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MTAP 1 - Clinical Chemistry 1

BS Medical Technology (University of the East Ramon Magsaysay)

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Basic Principles and Practices, Laboratory Mathematics,

Safety, and Method Evaluation, and Analytic Techniques CLCHEM
UERM CAHP 2023 First Semester LEC 10
Medical Technology Assessment Program 1 TOPIC 1
OUTLINE REFER TO FIGURE 1
I. Blood and Chemistry 1 ● The crest or peak is the highest point of the wave
II. Instrumentation 1 ● Light is measured in wavelength and expressed in
A. Photometry 1 nanometers (nm)
B. Spectrophotometer 1 o Wavelength – distance between two consecutive peaks
C. Other Light-Based Instruments 2 o Wavelength is inversely proportional to energy (light)
III. Quality Assurance 2 ▪ Longer wavelength = farther distance = lower energy
A. Pre-Analytical Phase 2 ▪ Shorter wavelength = closer distance = higher energy
B. Analytical Phase 3
C. Post-Analytical Phase 5 Table 1. History of Medical Technology
IV. Laboratory Mathematics 5 SPECTRUM OR RANGE OF LIGHT
A. Arterial Blood Gas 6 γ-ray ← X-ray ← UV ← Visible Light → IR → Microwave → Radio waves
V. Laboratory Safety 7 ● Light seen by the normal human eye
A. Chain of Infection 7 ● A.k.a white light because its original
B. Handwashing 7 color was technically white
C. HEPA Filters 7 o White is the most flexible color of all
D. Waste Segregation 7 time because all other colors are
E. Material Data Safety Sheet 7 derived from the white color
F. NFPA Hazards Identification Symbol 7 o Rainbow after the rain – the white
G. Fire 7 light coming from the sun passes
VISIBLE LIGHT through a rain drop which will act as
I. BLOOD AND CHEMISTRY (WHITE LIGHT) a prism and separate the white light
● Blood is made up of formed elements and fluid portion into seven different colors
o Fluid portion – known as plasma (anticoagulated or (ROYGBIV)
unclotted blood) or serum (coagulated or clotted blood) ● 400nm – 700nm
▪ Made up of 90% water and 10% solutes ● Colors – violet, indigo, blue, green,
● Solutes measured in chemistry – CHO, lipids, CHON, yellow, orange, and red
enzymes, NPNs, TDM, toxic substances, tumor markers, o Longest wavelength (700nm) –
vitamins, electrolytes, trace elements, bilirubin, etc. red
● Goal of chemistry – detect solutes or analytes o Highest energy (400nm) – violet
● 4nm – 400nm
II. INSTRUMENTATION ULTRAVIOLET o Gamma rays are smaller than UV
(UV) ● Called ultraviolet because it is smaller
A. PHOTOMETRY
than the color violet
● Involves measurement (-metry) of light (photo-)
● 700nm – 0.3cm
● Light – a form of energy that travels in waves
o Microwaves and radio waves are
o Light waves have a peak, trough, amplitude, and
INFRARED (IR) bigger than IR
wavelength
● Called infrared because it is bigger than
● Light follows the law of conservation of mass and energy
the color red
o Law of conservation of mass and energy – energy is
neither created nor destroyed, energy is already around
NICE-TO-KNOW
us, it can only be transformed from one form into another ● The eyes of cats are designed as such so that they can see light
o Energy is convertible which means light is also beyond the visible spectrum
convertible ● Humans who can see beyond the visible light (usually related to
▪ Light can be converted into heat energy (light bulbs paranormal activity) are known as humans with the third eye
in chicken coops) and electrical energy (solar panel)
o Photodetector – part of the spectrophotometry that B. SPECTROPHOTOMETER
converts light energy to electrical energy ● Measurement of light on a specific spectrum or range of light
● Light measurement is complicated because there is a lot of o Spectro (spectrum) + photo (light) + meter (measurement)
different types of light (Refer to Table 1) and light can be ● Follows the Beer-Lambert law – instructs us to find
configured absorbance through the use of the spectrophotometer to find
o Types of light – gamma rays, X-rays, UV, visible light, concentration
infrared, microwaves, and radio waves 𝐴 = 𝑎𝑏𝑐
o Light can be – absorbed (spectrophotometer and atomic o A = absorbance
absorption spectrophotometer), emitted (flame emission o a = absorptivity coefficient (constant)
photometer), fluoresce (fluorometer), scattered o b = path length (thickness of the cuvette or sample holder)
(nephelometer), blocked (turbidimeter), and reflected o c = concentration
(reflectance photometers) ● Absorbance is directly proportional to concentration
o Increased absorbance = increased concentration
o Decreased absorbance = decreased concentration

