted by the G cells of the
antrum of the stomach.
t is released in response to
vagal stimulation and food in the stomach.
t causes secretion of the HCl
by parietal cells in the body of the stomach.
t is the diagnostic marker for
Zolinger-Ellison syndrome (islet-cell tumor).
Major stimulus: presence of amino acids
Increased: ZES, achlorhydria, chronic renal failure
4. Serotomin (5-hydroxytryptamine)
t i s an amine derived from
hydroxylation and decarboxylation of tryptophan.
I t is synthesized by
argentaffin celis, primarily in Gl tract.
It also found in high concentrations in
pineal gland and CNS.
i t binds to platelets and released
during coagulation.
Urinary metabolite: 5-hydroxyindoleaceticacid (5-HIAA)
5-HIAA is a diagnostic marker
for carcinoid syndrome.
Plasma 5-HIAA may increase after ingestion of a fatty meal.
Serotonin-rich food (banana, pineapple, tomato and avocado) increase the
5-HIAA.
urinary excretion of
Test for 5-HIAA: Ehriich's aldehyde test (+) purple color
5. Somatostatin
It is also called growth inhibiting hormone.
It is found in the GIT, hypothalamus and the delta celis of
the pancreatic islets.
It is an inhibitor of growth hormone,
glucagons and insulin.
6. InhibinA
I t is a reproductive systern hormone which inhibits FSH activity; it is the 4th Down
marker syndrome

Methods:
in clinical
practice, it is desirable to measure multiple hormone levels in order to ruleout even
minor abnormalities that might be the only chemical
sign in the course of endrocrine diseases.
Diagnosis of endocrine disorders is made possible through the direct measurement of individual
hormones in serum.

209
Samplesfor Hormonal Assay:
1. Whole blood LH, testosterone
2. Plasma
EDTA (ACTH, ADH,
PTH) and heparin (catecholamines, cortisol, dopamine, FSH)
3. Serum aldosterone, androstenedione, DHEA, estrogen, FSH, GH, HCG, progesterone
4.Urine-for measurement of estriol
Boric acid in a concentration of 1 g/dL presenves urine elements such as estriol and estrogen for
up to 7 days.
For catecholamines,
vanilylmandelic acid (VMA), or 5-hydroxyindoleacetic acid (5-HIAA)
collections, 10 ml of 6N HCI is added to a 3 to 4 Lcontainer.
The HCl establishes a pH of approximately «3.0 that is good for chemical testing.

1. Classic Assays
a. Bioassoy
I t i s based on observations of physiological responses specific for the hormone being measured.
t involves the injection of test materials into
prepared animals.
b. Competitive Protein Binding (CP8)
it is based on competition for protein-binding sites between a known "tagged or labeled"
hormone and the unlabeled hormone in the patient's sample.
Measurement of total Ta is based on the specific binding properties
of TBG.
2. Immunologic Assays
These are widely used to quantitate hormones using
labeled-antibody with nonisotopic labels.
a. Radioimmunoassay (RIA)
It isCPB technique that utilizes radio-labeled hormone
a
the
as tagged hormone and antisera
prepared against the specific hormone as a binding site.
b. Immunoradiometric (RMA)
t is a radiolabeled substance is attached to the
antibody instead of the hormone; an antigen-
antibody reaction.
c. Enzyme-Linked Immunosorbent Assay (ELISA}
An ezyme is attached to an antibody.
The end product can be
d. Enzyme
measuredspectrophometricaily.
Multiplied immunosorbent Technique (EMIT)
Enzyme tags are used; the enzyme is attached to the hormone or drug being
The rate of NADH produced is proportional to the amount of drug tested. tested
Requires no separation of bound and free antigen.
This method is widely used also in TDM.
e. Immunometric
For TSH test; sensitive test

3. Fluorescent Techniques
Fluorescence Polarization Immunoassay (FPLA) fluorescein-labeled drug, serum, and antibody
are mixed and placed in the light path of a fluorometer.
Antibody-bound conjugate (polarized fluorescence) is inversely proportional to serum drug
concentration

210
4. High Performance Liquid Chromatography (HPLC)
between the mobile phase and the
I t is based on the differential partitioning of the compounds
stationary phase.

5. Colorimetry
a. Porter-Silber Method- for 17-0HCS
b. Zimmerman Reaction it those steroids with 17-keto structure
measures

c. Pisano Method for quantitating metanephrines and normetanephrines

reddish-brown color)
estrogen (H2SO4+ hydroquinone
=
d. Kober Reaction for
Teminoloaies
1. Amenorrhea occurred,
amenorrhea is menstruation having never
it is the absence of menstruation. Primary
menses for 6 months.
while secondary amenorrhea is the absence of
2. Carcinoid syndrome
diarrhea, flushing, tachycardia and hypotension,
I t is a constellation of symptoms consists of kallikrein and prostaglandins) into the
which are produced by the released of amines (histamine,
circulation.
3. Cushing's disease
increased secretion of ACTH resulting to
It is metabolic disorder characterized by abnormal
a
accumulations of fat on the chest, upper
increased cortisol production. t is characterized by
acne, facial hirsutism
back andface, muscle weakness, purplish striae on the skin, osteoporosis,
and hypergycemia.
4.Cushing's syndrome
I t is a metabolic disorder resulting from chronic excessive production of cortisol by the adrenal
cortex or by the administration of large doses of glucocorticoids.
the secretion of cortisol or
The syndrome also represent a failure in the body's ability to regulate
ACTH.
tumor that causes an increased
T h e most common cause of the syndrome is a pituitary
secretion of ACTH.
5. Gynecomastia
It is related to the balance of estrogen to
T h e development of breast tissue in males.
androgens.
6. Hirsutism
t is characterized by the excessive growth of hair in a female - the hair growth pattern is same

with maies.
it is the most common endocrine disorder in women.
7. Turner's syndrome
I t is a chromosomal abnormality (45X0) with physical features of short stature, "webbed neck",
low hairline on the neck and broad "shield-like" chest.
There is amenorrhea with increased levels of FSH and LH.
8. Virilization
t is a process in which secondary male sexual characteristic are acquired by a female, usually as
a resuit of adrenal dysfunction or hormonal medication.
9. Zollinger-Ellison sysndrome
I t is characterlzed by severe peptic ulceration of the stomach.

211
 THERAPEUTIC DRUG MONITORING
I t involves the
analysis, assessment and evaluation of
plasma or whole blood. circulating concentrations of drugs in serum,
It is a
quantitative procedure performed for drugs with a narrow therapeutic index.
It allows for the safe
use of
drugs that would otherwise be
TDM ensures that a
given drug produces maximal potentially toxic.
achieve a constant serum level of therapeutic benefit and minimal side effects; to
the drug that will be
Most drugs have a half-ife therapeutic.
The half-life of the independent of their concentrations.
drug determines the time to reach the
Only the free fraction of the steady-state or average concentration.
drugs can interact with the site of action and result in
response. a biologic
Mixed Function Oxidase
(MFO) system is the biochemical pathway responsible for the
portion of drug metabolism. greatest

Indications
1. The
for TDM:
consequences of overdosing and
2. There is a small difference
underdosing are serious.
between a therapeutic and toxic dose.
3. There is a poor
relationship between the dose of drug and circulating
correlation between circulating concentrations and concentrations but a good
4. There is a therapeutic and toxiceffects.
change in the patient's physiologic state that may
concentrations. unpredictably affect circulating drug
5. A drug interaction is or may be occurring.
6. It helps in
monitoring patient compliance.
Routes of Administration
The circulatory system is a convenient route that can effectively deliver most
action drugs to its site of
Intravenous route of
the
administration is associated with 100% bioavailab1lity- al the drugs enter
bioodstream (1.0 bioavailable fraction).
Orally administered drug should achieved 0.7 bioavailability
When drugs are intravenously (most immediate fraction; 10
elimination rates are constant.
route) administered, the distribution and
Routes: intravenous, oral, intramuscular,
and transcutaneous
subcutaneous, inhalation, suppository

Five pharmacological.parameters that determine serum drug

1. Liberation -is the release of the drug. .concentration:
2.Absoprtion is the transport of drug from the site of admin:stration to the blood.
3. Distribution refers to the delivery of the drug to the tissues.
4. Metabolism is the process
5. Excretion is the
of chemical modification of the drug by cels.
process by which the drug and its metabolites are excreted from the
body.