NICE-TO-KNOW
● It is important to determine the spectrum or range of light so that
you can determine which light source to use
● Caraway method – measures the formation of tungsten blue for uric
acid analysis
o A.k.a phosphotungstic acid because it is the same reagent used
o Positive result – formation of tungsten blue measured
spectrophotometrically in the range of 650nm – 700nm (660nm)
Figure 1. Transverse Wave o Light source used should be visible light source since the
wavelength is under the visible light
● Enzymatic method – measures uric acid directly
o Enzyme used is uricase
o Measured at 293nm which is under the ultraviolet spectrum
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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
● Dubowski – a.k.a condensation method or ortho-toluidine used for 2. SAMPLE PROBLEMS
glucose analysis
o Reagent used is ortho-toluidine + acetic acid + 100°C
o Positive result – bluish-green complex measured at 620nm –
LS M C D ROD
630nm (visible light)
● Cyanmethemoglobin (HiCN) method – measures the formation of
HiCN at 540nm (visible light) Visible Light 600nm Tungsten TL Telec #
● Optical density or absorbance (OD450) – used for measuring (400nm – 700nm) Blue
bilirubin in the amniotic fluid to assess the severity of HDN at 450nm
Figure 3. Measuring Tungsten Blue

1. PARTS OF THE SPECTROPHOTOMETER 1. Measuring tungsten blue

o Light source – visible light (since it ranges from 400nm –
700nm)
o Monochromator – 660nm (wavelength which is needed
LS M C D ROD
to measure the tungsten blue)
o Cuvette – holds the tungsten blue
▪ The tungsten blue will hold the absorbed light (light
Figure 2. Parts of the Spectrophotometer in Order of Appearance
hugged by the tungsten blue)
▪ Some of these absorbed lights will pass through or
Table 2. Parts of the Spectrophotometer
escape the tungsten blue (transmitted light)
PARTS DESCRIPTION
o Photodetector – converts light into electrical energy
● Serves as the source of light
o Read out device – converts electrical energy into
● Visible infrared (vis-IR) – tungsten
LIGHT readable numbers
halogen (iodide) lamp
SOURCE
● Ultraviolet – mercury arc lamp, xenon arc
2. What is the concentration of a glucose sample that has an
lamp, and deuterium discharge lamp
absorbance of 0.20, if a 100mg/dL glucose standard has an
● Isolates the wavelength of interest absorbance of 0.80?
MONO-
● Examples – prism, diffracting gratings,
CHROMATOR o (𝐴𝑢 /𝐴𝑠 ) = (𝐶𝑢 /𝐶𝑠 )
and interference filter
o (0.20/0.80) = (𝐶𝑢 /100)
● Holds the sample and contains the analyte o 𝐶𝑢 = (0.20/0.80)(100)
of interest
o 𝐶𝑢 = 25
● Transparent and free from scratches and
o 𝐶𝑔𝑙𝑢𝑐𝑜𝑠𝑒 = 25𝑚𝑔/𝑑𝐿
dirt
o For reflectance photometers,
transparent cuvettes are not allowed C. OTHER LIGHT-BASED INSTRUMENTS
CUVETTE OR because transparent cuvettes do not ● Double beam spectrophotometer – modified
SAMPLE reflect spectrophotometer
HOLDER ● Round end and square end cuvettes ● Fluorometer – light that is fluorescent
o Square ends are preferred over the ● Flame emission photometry – light that is emitted
round ends because on the “b” in the ● Atomic absorption spectrophotometry – light that is
Beer-Lambert’s law or path length or absorbed
thickness of the cuvette which should ● Reflectance photometry – light that is reflected
be constant (square ends have more ● Nephelometry – light that is scattered
equal thickness than round ends) ● Turbidimetry – light that is blocked
● Converts light into equivalent amounts of
electrical energy to make it easier to III. QUALITY ASSURANCE
measure ● Checks if the results generated are reliable
● Light is measured in wavelengths in ● A complete system of creating and following procedures and
PHOTODETE policies to aim for providing the most reliable patient laboratory
nanometer (too small) making it harder to
CTOR results and to minimize errors in the pre-analytical, analytical,
measure compared to electrical energy
● Examples – barrier layer cell, and and post-analytical phases
photomultiplier tube (most sensitive), and ● Quality control – an aspect of quality assurance that is used
photodiode to assess the analytical phase of patient testing
● Helps read the result by converting
electrical energy into digits or numbers A. PRE-ANALYTICAL PHASE
● Determines the magnitude of electrical ● Patient preparation – fasting, dos and don’ts, and instructions
energy to patients prior to the testing
READ OUT
● The number given by the ROD is the ● Time of collection – morning, night, random, and timed
DEVICE
transmittance light (%T) not the specimens
absorbance ● Specimen collection order – proper collection order
● The transmittance (%T) will be converted (venipuncture)
to absorbance using Beer-Lambert’s law ● Quality of specimen collected – hemolyzed, icteric, and
lipemic
a. BEER-LAMBERT’S LAW ● Specimen processing – transport and centrifugation
1. Convert transmitted light (%T) to absorbance ● Specimen storage – refrigeration, room temperature, frozen,
𝐴 = 2 − 𝑙𝑜𝑔%𝑇 and light sensitivity
2. Convert absorbance to concentration ● Specimen preservation – anticoagulants and preservatives
𝐴 = 𝑎𝑏𝑐
3. Beer’s law of unknown is directly proportional to the solution of
known concentration (known as the standard or reference
solution) by comparison
(𝐴𝑢 /𝐴𝑠 ) = (𝐶𝑢 /𝐶𝑠 )
o A = absorbance
o C = concentration
o u = unknown
o s = standard

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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
B. ANALYTICAL PHASE NICE-TO-KNOW
1. STATISTICAL TOOLS ● Prostate specific antigen (PSA) – more reliable in diagnosing
patients with prostate cancer because it is more sensitive and specific
● Measures of central tendency – mean, median, and mode than acid phosphatase (old method)
o Checks the tendency or desire of the values to cluster or ● How to know determine the diagnostic sensitivity and specificity of
aggregate in the middle or center PSA?
o Mean – most commonly used o PSA is used to patients confirmed with prostate cancer (positive
o Mean is usually seen in the middle in the Gaussian curve diagnosis) and confirmed without prostate cancer (negative
and Levey-Jennings diagnosis)
Get the patient’s laboratory results and correlate with the
● Measures of scatter or dispersion – standard deviation, o
diagnosis
variance, and coefficient of variance ▪ Diagnosis (+) and laboratory test (+) = true positive
o Checks how far and wide are the values are apart from the ▪ Diagnosis (-) and laboratory test (+) = false positive
value ▪ Diagnosis (+) and laboratory test (-) = false negative
o Always moving away from the mean or center ▪ Diagnosis (-) and laboratory test (-) = true negative
o Standard deviation – most commonly used
5. CHOOSING A GOOD METHOD OR ASSAY
2. STATISTICAL SIGNIFICANCE ● A good assay should be both sensitive and specific and at the
● t-test – statistical difference between two means same time accurate and precise
● F-test – statistical difference between two standard
deviations Table 4. Evaluating a Good Method or Assay
DESCRIPTION
3. GAUSSIAN DISTRIBUTION CURVE ANALYTICAL ● Ability to detect the smallest
● Combination of central tendency and dispersion SENSITIVITY amount of analyte
● Bell-shaped ANALYTICAL ● Ability to detect the specific analyte
● Normal distribution curve assumes that the mean = median = SPECIFICITY of interest
mode ● Ability to maintain both accuracy
RELIABILITY
o ± 1SD = 68.3% (inside) and 31.7% (outside) and precision
o ± 2SD = 95.5% (inside) and 4.5% (outside) ● Ability of the values to be close to
ACCURACY
▪ Confidence interval (usually used) the true or target value
▪ Not extremely small or big ● Ability of the values to be close with
PRECISION
▪ Used for reference ranges each other
o ± 3SD = 99.7% (inside) and 0.3% (outside) ● Aspects of precision
▪ Not really reliable because it is too big ● Closeness of the values after
repeating it using the same
conditions
REPEATABILITY
● Example – a specimen for glucose
value is repeated by the same RMT
with the same machine, pipette,
sample, and temperature
● Aspects of precision
● Closeness of the values after
repeating it using changed
conditions
REPRODUCIBILITY
● Example – a specimen for glucose
value is repeated by a different RMT
with a different machine, pipette,
sample, and temperature
● How practical a method is
● How easily a method is repeated
Figure 4. Gaussian Curve ● Example – one step pregnancy test
PRACTICABILITY
o Biological tests for pregnancy
4. STATISTICAL BINARY CLASSIFIERS are not practical (use of frogs
● Tools for evaluating diagnostic efficiency (how helpful a method and rabbits)
is in diagnosing a disease)
o Diagnostic sensitivity and diagnostic specificity – 6. HOW TO CHECK FOR ACCURACY AND PRECISION
mostly important and used by medical technologists ● Calibrating the instruments – usage of standards (solutions
▪ Important in promoting a product, test, and method of known concentration)
(high sensitivity and specificity = good and helpful) ● Internal quality control – done at least on a daily basis by
o False negative rate and false positive rate RMTs in their own laboratory alone (no outside forces or
o Positive predictive value and negative predictive value agencies involved)
o False discovery rate and false omission rate o Good for accuracy
● External quality control – done by outside forces or agencies
Table 3. Diagnostic Sensitivity and Specificity o Outside agencies sends a sample for the laboratory to run
SENSITIVITY SPECIFICITY o The results from different laboratories will be compiled
DIAGNOSIS (+) DIAGNOSIS (-) within the geographical area and evaluate them with each
LABORATORY other
True Positive (TP) False Positive (FP)
RESULT (+) o Good for precision
LABORATORY
False Negative (FN) True Negative (TN)
RESULT (-)