212
Absorption
For a drug to achieve therapeutic dose, it must be at the proper concentration at its site of
action.
Most drugs are absorbed by passive diffusion - the drug must be in a hydrophobic state

(nonionized).
Some drugs require uptake by transport mechanisms intended for dietary constituents.
Drugs absorbed from the Intestine enter the hepatic portal system- all the blood from the GIT
is routed thrcugh the liver before it enters into the general circulation.
Tablets and capsules require dissolution before being absorbed; liquid solutions are rapidly
absorbed.
Weak acids are absorbed in the stomach; weak bases in the intestine.
Changes in the absorptive characteristic of the drug may be due to age, pregnancy or pathologic
conditions.
Factors affecting absorption: changes in intestinal movement, pH, inflarmmation,and the

presence of food or other drugs

Distribution
The locations where the drugs are effective are in the body tissues, not generally in the blood.
than
Drugs diffuse widely through the body, frequently reaching higher concentrations in tissues
in blood.
The relationship between tissue and blood levels is termed the distribution space.
A large distribution space indicates that much of the drug moves into the tissues than stays in

the circulation.

Excretion
The rate at which a particular drug is cleared from the circulation is dependent not only on the
type of drug itself, but also on a patient's capacity to metabolize and excrete it.
All drugs are excreted- either unchanged in the urine, or excreted as metabolites of the parent

drug.
Conversion of a parent drug to its metabolites occurs in the liver (extramitochondrial
microsomal system of the hepatocytes-cytochrome P450 enzymes).
Some drugs enter the enterohepatic circulation and excreted in stool.

Causes of Drug Toxicity:

1. Elevated concentration of free drug.
2. Abnormal response to drug after administration.
3. The presence of active drug metabolites.

Terminologies
1. Bioavailabiefraction (f -is the fraction of the dose that reaches the blood
represents the dilution of the drug after it has been distributed in the body. It is used
2. Va of a drug
to estimate the peak drug blood level expected after a loading dose is given. It is the
principal determinant of the dose
3. First-poss hepatic metobolism drugs that are transported to the liver lost a fraction of its
bioavailability before the drug reaches the general circulation
a. First-order elimination represents a linear relationship between the amount of drug
eliminated per hour and the blood level of a drug

213
 5. Half-life (tyn) the time required to reduce a drug level to half of its initial value
6. Peak concentration is the highest concentration of a drug obtained in the dosing interval
7. Pharmacodynamics is the relationship between the drug concentration at the target site and
ox com Togctutes response of the tissues
8. Pharmacogenomics - refers to the study of genes that affect the performance ofa drug in an

individual
9. Pharmacokinetics is the mathematical expression of the relationship between drug dose and
drug blood level
10. Therapeutic indexthe ratio between the minirnum toxic and maximum
-

therapeutic serum
Concentration
11. Therapeutic range the difference between
highest and lowest effective dosages
-

12. Trough concentration i s the lowest

concentration of a drug obtained in the dosing interval

A. CARDIOACTIVE DRUGS
These drugs are used for treatment of arrhythmias and
congestive heart failure (CHF).

Classification of Cardioactive Drugs:

ClassI (rapid sodium channel blockers) Quinidine, Procainamide, Lidocaine
Class II (beta receptor blockers) Propranolol
Class Iil (K channel blockers) - Amiodarone
Class V(calcium channel blockers) Verapamil
1. Digoxin ats cardiou lons
i t is a cardiac glycoside for treatment of atrial
arrhythmia and CHF
the therapeutic actions and toxicities can be influenced by serum
eiectrolytes.
it inhibits membrane Na-K-ATPase, thus it decreases K' and
Mg, and increases Ca(cardiac
contractility-inotropic effect, 0.8-2ng/ml).
25% is protein bound; free (nonbound) form is sequestered into muscle cells.
GIT absorption is variable; change in GFR affect serum concentration.
hyperthyroid individuals are resistant to the action of digoxin.
the timing of blood collection after last dose can be critical to interpretation of the drug levels.
elimination: renal filtration of the plasma free form
peak serum leve: 8 hours after an oral dose
half life: 38 hours (average adult)
therapeutic level: 0.5-2 ng/mL
toxic leve: >2 ng/mL
toxic effects: nausea, vomiting, visual disturbances, premature ventricular contractions and
atrioventricular node blockage

2. Lidocaine (Xylocaine)
i t is used to correct ventricular arrhythmia for treatment of acute myocardial infarction.
t itisadministered by continuous IV infusion after a loading dose.
it can be used as a local anaesthetic.
it is bound to albumin and AAG.
it cannot be administered orally due to almost complete hepatic removal of
the absorbed drug.
elimination:by hepatic metabolism; changes in renal function have little effect.

214
 primary product of hepetic metabolism: monoethylglycinexylidide (MEG)x)
therapeutic level: 1.5-4.0 ug/ml
toxicity levet: > 4.0 ug/ml
CNSdepression: >4-8 ug/ml
seizure and Vbp and cardiac output:> 8 1ug/ml.
toxic effect: CHF and heart block

3. Quinidine bioOa aysurasiq

it is a naturally occurring drug for the treatment of arrhythmia.
85% protein-bound; GIT absorption is complete and rapid for the sulfate.
elimination: hepatic metabolism
route of delivery: oral administration
common fomulations: quinidine sulfate and quinidine gluconate
peak serum level: 2 hours after an oral dose (sulfate); 4-5 hours (gluconate)
therapeutic level: 2.3-5 ug/mL
toxic level: »5 ug/ml
toxic effects: cinchonism, blood dyscrasia and hepatitits

4. Procainamide (Pronestyl)
it is used to treat ventricular arrhythmia.
GIT absorption is rapid and complete
20% protein-bound; eliminated by renal fitration and hepatic metabolism.
common route: oral
hepotic metabolite: N-acetyl procainamide (NAPA)
peak serum leve!: one hour after the dose
therapeutic level: 4-10 ug/mL
taxicleve: >12 4g/mt
toxic effects: reversible lupus-like syndrome( ANA), nephrotic syndrome, urticaria

5. Disopyramide
i t is used to treat cardiac arrhythmias; used as a substitute for quinidine.
it is administered oraily; GIT absorption is complete and rapid.
it has anticholinergic effects dry mouth and constipation ( 4.5 Hg/ml).
elimination.renal filtration
therapEutic level: 3-5 HE/mi
toxic level: 10 ug/mL
toxic effects: bradycardia and atrioventricular node blockage

6. Propranolol
. i t is a beta-receptor blocking drug; used in the treatment of angina pectoris, hypertension,
coronary artery disease.
i t suppresses the conversion of Ta to T- it is used in the treatment of thyrotoxicosis.
theropeutic range: 50-100 ng/mt
toxic effects: bradycardia, arterial insufficiency (Raynaud's type), platelet disorder and
pharyngitis

215
 7. Amiodarone (Cordarone)

it blocks potassium channels in the cardiac muscle; use for treatment of ventricu
it is an iodine-containing drug which can cause hyperthyroidism or hypothyroidis
therapeutic level: 1.0-2.5 ug/ml
toxicleve: >2.5 ug/mL
toxic effects: bradycardia, hepatitis, photodermatitis and thyroid dysfunction

8. Verapamil
i t i s usedfor treatment of angina, hypertension and supraventricular arrhythmias.
therapeutic range: 80-400 ng/mL
toxic effects: hypotension, peripheral edema and ventricular fibrillation

B. ANTIBIOTICS nctmcin
1. Aminoglycosides (gentamicin, tobramycin, amikacin, kanamycin, neomycin, streptomycin)
it is used for treatment of Gram-negative bacterial infections; not given to outpatient.
tt is administered IM or V; it is not well absorbed from the GIT.
it may cause damage to the 8th cranial nerve at toxic levels (hearing loss).
it requires trough and peak measurements.
elimination.renal filtration
toxic level:
30 4g/mL ( amikacin and kanamycin)- peak levels
12-15 Hg/ml (gentamicin and tobramycin) - peak levels
foxic effects: nephrotoxicity and ototoxicity hcaríng toss h cranial NenNe
dgnaqes tho
2. Vancomycin
i t i s a glycopeptide effective against Gram-positive cocci and bacilli.
it has poor oral absorption; administered by V infusion.
toxic side effects occur in the therapeutic range (S-10 ug/mi).
Me Only the trough levels are monitored to ensure the serum drug concentration is within the
therapeutic range.
eliminotion: renal filtration and excretion
toxic effects: "red-man syndrome", nephrotoxicity and ototoxicity
toxic leve: > 10 ug/mL nephrotoxicity
>40 Hg/mL ototoxicity

3. Chloramphenicol
it distributes to all tissues, and it concentrates in the CSF.
50% protein bound; rapidly absorbed in the GIT.
toxic effects: blood dyscrasia, cytoplasmic vacuolation (erythroid & myeloid cels)
toxic level:> 25 ug/mL

216
 : StIURES
C. ANTIEPILEPTIC DRUGS
1.Phenobarbitat r tohmon
i t is a long-acting barblturate that controls grand mal tonic-clonic seizure and focal epileptic; not
used for petit mal seizure.
it is used for treating withdrawal symptoms in infants mothers are addicted to opiate or

barbiturate.
it is used to treat cases of congenital hyperbilirubinemia - this drug enhances bilirubin

metabolism.
absorption is slow but complete; 50% protein-bound; majority is stored in the brain.
renal impairment slows down elimination process.

only trough levels are evaluated unless there is toxicity.

elimination: hepatic metabolismn
hours
inactive proform: primidone (mysoline); half life: 70-100
peak serum level: 10 hours after an oral dose

therapeuticlevel: 20-40 ug/ml (phenobarbital)/5-12 ug/ml (primidone)

toxic effects: nystagmus, stupor, ataxia and respiratoy depression

2. Phenytoin (Dilantin)
in
i t controls seizures (tonic-clonic, simple partial seizures); a short-term prophylactic agent
brain injury. can inihat StiRURE ( OCIdone)
it isinot'used for petit mal and atomic seizures.
it decreases sodium and calcium influx into hyperexcitable neurons.

V administered; GIT absorption is incomplete.

87-97% protein-bound; free (unbound) form is the biologically active portion.
toxicity may be seen at the level of the therapeutic range.
elimination: hepatic pathway (zero-order kinetics)
major toxicity: initiation of seizures; teratogenic action (cleft lip and palate) andnystagmus
therapeutic range: 10-20 ug/mL ; 1-2 Hg/ml (free form)
toxic level:> 20 ug/ml
injectable proform: fosphenytoin

3. Valproic acid (Depakene) (PNcRPaTI T1S

it is used for treatment of petit mal (absence seizure), atomic seizure and grand mal.
oraily administered; GIT absorption is rapid and complete.
it is highly protein bound (93%).
hepatic dysfunction is observable which requires monitoring after 6 months of therapy.
elimination:hepatic metabolism
therapeutic level: 50-100 ug/ml
toxic level:> 100 ug/mi (nausea, iethargy and weight gain)
200 ug/ml (pancreatitis, hallucinations and hyperammonemia)

Carbamazepine (Tegretol)
it is a tricyclic compound related to imipramine (TCA).
it effective for grand mal seizures and treating selzures accompanied by pain.
it has antineuralgic action; 70-80% protein-bound.
it has serious toxic effects and not frequently used; orally administered.
elimination:hepatic metabolism

217
 idiosyncratic effects rashes, leukopenia, nausea, vertigo, febrile reactions
therapeutic level: 4-16 ug/ml
toxicleve: >12 ug/ml
toxic effects: hematologic dyscrasias, aplastic anemia, iregular pulse and ataxia

5. Ethosuximide (Zarontin)
the drug of choice for controlling petit mal (absence seizure); oraly administered.
S mI it is free in serum and not protein bound.
therapeutic level: 40-100 ug/mt
toxic level:> 100 ug/ml
toxic effects: GI disturbances, ataxi, SLE, aplastic anemia and pancytopenia

6. Gabapentin (Neurontin)
i t is chemicaly similar to neurotransmitter gamma aminobutyric acid (GABA).
i t is used for partial seizures; for adiunctive therapy
it is administered orally; it is unbound to plasma proteins.
it is excreted unchanged in the urine; not metabolized in hunmans.
adverse effects: diziness, ataxia, fatigue and nystagmus
theropeutic level: 2-15 ug/mt
toxic effects: ataxia and somnolence (drowsiness)

T. Toptramate- used as an adjunct drug for partial seizures

8. Lamotrigine (Lamictal) used as an adjunct drug for partia! seizures
9. Felbamate

D. PSYCHOACTIVE DRUGS AUT 0EPRES90N

1. Lithium
it is used for treatment of manic-depressive illness (bipolar disorders).
it is the drug of choice for the prevention of chronic ciuster headache.
it inhibits thyroid hormone synthesis and release- inhibits iodine uptake.
it is a cationic metal that does not bind to proteins.
it is orally administered, absorption is rapid and complete.
the distribution of this drug is uniform throughout the body water.
it is subject to reabsorption.
lithium and demeciocycline inhibit the effect of ADH on the kidney.
elimination:renai filtration
therapeutic range: .0.8-1.2 mmol/L
toxic effects: severe dehydration, nephrotoxicity and hypothyrcidism
toxic level: 1.2-2 mmol/L apathy, iethargy, speech difficulties
2 mmol/L seizures/muscle rigidityand coma
-

2. Tricyctic Antidepressants (TCAs)

they are used for the treatment of depression, insomia, extreme apathy and loss of ibido.
theyare orally administered with variability in absorption (slow gastric emptying and intestinal
motility)
examples: imipramine, amitriptyline, doxepin, nortriptyline, trazadone
eliminotion: hepatic metabolism
toxic effects: drowsiness, blurred vision,memory loss,eizure, cardiac arrhythmia, parkinsonian
syndrome and unconsciousness