𝑇𝑃
𝐷𝑖𝑎𝑔𝑛𝑜𝑠𝑡𝑖𝑐 𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 (𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑅𝑎𝑡𝑒) = 𝑥 100
𝑇𝑃 + 𝐹𝑁
𝑇𝑁
𝐷𝑖𝑎𝑔𝑛𝑜𝑠𝑡𝑖𝑐 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦 (𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑅𝑎𝑡𝑒) = 𝑥 100
𝑇𝑁 + 𝐹𝑃

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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
a. INTERNAL QUALITY CONTROL 4. EVALUATE CHART USING THE WESTGARD
1. RUN CONTROLS MULTIROLE TO DETECT ERRORS
● At least once a day or when a new test kit or reagent is used
Table 5. Westgard Control Rules
2. RECORD AND PLOT CONTROLS USING RULE DESCRIPTION
LEVEY-JENNINGS’S CHART ● One control result exceeds the mean ± 2SD
12s
● Shewhart plot – most common QC chart used ● Random errors
o Day or time are plotted in the X-axis ● One control result exceeds the mean ± 3SD
13s
o Analyte concentrations are in the Y-axis ● Random errors
● The acceptable range or value of every analyte when ● Last two or any two control result exceeds the
performing the quality control should fall within ± 2SD 22s mean ± 2SD
● Acceptable limits of the variations and results in the context of ● Systematic errors
the upper and lower limits ● Last four or any four consecutive control result
o Sample – a control has a mean of 100mg/dL and an SD 41s exceeds the mean ± 1SD
of 2, what is the control limit? ● Systematic errors
▪ ±1𝑆𝐷 = 𝑚𝑒𝑎𝑛 ± 1(𝑆𝐷) ● Range or difference between the highest and
→ ±1𝑆𝐷 = 100 ± 1(2) R4s lowest control result exceeds ± 4SD
→ ±1𝑆𝐷 = 98𝑚𝑔/𝑑𝐿 𝑎𝑛𝑑 102𝑚𝑔/𝑑𝐿 ● Random errors or increased imprecision
▪ ±2𝑆𝐷 = 𝑚𝑒𝑎𝑛 ± 2(𝑆𝐷) ● 10 consecutive results are on the same side of the
→ ±2𝑆𝐷 = 100 ± 2(2) 10x
target mean
→ ±2𝑆𝐷 = 96𝑚𝑔/𝑑𝐿 𝑎𝑛𝑑 104𝑚𝑔/𝑑𝐿 ● Systematic error
▪ ±3𝑆𝐷 = 𝑚𝑒𝑎𝑛 ± 3(𝑆𝐷) ● No requirement for SD size
→ ±3𝑆𝐷 = 100 ± 3(2)
→ ±3𝑆𝐷 = 94𝑚𝑔/𝑑𝐿 𝑎𝑛𝑑 106𝑚𝑔/𝑑𝐿

Figure 7. Westgard Rules

Table 6. Systematic vs Random Errors

ERROR DESCRIPTION
Figure 5. Shewhart Levey-Jennings Chart ● Errors that are predictable because
they have a pattern or system
3. EVALUATE CHART FOR TRENDS AND SHIFTS ● Aging reagents
● Technologist interactions
(LEVEY-JENNINGS)
● Optical changes
● Trends – gradual change above or below the mean
SYSTEMATIC ● Reagent lot variability
o Increase or decreased control values for six consecutive
● Instrument wear and tear
days
● Aging calibrators
● Shifts – sudden or abrupt change in the data
● Instrument components
o Establishes a new distribution pattern above or below the
● Voltage fluctuations
established mean
● Calibration differences
▪ Control values that distribute themselves on one
● Errors that are not predictable or due to
side of the mean for six consecutive days
chance
● Reagent dispensing
● Temperature or analyzer
● Calibrator reconstitution
● Instability of instrument
RANDOM
● Variation in handling techniques
(pipetting, mixing, and timing)
● Sample evaporation
● Electrooptical mechanism
● Environmental conditions
● Variation in operators

7. PROBLEM SOLUTION FOR QUALITY CONTROL

PROBLEMS
1. Repeat the control again after mixing
2. Repeat the control using a fresh vial or another lot number
3. Recalibrate the assay
4. Replace the reagents
5. Perform necessary maintenance
6. Consult the troubleshooting guide, supervisor, or service
Figure 6. Trend (Top) and Shift (Bottom) representative