218
 peak serum concentrotion: 2-12 hours
therapeutic levet: 100-300 ng/mL
major metabolite: desipramine

3. Fluoxetine (Prozac)
it blocks the re-uptake of serotonin in central serotonergic pathways.
it is also used for treatment of obsessive-compulsive disorders.
toxic effects:attempted suicide, decreased libido and sexual function
therapeutic level: 90-300 ng/ml

E. BRONCHODILATOR

Theophyline
it belongs to the methylated xanthine class.
its action is specific to the relaxation of bronchial smooth muscle.
it is used in the treatment of asthma and chronic obstructive pulmonary disease.
drug for primary apnea of prematurity-absence of respiratory effort in newborn infants
its action is inhibitory to the release of histamine and other proinflammatory agents.
S0% protein-bound; initialy administered intravenously then oraly.
it crosses the placenta and may be teratogenic in pregnant females.
t h e best predictor of toxicity is the blood level of the drug and not early signs or symptoms of
toxicity.
elimination: renal filtration and hepatic metabolism
theropeutic level: 10-20 ug/mt
toxic level:> 20 ug/mL
toxic effects: Gl bleeding, seizures,
tachycardia and syncope
CAROIMRTHNTMMA ChRIAG PAAALY SIS
F. IMMUNOSUPPRESTVE DRUGS OAt WioE
1. Cyclosporine t arIIN 10 RED EL?
bLono
i tinhibits the cellular immune response by blocking production of interleukin-2.
it is used to prevent rejection of allogenic organ transplants;
it is used for suppression of acute graft-versus-host disease
(GVHD).
the organs such as the heart, liver and pancreac require high dosage (300
ng/mL).
it has marked affinity with RBC;
RBC-cyclospororine is temperature dependent.
orally administered with 5-50% absorption
elimination: hepatic metabolism
specimen of choice: whole biood (with lysis of RBC to yield the total amount)
toxic tevet >500 ng/ml
toxic effects: renal tubular and glomerular dysfunctions, Gi disturbances,hirsutism and
hematologic dyscrasia
Renal Inpormrd
2. Tacrolimus (Prograf/FK-506)
it is 100x niore powerful than cyclosporine.
it is a macrolide lactone antibiotic; GIT
uptake is variable.
elevated levels are observed in cholestasis.
elimination: hepatic metabolism
specinen of choice: whole biocd
toxic effects: thrombus formation,
CLQT FOMRTION
nephrotoxicity and neurotoxicity

219
 3. Rapamycin (Sirolimus)
i t is
similar to tacrolimus; major side effects are lipid
4. Mycophenolate mofetil
abnormaities andthroinbocytopenia.
i t decreases renal allograft
rejection.
5. Leflunomide (LFM)
i t inhibits
lymphocyte proliferation; for
treatment of rheumatoid arthritis.
G. ANTINEOPLASTIC DRUGS
Methotrexate
che it is effective therapy for a
an

i t inhibits ONA synthesis in all variety of neoplastic

conditions; also an
cells,
leucovorin is used to reverse the by blocking dihydrofolate reductase.immunosuppressive agent.
toxic level: 0.01 action of methotrexate leucovorin rescue.
umol/L
-

toxic effects: leucopenia, Gl

LGG T WBC
ulceration, thrombecytopenia, cirrhosis
2. Busulfan
i t is an
alkalyting agent used to
treat leukemias
transplantation. and lymphomas prior to bone marrow
overdosage may cause hepatic occlusive
CYTO TOC TOo. disease.
H. ANTI-INFLAMMATORY/ANALGESICS
1.
Salicylates/Asptrin
it is (acetylsalicylic acid)
commonly used analgésic, antipyretic
a
it is a direct
peRe
and
it has an
stimulator of the respiratory anti-inflammatory drug.
system and an inhibitor of the
anticoagulant property (antiplatelet activity) Kreb's
function: it decreases thromboxane by inhibiting the action ofcycie.
cyclooxygenase
and cyclooxygenase.
prostaglandin formation through inhibition of
acute aspirin intoxication is a common
-

of fatal drug
side effects: gastrointestinal disturbance cause poisoning in children
and interference with
therapeutic levet: 5 mg/dL (treatrment of platelet aggregation
toxiclevel: >30 headache)
toxic
mg/d
effects: mixed acid-base disturbance
(metabolic acidosis and respiratory
hypoglycemia
method: Trinder assay;
and Reye's
syndrome alkalosis),
enzymatic assay (salicylate hydroxylase);
2.
HPLC; EMIT
Acetaminophen
i t is an inhibitor
(Tylenol) Mpyo soxic
it is
of prostaglandin metabolism.
commonly used as analgesic and
overdosage of this drug leads to antipyretic drug.
h theroapeutic level: 25 hepatoxicity.
toxic level:> 50
u8/ml
ug/mL
100-300 ug/mi.
toxic effect: cyanosis due to(hepatic necrosis)
method: HPLC methemoglobinemia, CNS depression and seizure

220
3. Ibuprofen
i t has analgesic and anti-inflammatory actions.
it has a lower risk of toxicities than salicylates and acetaminophen.
toxic effects: nausea, vomiting, biurred vision, abdominal pain, edema
therapeutic level: 10-50 ug/ml
toxiclevel:>100ug/mL
I. NEUROLEPTICS (Antipsychotie major traquilizers)
it blocks the action of dopamine and serotonin in the limbic system.
i t is used in the treatment of acute schizophrenia anm ogPRE$^1ON uKE)
is difficult due to abundant metabolites for
each drug
monitoring of these drugs in serum

resulting to extensive metabolism in the liver.

(haloperidol)
2 classes: phenothiazines (chlorpromazine) and butyrophenones
examples: Afisperdal, olonzapine (Zyprexa), quetiapine (Seroquel), aripiprazole{Abilify)
toxic effects: cholestasis, orthostatic hypotension,éplastic anemia, muscle rigidity
auNTI TAvE GC MS
Methods for TDM: GEL; $ST
Specimen ofchoice: serum or plasma (Rcd Tap mho ThyNehpio
Whole blood EDTA sample is required for cyclosporine and
tacrolimus tests. (nMUNOSUPRES99(VE D«)
TIMING & CLECTION
Timing of specimen collection is the single mestimportant-factorinFBM.
:

s o m e gels absorb
TDM samples should not be collected in tubes with gel separators or SST
certain drugs (phenytoin, phenobarbital, lidocaine, quinidine, and carbamazepine) causing
a

falsely low result.

No changes occured in theophyiline and salicylate levels when blood is collected in serum

separator tube (SST).

Measurement of serum concentrations should be done only after steady state has been

achieved.
HLL TOM : PRESCRIPTEDH
Specimen Considerations the next dose.
1. Trough concentrations are drawn immediately for 30 minutes) before
in the blood.
Trough concentration reflects the lowest level of drug
.The trough level is affected by the drug clearance rate if clearance increases, then trough level
decreases.
2. Peak concentrations are drawn one hour after an orally administered dose (except digoxin).
T h e peak blood sample (sample after absorption and distribution is complete) is the best
for initial investigation of therapeutic drug toxicity it is most likely to exceed the
-

specimen
upper therapeutic limit.
For V drugs, peak levels are determined after the infusion is completed.

Increasing the dose rate may resut in peak drug leyels in the toxic range.
MOMIN )
4K FTER «pMANIGKHTioN CoiG OxiN hrs QFTER

1. Colorimetry
Acetaminophen in urine is detected by boiling to form p-amphenol which then reacts with o-
cresol to form indophenol blue.
Trinder assay for salicylate using ferric nitrate forming a (+) colored complex.

221
 2. Immunoassay Methods
it provides rapid analyses of blood and urine sarnples.
It can detect drug levels in the nanomolar range; sensitive and
specific methods.
. I t uses an antibody specifically reactive with a particular drug- the drug must must bind to
antibody before It is identified.

a. Enzyme-Mediated (Multiplied) Immunologic Technique (EMIT)

The amount of enzyme activity is directly proportional to the amount of drugs present in the
sample.
b. Fluorescence Polarization Immunoassay
(FPIA)
The binding of the marker drug to antibody can be quantitated by the angle at wbich the
emissions ocCur.
The drug is attached to a fluorescent label or fluorophore.
3. Chromatographic methods
Best specimen: urine
a. Thin layer
Chromatography
This method uses serum, urine or
gastric fluid for analysis for toxicology screening.
It can demonstrate the presence of a multitude of abuse drugs.
It qualitatively identifies drugs by means of their Re values.
Drugs are identified according to how far they have travelled or separated and to how they
appear with each of the stains.
Extraction of drugs is pH dependent acidic drugs at pH 4.5 and alkaline drugs at pH 9.0.

b. High Peformance Liquid Chromatography (HPLC)

Is a highly quantitative procedure.
Measurement depends on the type of column used, the solvent and dectector systems
It is ideal for separation of tricyclic antidepressants and its metabolites.

c. Gas Chromatography-Moss Spectrometry (GC-MS) - gold standard

It is used for the quantitation of many drugs.
Drugs must be volatile in form or can be chemically derivatized into volatile form.
MS can be added onto effluent end of GC, enhancing its capability to quantify drugs.

Note: For complete explaination on the above-mentioned chromatographic principles, please refer
to the previous discussions on analytical methods.

Notes to Remember.
ndividual differences alter pharmacokinetics, causing lack of correlation between dose and drug
level.
Altered pharmacokinetics may result in toxicty even when the dose of drug is within accepted
therapeutic range cause by the presence of unmeasured metabolites that are physiologically
active and the presence of a higher than expected concentration of free drug.
Some drugs with wide therapeutic index are potentially toxic because they may be ingested in
great excess wth littie or no initial toxicity
Most drugs fllow first-order pharmacokinetics theclearance of the drug is linearly related to
the dose of the drug.
Trough and peak levels are characteristics of intermittent dosing regimerns.

222
 Steady-state drug level is reached when drug in the next dose is sufficient only to replace the
day eliminated since the last dose.
Steady-state drug level can be measured after five drug half-lives because blood levels will have
reached 97% of steady state.
f the steady-state is too high, decrease the dose.

Formulae:
1. Bioavailable froction for oral drug ()
f peak blood concentration after oraladministration
peak drug concentration after IV administration

2.Estimate dosage to give a steady-state blood level

Dose/hour clearance (mg/hour) x averaReconcentrationat steady state
f
3.V Xo Co

4. Peak blood leve! Dose x f xVa

5. K 0.693 halflife(tu2)
6. Dosage adjustment to achieve the desired blood leve!
New dose Current dose x desired concentration
concentration at steady state

223
 TOXICOLoGY
Toxicology is the study of substances toxic to the body.
Absorption of toxins in the GIT is by passive diffusion this process requires that the substance
cross cellular barriers.
Toxins that are not absorbed from the GIT do not produce systemic effects but may produce local
effects diarrhea, bleeding and malabsorption of
In cases of
nutrients.
drug overdose, CBC, serum electrolytes, BUN, glucose, urinalysis and blood gas must be
determined.
Common substances causing acute toxicity: alcohol, acetaminophen, salicylate, abuse substance
and carbon monoxide
Routes of exposure: ingestion, inhalation and transdermal absorption
Teminologies
1. Acute toxicity single, short-term exposure to a substance
2. Chronic toxicity repeated exposure for extended of tiine
period
3. TDso is the dose that would be predicted to produce a toxic response in 50% of the
population
4. LDso i s the dose that would
predict death in 50% of the pepulation
5. EDso i s the dose that would be
predicted
to be effective or have a therapeutic
benefit in 50% of the population
ACCTMINDDwEN, ShuCItYLATES
L TOXIC AGENTS TOXA THERAPeUTIG OKX)
A. ALCOHOL (MOST tOMMON)
it is a common CNS depressants
cause disorientation, euphoria, confusion and may progress to unconsciousrress, paralysis and
even death.
symptoms of alcohol intoxication begin when the concentration is > 0.05% w/v (> S0
blood alcohol). mg/dl

1. Ethanol (grain alcohol) D: 0-40mL

i t is the most common abused drug; a CNS depressant.
i t causes diuresis by inhibiting ADH.
i t is readily absorbed in the GIT and diffuses easily in tissues.
ethanol abuse causes acidosis through accumulation of ketones and lactate and also
through
direct generation of hydrogen ions as alcohol is oxidized; it also adds
to osmolality of blood.
quid symptoms of intoxication: blurred vision, incoordination, slurred
OuLpttd ntd
speech and coma;
"hangover symptoms" are due to the effects of acetaldehyde
ntidote for chronic intoxication: diazepam (for alcoholic mania)
specimen precoution: specimen must be capped at all times to
avoid evaporation of alcohol;
prior to blood collection, alcohol-free skin cleanser must be used
of isopropanol
instead
specimen: serum (capilary and arterial blood samples are preferred, it reflects the
concentrationof ethanol in the brain)
major metabolic pathywoy: conversion of ethanol to acetaldehyde and
acetyl coenzyme A
by hepatic alcohol dehydrogenase

224
 methods for testing: enzymatic, gas-liquid chromatography and electrochemical oxidation
preferred method: enzymatic using alcohol dehydrogenase reagent
common laboratory results: elevated GGT, AST, AST/ALT ratio (> 2.0), HDL and MCV

fotal duse: 300-400 mL of pure alcohol consumed in less than one hour
peak blood level: withinan hour after intake of alcohol
toxic bloodlevel:> 400 mg/dt
500 mg/dL (for hemodialysis)
2. Methanol (wood alcohol) ro: G0 150nL
i t is a commonly used solvent and a contaminant of homemade liquors.
it is converted first to formaldehyde, then finally to formic acid in the liver by alcohol
dehydrogenase. ORROSS NE McOHoL
acidosis
symptoms of intoxication: frank blindness (ocular toxicity) and metabolic
screening test: computation of osmolal gap
preferred method: GC-MS
fatal dose: 60-250 mL
toxic blood level: > 50 mg/dl