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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
C. POST-ANALYTICAL PHASE 9. How many grams of sulfuric acid should be weighed to prepare
● Reference ranges 2L of 4N sulfuric acid?
● Delta check – checking for any changes Atomic weights – H = 1, S = 32, O = 16
o Used to check the patient’s current results with their
𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
previous results o Formula: 𝑁 𝑜𝑟 𝐸𝑞/𝐿 = 𝑀𝑊
( ) 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● Alarms and flags (critical values) 𝑣𝑎𝑙𝑒𝑛𝑐𝑒
𝑀𝑊
● Recording and reporting of results o 𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = (( ) 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) 𝑁
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
● Calculations
o Calculate the molecular weight of H2SO4:
▪ H=2x1=2
IV. LABORATORY MATHEMATICS ▪ S = 1 x 32 = 32
1. Convert 444mm into meters
▪ O = 4 x 16 = 64
o Conversion factor: 1m = 1,000mm
1𝑚 ▪ Total MW = 98
o 444𝑚𝑚 𝑥 = 0.444𝑚 98
1,000𝑚𝑚 o (( ) 𝑥 2𝐿) 4 = 392𝑔 𝑜𝑓 𝐻2 𝑆𝑂4
2
2. Convert 100mg/dL to g/dL
o Conversion factor: 1g = 1,000mg 10. A serum sample shows a chloride concentration of 500mg per
100𝑚𝑔 1𝑔 100mL, which is the same as ___ mEq/L
o 𝑥 = 0.10𝑔/𝑑𝐿
𝑑𝐿 1,000𝑚𝑔 Atomic weight of chloride – 35.5

3. A patient’s weight is 154lbs, express his weight in kilograms 𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

o Formula: 𝑚𝐸𝑞/𝐿 = 𝑀𝑊
o Conversion factor: 1kg = 2.2lbs ( ) 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
1𝑘𝑔 500𝑚𝑔
o 154𝑙𝑏𝑠 𝑥 = 70𝑘𝑔 o 35.5 = 140.85𝑚𝐸𝑞/𝐿
2.2𝑙𝑏𝑠 ( ) 𝑥 0.1𝐿
1

4. What is the %v/v if you mixed 50mL of sulfuric acid with 50mL
11. What is the equivalent molarity of a 0.5N H2SO4?
of water? 𝑁
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 o Formula: 𝑀 =
o Formula: %𝑣/𝑣 = 𝑥 100 0.5𝑁
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
50𝑚𝐿 o = 0.25𝑀
o 𝑥 100 = 50%𝑣/𝑣 𝑜𝑓 𝑠𝑢𝑙𝑓𝑢𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 2
50𝑚𝐿+50𝑚𝐿
12. How much 95% alcohol is required to make 200mL of 5%
5. What is the %w/w if you mixed 30g of HCl with 70g of water? alcohol?
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
o Formula: %𝑤/𝑤 = 𝑥 100 o Formula: 𝐶1 𝑉1 = 𝐶2𝑉2
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
30𝑔 o 95 (𝑥) = 5(200)
o 𝑥 100 = 30%𝑤/𝑤 𝑜𝑓 𝐻𝐶𝑙 5(200)
30𝑔+70𝑔
o 𝑥=
95
6. If 1g of potassium iodide is used to make up to a total volume o 𝑥 = 10.53𝑚𝐿
of 100mL, calculate the %w/v o TIP – the word “of” serves as the clue to which is partner
𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 with which
o Formula: %𝑤/𝑣 = 𝑥 100
𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
1𝑔
o 𝑥 100 = 1%𝑤/𝑣 𝑜𝑓 𝑝𝑜𝑡𝑎𝑠𝑠𝑖𝑢𝑚 𝑖𝑜𝑑𝑖𝑑𝑒 13. If directions for a reagent specify 8.0g of Na2SO4 (anhydrous),
100𝑚𝐿
but the available salt is Na2SO4 • 10 H2O, how much of the
7. Determine the molarity given the following data: hydrated salt will use?
Mass of NaOH – 120g Atomic weights – Na = 23, S = 32, O = 16, H = 1
𝑔 𝑜𝑓 ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 𝑀𝑊 𝑜𝑓 ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑
MW of NaOH – 40 o Formula: =
𝑔 𝑜𝑓 𝑎𝑛ℎ𝑦𝑑𝑟𝑜𝑢𝑠 𝑀𝑊 𝑜𝑓 𝑎𝑛ℎ𝑦𝑑𝑟𝑜𝑢𝑠
Volume of solution – 750mL o Calculate the molecular weight of anhydrous Na2SO4:
▪ Na = 2 x 23 = 46
𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
o Formula: 𝑀 𝑜𝑟 𝑚𝑜𝑙/𝐿 =
𝑀𝑊 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
▪ S = 1 x 32 = 32
𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ▪ O = 4 x 16 = 64
o To convert mol/L to mmol/L: 𝑚𝑚𝑜𝑙/𝐿 =
𝑀𝑊 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ▪ Total MW of anhydrous Na2SO4 = 142
o Conversion factor: 1L = 1,000mL o Calculate the molecular weight of hydrated Na2SO4:
1𝐿
o 750𝑚𝐿 𝑥 = 0.75𝐿 ▪ H = 10 x 2 x 1 = 20
1,000𝑚𝐿
120𝑔 ▪ O = 10 x 16 = 160
o = 4𝑀 𝑜𝑟 4𝑚𝑜𝑙𝑒𝑠/𝐿 ▪ 180 + 142 = 322
40 𝑥 0.75𝐿
▪ Total MW of hydrated Na2SO4 = 322
8. Determine the normality given the following data: 𝑔 𝑜𝑓 ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 322
o =
Mass of NaOH – 120g 8.0𝑔 142
322
MW of NaOH – 40 o 𝑔 𝑜𝑓 ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 =
142
𝑥 8.0𝑔
Volume of solution – 750mL o 𝑔 𝑜𝑓 ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑑 = 18.14𝑔 𝑜𝑓 𝑁𝑎2 𝑆𝑂4
𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
o Formula: 𝑁 𝑜𝑟 𝐸𝑞/𝐿 = 𝑀𝑊 14. Determine the anion gap
( ) 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑣𝑎𝑙𝑒𝑛𝑐𝑒 Sodium – 140
▪ Valence – the subscript of the first element Potassium – 5
▪ Example – NaOH (valence is 1), H2SO4 (valence is Chloride – 87
2), and H3PO4 (valence is 3) Bicarbonate – 35
o To convert Eq/L to mEq/L:
𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝑚𝐸𝑞/𝐿 = 𝑀𝑊 o Formula: 𝐴𝑛𝑖𝑜𝑛 𝐺𝑎𝑝 = (𝑁𝑎 + 𝐾) − (𝐶𝑙 + 𝐻𝐶𝑂3 )
( ) 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
o Which parameter can you remove? Potassium is the least
o Conversion factor: 1L = 1,000mL
1𝐿 likely to affect the anion gap because it is the major
o 750𝑚𝐿 𝑥 = 0.75𝐿 intracellular cation (only exists in small amounts in the
1,000𝑚𝐿
12𝑔 plasma)
o 40 = 4𝑁 𝑜𝑟 4𝐸𝑞/𝐿
( ) 𝑥 0.75𝐿 o Modified Version (if K is not given): 𝐴𝑛𝑖𝑜𝑛 𝐺𝑎𝑝 = 𝑁𝑎 −
1
(𝐶𝑙 + 𝐻𝐶𝑂3 )
o (140 + 5) − (87 + 35) = 23