3. Isopropanol frubbing alcohol)

i t is rapidly absorbed by the GIT.
it is metabolized by hepatic alcohol dehydrogenase to acetone.
symptoms ofintoxication: CNS depression and hypertension
indication of toxicity:. elevated levels of acetone in the blood and urine
preferred method: gas chromatography
antidote: activated charcoal
fatal dose: 250 ml
HomicaDE SUNOE
4. Ethylene glycol (1,2-ethanediol) catx tmonehyaate)
it is a common constituent of hydraulic fluid and antifreeze.
it is converted to oxalic acid and ghycolic acid (toxic products) by hepatic alcohol dehydrogenase.
symptoms of intoxicotion: metabolic acidosis, depressed reflexes, anuria and necrosis
indicction of toxicity: deposition of calcium oxalate crystals in renal tubules
mode of treatment: inhibit the action of alcohoi dehydrogenase
major metabolite: glycolic acid (cause of acute toxicity and death)
preferred method: HPLC
fatal dose: 100 grams
IN SERT MUooL LevELS 5ymPRnS!
B. CARBON MONOXIDE (CO)
it is a colorless, odorless, tasteless gas; very toxic substance.
it is produced by incomplete combustion of carbon-containing substances like gasoline engines,
organic materials in fire and cigarette smoke.
it binds with heme prateins (cytochromes, hemoglobin and myoglobin) - binding ot co to

cytochrome A3 results to inhibition of cellular respiration and electron transport whereas

binding to hemoglobin and myoglobin reduces oxygen supply to cardiac and skeletal muscles,
and direct damage to the muscles, respectively.
it has higher affinity for hemoglobin than does oxygen (200x faster than oxygen) - impairs

oxygen transport by binding to hemoglobin producing carboxyhemoglobin.

it stimula production of nitrous oxide resulting to hypotension and neurologic changes.
Binds neChy5)
225
Chemy Red
 major toxic effect: diminish available
the oxygen to the tissues or tissue hypoxia due to inhibition of
oxyhemoglobin saturation (shift to the left of the oxygen dissociation
curve
toxic level: 20% CO
susceptible organs: brain and heart
indication of acute toxicity:
"cherry-red" color of the face
sample for testing: EDTA whole blood
definitive method for testing: cooximetry
(carboxyhemoglobin measurernent)
C. CYANIDE PAST MCTING POGONING!
i t can exist as a solid,
gas or in solution.
it is a super toxic substance
it is a component of
(fast-acting toxin) and death may occur less than an hour.
insecticides and rodenticides; common suicidal
it is also a
pyrolysis product burning agent.
it expresses its of plastics.
toxicity by binding to iron (ferric and ferrous
hemoglobin and cytochrome oxidase resulting to tissue and cellular forms) containing substances like
it inhibits cellular hypoxia.
respiration, electron transport and ATP formation by
cytochrome A3 inhibition of celular respiration leads to preventing reoxidation of
lactic concentration in the blood. metabolic acidosis due to increased
toxic effect: inhibition of the electron
transport chain and cell death
indication of toxicity: "odor of bitter almonds"
breath and altered mental status
antidote: sodium thiosulfate, amyl and sodium
nitrite
toxic symptoms:
tachypnea, convulsions and coma
toxic levels: >2 ug/mlL

D. METALS
All metals can be toxic if ingested in
large quantities and absorbed in their ionized forms.
1. Arsenic
OP it is a
component of ant poisons, rodenticides, paints and metal alloys.
t is common homicide or suicide agent; common agent of
a

i t inhibits sulfhydryl enzymes throughout the body; it crossesheavy

metai poisoning.
the placenta.
it expresses its toxicity by high
affinity binding to the thiol groups in proteins.
the used of hair and nalls ("Mees
lines") as specimens are important in the evaiuation of
erm (chronic) exposure. long-
blood and urine specimens are for assessment of short-term
(acute) exposure.
toxicforms: sodium arsenate, copper arsenite, carbarsone and tryparsamide
most toxic form: arsine gas
symptoms of intoxication: hyperpigmentatiori, dryness of the mouth,
swaliowing, anorexia and bloody diarrhea difficulty in
indication of toxicity: "odor of garlic" breath and metallic taste
tonic effects intravascular hemolysis,
involvement
hemoglobinemia,nephrotoxicity and multi-organ
acute fatol dosage: 120 mg (arsenic trioxide) and 30 ppm (arsenic
gas)
antidotearitish anti-tewisite (BAL)- for "arsenic-rescue" of affected cells
WE, AK, S Chanit
226
 method: Reinsch test, atomic absorption spectrophotometry

2. Cadmium
i t is utilized in electroplating and galvanizing.
it is a significant environmental pollutant pigment in paints and plastics.
poisoning can result from ingestion of acidic foods stored or prepared in metal containers made
up of cadmium.
toxicty may result to destruction of type 1 epithelical cells in the lung and decreased resistance
to bacterial infections.
it may also accumulate in rena! tubules causing tubular damage.
toxic renal indicator: (+} GGT in urine sample

3. Lead epoe: knenia,t dlo. ADHO, »i ta chwmagenic dye

i t is a potent enzyme inhibitor- it blocks delta aminolevulinic acid (ALA) synthetase, pyrimidine
S'-nucleotidase and Na-K-dependent ATPase.
source: paints and gasoline
mode of acquisition: ingestion or inhalation
susceptible areas: central and peripheral nervous systems
indications of toxicity: urinary amipgleyulinic acid, free RBC proporphyrin and presence of
basophilic stippling in RBC
toxic dose: >0.5mg/day Eqnuy moRM URINe
fatal dose: 0.5g baxap hilic Shppaiog
CDCcutoff level in children: <10 ug/dL
toxic blood levek: > 70 ug/dL (definitive lead poisoning) USE: Mgbi STKIaN ETRY
requires chelation therapy (children): <25 ug/dL
lead chelators: EDTA and dimercaptosuccinic acid (DMA)
T8L: oug/d (deinl sohing)
ToxicEffects:
it interferes with vitamin D and heme synthesis pathways by inhibiting delta aminolevulinic acid
(ALA) synthetase, producing anemia.
i t inhibits pyrimidine-5'nucdeotidase and Na-K-dependent ATPase resulting to diminished
integrity of the red cell membrane.
it combines with the matrk of bone and persists in this area for a long time (half life- 32 years).
low-level exposure may cause behavioral changes hyperactivity and attentional deficit
disorder, and also affects intelligence quotient scores (decrease score).
it has a characteristic "wrist drop or foot drop" manifestation (peripheral neuropathy).
.toxic eftects: encephalopathy, nephrosis, anorexia, perlpheral neuropathy, birth defects,
anemia, behavioral changes and compromised immunity

Methods:
Samples: whole blood, urine and hair
whole blood is the sampie of choice for quantitative testing because lead is bound to the red
blood cells, and it will produce the greatest sensitivity.
urine is used for assessment of recent lead exposure.
testing for the diagnosis of lead poisoning should include analysis of morning urine for delta
ALA.
serum or plasma should not be used because iead is rapidly eliminated from plasma.