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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
15. Determine the creatinine clearance 1. SAMPLE PROBLEMS
Urine creatinine – 60mg/dL 1. pH = 7.29
Plasma creatinine – 1.73mg/dL pCO2 = 68.2mmHg
Body surface area (BSA) – 1.20 HCO3 = 26mEq/L
Urine volume – 2,880mL/24 hours
𝑈 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 (24ℎ𝑟 𝑢𝑟𝑖𝑛𝑒 𝑚𝐿/𝑚𝑖𝑛) 1.73
pH ❌ 7.35 7.45
o Formula: 𝐶𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 = 𝑐𝑟𝑒𝑎 𝑥 pCO2 ❌ 45 35
𝑃𝑐𝑟𝑒𝑎 𝐵𝑆𝐴
o Urine should be a 24-hour urine to standardize the test HCO3 22 ❌ 26
because creatinine is the end product of metabolism
Respiratory
(highly dependent in the activity of a person) Acidosis Uncompensated
o Body surface area is used to adjust for the muscle mass
of a person 2. pH = 7.49
▪ Increased muscles = increased creatinine pCO2 = 24mmHg
o Conversion factor: 1 hour = 60 minutes HCO3 = 22mEq/L
2,880𝑚𝐿 1 ℎ𝑜𝑢𝑟
o = 2𝑚𝐿/𝑚𝑖𝑛
24 ℎ𝑜𝑢𝑟𝑠 60 𝑚𝑖𝑛𝑠
60𝑚𝑔/𝑑𝐿 𝑥 2𝑚𝐿/𝑚𝑖𝑛 1.73 pH 7.35 7.45 ❌
o 𝑥 = 100𝑚𝐿/𝑚𝑖𝑛
1.73𝑚𝑔/𝑑𝐿 1.20 pCO2 45 35 ❌
HCO3 22 ❌ 26
16. Determine the LDL cholesterol level
Total cholesterol – 250mg/dL Respiratory
Uncompensated
Triglycerides (TAG) – 140 mg/dL Alkalosis
HDL-C – 45mg/dL
3. pH = 7.5
𝑇𝐴𝐺 pCO2 = 41mmHg
o Formula (Friedewald): 𝐿𝐷𝐿 (𝑚𝑔/𝑑𝐿) = 𝑇𝐶 − 𝐻𝐷𝐿 − HCO3 = 29mEq/L
5
𝑇𝐴𝐺
▪ 𝑉𝐿𝐷𝐿 =
5
140𝑚𝑔 pH 7.35 7.45 ❌
o 250𝑚𝑔/𝑑𝐿 − 45𝑚𝑔/𝑑𝐿 − 𝑑𝐿 = 177𝑚𝑔/𝑑𝐿 pCO2 45 ❌ 35
5
HCO3 22 26 ❌
17. Given the following data, determine the carbonic acid level
Uncompensated
pH – 7.07 Metabolic Alkalosis
pCO2 – 29.7mmHg
HCO3 – 8.3mmol/L 4. pH = 7.3
Delta content – 22.6 pCO2 = 63mmHg
HCO3 = 29mEq/L
o Formula: 𝐻2 𝐶𝑂3 = 𝑝𝐶𝑂2 𝑥 0.031
o 29.7𝑚𝑚𝐻𝑔 𝑥 0.031 pH ❌ 7.35 7.45
o 𝐻2 𝐶𝑂3 = 0.9207
pCO2 ❌ 45 35
18. Determine the free thyroxine index (FT4I) using the given data HCO3 22 26 ❌
Total T4 – 10ug/dL Respiratory Partially
Resin T 3U of patient – 27% Acidosis Compensated
Resin T 3U of serum pool – 30%
5. pH = 7.29
𝑇3 𝑈 𝑜𝑓𝑝𝑎𝑡𝑖𝑒𝑛𝑡 pCO2 = 41.2mmHg
o Formula:𝐹𝑇4 𝐼𝑛𝑑𝑒𝑥 = 𝑇𝑜𝑡𝑎𝑙 𝑇4 𝑥 ( )
𝑇3 𝑈 𝑜𝑓𝑐𝑜𝑛𝑡𝑟𝑜𝑙 HCO3 = 15mEq/L
▪ T3U = T3 uptake
▪ Control = a.k.a serum pool pH ❌ 7.35 7.45
▪ Thyroid hormone binding ratio (THBR) =
𝑇3 𝑈 𝑜𝑓𝑝𝑎𝑡𝑖𝑒𝑛𝑡 pCO2 45 ❌ 35
𝑇3 𝑈 𝑜𝑓𝑐𝑜𝑛𝑡𝑟𝑜𝑙 HCO3 ❌ 22 26
27
o 10𝑢𝑔/𝑑𝐿 𝑥 ( ) = 9 Uncompensated
30
Metabolic Acidosis

A. ARTERIAL BLOOD GAS 6. pH = 7.3

● Draw a tic tac toe like box with the reference ranges pCO2 = 30.7mmHg
o pH = 7.35 – 7.45
HCO3 = 13mEq/L
o pCO2 = 35 – 45
o HCO3 = 22 – 26
pH ❌ 7.35 7.45
● When given the pH, pCO2, and HCO3 fill in the corresponding
boxes depending on where they lie in the reference range pCO2 45 35 ❌
● Interpret the results HCO3 ❌ 22 26
o pH = 7.35 (acidosis) – 7.45 (alkalosis) Partially
o pCO2 = respiratory Metabolic Acidosis Compensated
o HCO3 = metabolic
● To determine if the ABG result is fully compensated, partially
compensated, or uncompensated: 7. pH = 7.38
o Fully compensated – pH is normal pCO2 = 60.8mmHg
o Partially compensated – all values are abnormal HCO3 = 29mEq/L Acidosis (closer to 7.35)
o Uncompensated – one value (either pCO2 or HCO3) is
normal pH 7.35 ❌ 7.45
pCO2 ❌ 45 35
HCO3 22 26 ❌
Fully
Respiratory Compensated

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10.1. Basic Principles and Practices, Laboratory Mathematics, Safety, and Method, and Analytic Techniques CLCHEM
IV. LABORATORY SAFETY
A. CHAIN OF INFECTION
● 6 components – pathogen, reservoir, portal of exit, mode of
transmission, portal of entry, susceptible host

B. HANDWASHING
● When done correctly, is the most effective way to prevent the
spread of communicable diseases in all settings (CDC)
● Happy birthday song is the handwashing song or twinkle twinkle
little star (WHO)

C. HEPA FILTERS
● High efficiency particulate air filters
● Used in biological safety cabinets (BSC)

D. WASTE SEGREGATION
● Black – dry and non-infectious wastes
● Green – wet and non-infectious wastes
● Yellow – wet and infectious wastes
● Red – sharps (puncture proof)
● Orange – radioactive wastes

E. MATERIAL SAFETY DATA SHEET

● Major source of safety information for employees who may use
hazardous materials in their occupations

F. NFPA HAZARDS IDENTIFICATION SYMBOL

● A.k.a safety diamond
● Color – blue (health hazard), red (fire hazard), yellow (instability
hazard), and white (specific hazard) quadrants Figure 8. Fire Safety
● Number (0 – 4) – indicates the magnitude of the hazard