227
Laboratory Iests:
1. Screening test
a. Zinc protoporphyrin test (Flurometric test)
.ALAD (6-ALA dehydratase) test -sensitive method; decreased urine ALAD activity in iead poisoning
2. In-vivo x-roy fluorescense of bones- to determine lead burden
3. Atomic absorption
spectrophotometery
4. Inductively coupled plasmo emission
spectrophotometry
5. Anodic stripping voltammetry
4. Mercuryy
i t binds with sulhydryl proteins.
t is a potent enzyme inhibitor - it inhibits catecholamine-O-metryltransferase, an enzyme
"essential in the metabolism of catecholamines.
it has the ability to "amalgamate" mix or merge with other substances.
-

forms of mercury: elemental or metallic mercury, mercurous, mercuric and alkyl mercury
modes of acquisition: inhalation, skin absorption and ingestion
symptoms oftowicity: hypertension, tachycardia and sweating -"cardinal signs" of
hare pheochromocytoma or mimics that adrenal gland disorder
general toxic effect: prgan dysfunction- lungs, kidney and CNS
major toxic effect of elemental mercury: pink disease (acrodynia) and erethism
major toxic effect ofäikyl mercury: congenital Minimata disease neiroltgical dlo
major route of excretion: through the bile (part of the biie fluid
samples: whole blood and 24 hour- urine
method: Reinsch test
reference levet: <10 g/dl
significontexposure: >50 ug/dt (whole blood)

Exposureamd Route of Absorption.

smalil drops of mercury on benchtops and floors can poison the environment in a poorly
ventilated room.
tf inhaled or absorbed through the skin it can pass through the blood-brain barrier, and can
accumulate in the CNS.
the presence of this substance in blood may result to loss of
glomerular integrity.

228
L. DRUGS OF ABUSE
Almost alf drugs of abuse are basic drugs (amine derlvatives) which contain benzene rings;
barbiturates are acidic drugs.
Many of the abused drugs act directly on dopaminergic neurotransmitter systems, especially the
limbic system (smell brain).
A pasitive drug screening test cannot differentiate casual user from chronic or habitual user,
ikewise detect the time frame of using the drug or dose of the drug taken.
Designer drugs are modified forms of established drugs of abuse
NAKLOLEPSY Lunonto wcd ooping)
1. Amphetamines
t i s a therapeutically used for treatment of narcolepsyand attentional deficit disorder.
t inereases mentelalertness and physleal-capacity, and hasanerectieproperty. iur. (o aPpcthe)
tis structurally related to dopamine and cathecolamines.
t cause the release (together with cocaine) of dopamine from the brain leading to a "pleasant
feeling" (so called "high') among users. DESINER OX;
3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy), a derivative of methamphetamine
is a popular recreational abused drug (designer drug); has psychedelic effects.
Examples: amphetanine, methamphetamine and methylphenidate (Ritalin, for treatment of
hyperactive children)
Amphetomine-like compounds: ephedrine, pseudoephedrine and phenylpropanolamine
Cause offaise-positive reaction: presence of antihistamine (diphenhydramine)
Sign of acute intoxication: hyperpyrexia
Acute psychotic syndromes: auditory and visual hallucinations, suicidal tendency and paranoia
Toxic effects: palpitation, hypertension, cardiac arthythmias, convulsions, psncytopenia,
mentai impairment and teeth grinding
Dx gmnt mduo vt
2. Anabolic sterolds (ESSTEEONs)
These are chemically associated to the male hormone testosterone (dihydrotestosterone and
testosterone).
t improvesetheletic performance by increasing muscle rnass.
Toxic effects: chronic hepatitis, atherosclerosis, abnormal platelet aggregationand cardiomegaly

3. Cannabinoids
Naturally occurring cannabinoids: marijuana and hashish
Tetrahydrocannabinol (THC), is the most potent component or the psychoactive substance of
marijuana.
THCa lipophilic substance, distributes in the adipose tissues; it easily enters the brain; it induces
a sense of well-being and euphoria; it is a hallucinogen.
THC is also associated with impairment of memory and intellectual functions.
After a single use, THC-CoOH can be detected in urine for 3-5days; up to 4 weeks for chronic
user. (LONGEGT +)
Principal psychoactive agent: deita-9-tetrahydrocannabinol
Orinary metabolite: 11-nor-deltatetrahydrocannabinol (THC-COOH)
Physiologic efects: reddening of the conjunctiva and increased pulse rate
Toxic effects: paranoia, disorientation, altered physical senses and bronchopulmonary disorders
POK MEMOoy,bs thecn Pa, REb eTE.
Lan 6UT45 Dh1S
Marijuafa
CtmONiC5 mawrhs 229
 4. Cocaine (Crack) NOT TRUE toaU TINE %"
it is an alkaloid salt
(ecgonine) that can be taken directly (insufflation of V) or by
inhalation/snorting.
uis derived from coca plant (Erythroxylon) and used as additive to some foods.
Is used as local anesthetic for
nasopharyngeal surgery.
A potent CNS stimulant that elicits a
sense of excitement and euphoria;
increasesphysical
activity
It has not beenconsidered as an addictive drug it does not reflect the true dependence
-

commonly seen in abusers of barbiturates and opiates.

t easily passes the
placenta and mammary glands (readily passed from mothers to infants)
resulting to mental retardation, slow mental development and drug dependence in newborns.
It can caused malformations in uterus.
It can cause sudden death due to direct
toxicity on myocardium (cardiac toxicity)- it
induces vasoconstriction, platelet aggregation and
synthesis of plasminogen activator inhibitor.
Overdosage of this drug may result to violent behavior; high abuse potential.
For single use, it can be detected in urine for
up to 3 days; up to 20 days for chronicusers.
Inhibitor: Prozac
Treatment for cocaine addiction:
Benzodiazepine
Toxic effects: hypertension, arrhytmias, seizures and
myocardial infarction
Urine metabolite: benzoylecgonine (sensitive and specific indicator)
4. Opiates
They are capable of analgesia, sedation and anesthesia.
They are derived chemicaly from opium poppy.
Naturally occurring opiates: opium, morphine and codeine
Chemically modified opiates: heroin, hydromorphone and oxycodone (Percodan)
Common synthetic opiates: meperidine (Demerol), methadone
(Dolophine), propoxyphene
(Darvon), pentazocine (Talwin) and fentanyl (Sublimaze)
Codeine is an antitussive drug.
Methadone is a nonbicyclic drug that binds with morphine in the brain.
Fentanyl "lolipops" or "patches" are more potent analgesics than morphine.
Commonly tested opiates:morphine and codeine
Mojor couse of drug-related death: darvon overdose combine with alcohol
Major metabolites of heroin: N-acetyimorphine (heroin) and morphine
Withdrawal symptoms of heroin: cold sweats, nightmares and
hypothermia
Antagonist for opiate overdose: naloxone (narcan)
Toxic effects: respiratony acidosis, myoglobinuria, cardiopulmonary failure and
pupillary constriction ("pin-poit pupils")
OX
MHLy Moo TIvE
Propertiesof Morphine and Heroin
Morphine is a metabotite of heroin; a powerful analgesic; used in the treatment of acute
congestive heart failure.
Morphine binds to mu-receptors in the limbic systern (CNS) producing analgesic effect.
Morphine and meperidine increase liver and pancreatic enzymes.
Heroin is highly addictive (true physícal dependencel;
Heroin crosses the blood-brain barrier- elevated levels in the CNS.

230
 Heroin is taken by Vadministration.

5. Phencyclidine (angel dust or angel hair)

* I tis a depressant, stimulant, and has hallucinogenic and anaesthetic properties.
It can be ingested or inhaled by smoking.
About 10-15% is unchanged when excreted in the urine acidification of the urine helps in
immediate excretion.
Physiologic effects: analgesia and anaesthesia
Major metobolite: Phencyclidine HCI
Mode of Treatment: Isolation (kept in quiet, dark room)
Toxic effects: tachycardia, seizure and coma (eventually death)

6. Sedatve Hypnotics
They have therapeutic roles and they are CNS depressants.
They are used also to potentiate the effects of heroin.
Examples: barbiturates and benzodiazepines
Commonly abused barbiturates: secobarbital, pentobarbital, phenobarbital andthiopental
Commonly used benzodiazepines: diazepam (Valium), chlordiazepoxide (Librium), &lorazepam-4Ativan)
Barbiturates are condensation products of urea and malonic acid.
Benzadiazepines are usedfortreatmemt ofcocaine addictiony
Diazepam has a clinical use for rapid control of acute seizure activity; a minortranquilizer.
Phenobarbital structurally resembles phenytoin (Dilantin).
Toxicity of this agents is initiated by ethanol.
Major metabolite: secobarbital (barbiturates)
Barbiturate chemoadsorbent:activated charcoal
Toxic effects: Cheyne-Stokes respiration, depression,cyanosis, arefiexia, stupor, coma

7. Lysergic Acid Diethylamide (L.SD, LYsergide)

I t i s a semisynthetic indolalkylamine; a hallucinogen.
it is one of the most potent pharmacologic materials known.
It produces effects at low doses- 20ug (N or ingestion).
Most common odverse reaction: panic reaction "bad trip"
Toxic effects: blurred or "undulating vision" and synesthesia

8. Methaqualone (Quaalude)
I t is a 2,3-disubstituted quinazoline with anesthetic, antihistamine and antitussive properties.
t alsc
has sedative-hypnotic
properties; a hatucinogen.
This abused drug has similar symptoms of toxicity to barbiturates, as well as pyramidal signs
(hypertonicity, hyperreflexia and myoclonus).
Chemoodsorbent: activated charcoal

9.Piperazines
They produced the same "pleasant feeling" observed in amphetamines.
N o available screening nor confirmatory
test for identification of these drugs
Major derivatives; N-benzyipiperazines (82P or A2) and phenylpiperazines
Minor derivotives: 1-13-4-methylene-dioxybenzy})
piperazine (MDMP), 1-43
trifluorometyiphenvi) piperazine (TFMPP or Molly)
Popular piperazines: BZP and TFMPP

231
 Toxicefects: tachyeardia, hypertension, hypertherma, psychomotor agitation and sore nasal
and throat passages
10. Tryptamines &unNE% naws LuNCH
These are derivatives of serotonin; some compounds are present in plants.
Examples: N,N-dimethyltryptamine (DMT), psilocin
DMT is a
short-term hallucinogen ("businessiman's !unch"); taken by smoking.
daubic M
Psilocycin is a component of "magic mushrooms" (psilocybe); a hallucinogen.
Monoamine oxidase inhibitor (B-carbolines) enhances the
M hallucinogenic effect of tryptamines.
Ayahuasca is a tea which contains tryptamines.
-

Antagonist: benzodiazepines
Toxic effects: tachycardia, hypertension, dystonia, seizures,
rhabdomyolysis and paralysis

Specimen Considerations:
1. Alcohol in blood samples may be analyzed even after a moment of
delay provided the
samples in tubes remain sealed, because blood lacks the enzyme that metabolize it.
2. Urine temperature is a vital factor to assure
that it is freshly
voided (abused drug).
3. Aspiration of gastric contents or vomitus may reveal tablets or capsules from which the
ingested drug can be determined.

Methods for identifying and measuring abused drugs:

Sample requirement: urine, serum, hair, nails, whole blood or plasma (alcohol), sweat, saliva and
exhaled breath (alcohol intoxication)
i h e sample for toxicology assay has the advantage of knowing the complete composite of
drugs that have been ingested over a longer period than blood
samples.
Once a urine specimen collected, it is subjected to concentration and extraction procedures.
In extractionprocedures, acidic drugs are separated from basic ones.
Examination of blood has the advantage of
identifying currently circulating drugs and to design
treatment plan and monitoring the success of treatment.
Drugs when deposited in hair, are generally present in relatively low levels.
oSFor sweat testing, parent drug is increased
than metabolites.
For saliva testing, drug concentration in it reflects the
free or active fraction.
2 basic techniques for identification of drugs: immunochemical and chromatograpy
1. Enzymatic test
Alcohol is measured from blood using alcohol
It quantitates the sum of all alcohols
dehydrogenase as reagent.
in
present a sample.
It does not distinguish alcohols from its metabolites
during quantitation.
2. Capillary Electrophoresis
Different analyte selectivity is based on different
physicochemical principles of separation
without changes in instrumental hardware, a distinct
advantage of this technique.
Recent variant of TLC that includes the advantages of HPLC.

3. Homogenous Immunoassay
This assay is done in one solution without
separation.

232
Enzyme Mediated Immunologic Technique (EMIT)
This method uses enzyme-Jabeled drug that competes with the drug in the sample.
In this reaction, the active site of the enzyme is blocked with the antldrug antibody, resulting to
decreased enzymatic activity.
The free drug (analyte in the serum or urine) competes with the antibody-drug-enzyme
complex, separating the

4. Chromatographic methods
a. Thin layer chromatography (TLC)
This method uses serum, urine or gastric fluid for analysis.
Extraction of drugs is pH dependent - acidic drugs at pH 4.5 (barbiturates) and alkaline drugs
(opiates, amphetamines) at pH 9.0.

b. Liquid Chromatography-Mass Spectrospcopy (LC-MS)

tcan be used to confim positive test results froma screening assay (nonvolatile
Compounds).
t also be used for detection of poisons in acute or chronic intoxication,
can

therapeutic drug identification and quantitation, pharmacokinetics and drug

metabolism studies.

cHigh performance liquid chromatography (HPLC)

I t allows quantitative measurement of drugs as well as separation of these same drugs,

especially tricyclic antidepressants including its active and inactive metabolites.

It may be used as an aternative to GC-MS in definitive identification of drugs.

d. Gas Chromatography
G a s Liquid Chromatography (GLC) - is the legally accepted method for ethanol testing
GC with Infrared Spectroscopy-for detection of amphetamines
as Chromatography-Mass Spectroscopy (GC-MS) CowPeATORY
is the gold standard for confirmation ofscreening methods such as TLC and EMIT.
- it allows for detection of low levels of drugs like cocaine and drug metabolites.

Note: For complete explaination of the above-mentioned methods, please refer to theprevious
discussions on analytical methods.

Urine isthe prefered sample especialy in drug testing because ofthe foilowing reasons:
1. Drugs and their metabolites are present in higher concentrations in urine than in blood.
2. Larger sample volumes are easilycollected
3. There is no pain or discomfort when collecting the sample.
4. The process of obtaining the sample is noninvasive.

Factorsresuttingtoincorrect drugtesting results

1. Presence of detergents will result to alkaline ph.
2. Use of sodiurn chioride (table salt).
3.Low specific gravity (diluted urine)
4.High pHfalkaline urine)
233
5. Blood in urine
(hematuria)
Notes to Remembe
Urine drug detection is from about 12hours
in 2-5 days.
to three weeks- but most drugs are undetectable
Drug detection in human hair has longer positivity (mornths).
Whole blood is the best specimen for alcohol
determination; breath test is also used.
Adulteration is a means of
tampering the specimen for drug testing, to make the specimen
falsely negative for drugs.
Any confirmatory method is valid, provided it is a
one (TLC can confirm
completely different method from the primary
EMIT).
DX ur or VmuES CAg Im
eMr
AnPeSTHAmnNE

CocME
0ANTE
PRENLCYuDINE
amgel dust f angelai

234