Figure 8. NFPA Diamond

G. FIRE
● RACE – things to do when fire is discovered
● PASS – how to use the fire extinguisher
● Types of fire extinguisher – water, halogen, carbon dioxide,
and dry chemicals

Table 7. Classes of Fire

CLASS TYPES OF FIRE EXTINGUISHER
● Ordinary combustibles ● Water
● Cloth, paper, and wood ● Foam spray
A
● ABC powder
● Wet chemical
● Flammable liquids ● Foam spray
● Grease, oil, paint, and ● ABC powder
B
solvents ● BC powder
● Carbon dioxide
● Live electrical ● ABC powder
equipment ● BC powder
C
● Electrical panel, motor, ● Carbon dioxide
wiring
● Combustible metal ● ABC powder
D
● Magnesium, aluminum
● Commercial cooking ● Wet chemical
equipment
K
● Cooking oils, animal
fats, vegetable oils
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Proteins and Non-Protein Nitrogen Compounds CLCHEM

UERM CAHP 2023 First Semester LEC 10
Medical Technology Assessment Program 1 TOPIC 2
OUTLINE 3. OTHER FUNCTIONS
I. Proteins 1 ● Coagulation cascade – fibrinogen (factor I)
A. Functions of Proteins 1 ● Complement fixation – C1 – C9
B. Protein Metabolism 1 ● Serves as biocatalysts – enzymes
C. Composition of Proteins 1 ● Maintenance of acid-base balance – proteins act as buffers
D. Aminoacidopathies 2 ● Immunologic functions – antibodies and immunoglobulins
E. Analysis of Amino Acids 2 ● Important for tissue repair – collagen
F. Structures of Proteins 2 ● Produce motility though contractile elements – actin and
G. Types of Proteins 2 myosin for muscular movement
H. How to Differentiate Proteins 2 o Bacteria – contains the protein flagellin which enables the
I. Different of Proteins 3 flagella to move
J. Proteins in Other Specimens 3 ● Form many important intracellular and extracellular structures
K. Methods for Protein Analysis 3 ● Generate energy through catalysis and electron transfer
II. Non-Protein Nitrogen Compounds (NPNs) 3 ● Assemble molecules and serve as ion channels and pumps for
A. Urea 3 sodium and potassium to enter and exit
B. Uric Acid 3 ● Serve as the following for intracellular regulation
C. Creatinine 4 o Receptors – insulin receptor, ADH receptor, and TSH
D. Ammonia 4 receptors
o Hormones
I. PROTEINS o Cytokines – used for intracellular regulation so that WBC
● Contains C, H, O, and N can talk to NK cells for them to send out messenger
o Nitrogen is the element that distinguishes protein from proteins
other macromolecules ● Constitute signaling networks for intracellular regulation
● Amphoteric – can yield a positive and negative charge
o Donate hydrogen ions and accept hydrogen ions which B. PROTEIN METABOLISM
makes them good buffers to regulate the pH 1. Mainly happens in the stomach
● Mostly synthesized by the liver o The stomach contains HCl which will denature the protein
o Exceptions – antibodies or immunoglobulins which are making them susceptible to enzyme digestion
produced by the B-cells or plasma cells 2. Pepsin – an enzyme in the stomach responsible for digesting
o Liver disease – decrease in proteins proteins into amino acids
● Made up of amino acids which are the building blocks of 3. Amino acids are absorbed from the small intestine into the
proteins blood and then transported to the liver
o Amino acids are joined by peptide bonds o The liver will then turn the amino acids into new proteins
● Only macromolecule expressed in g/dL because of their
abundancy C. COMPOSITION OF PROTEINS
o Glucose, TAG, and cholesterol are all expressed in mg/dL ● Amino acids – building blocks of proteins
o Have an amino group (-NH2) and a carboxylic acid group
A. FUNCTIONS OF PROTEINS (-COOH)
1. REGULATE COLLOIDAL ONCOTIC PRESSURE o Deaminase – enzyme that removes the amino group
● Albumin (plasma) maintains colloidal oncotic pressure which terminal end
keeps the water within the circulation o Decarboxylase – enzyme that removes the carboxylic
● Blood becomes liquid because of its water content which allows group terminal end
circulation ● Peptide bonds – a.k.a amide linkages which hold amino acids
● Without albumin (deficiency) water goes out the blood vessel together
and accumulates in the connective tissue causing edema ● Essential amino acids – amino acids that are important for
protein synthesis
2. ACT AS CARRIER FOR TRANSPORT OF DIFFERENT o They are not produced in the body and therefore an
SUBSTANCES essential constituent of the diet
Table 1. Transport Carriers
CARRIERS DESCRIPTION Table 2. Essential and Non-Essential Amino Acids
HAPTOGLOBIN ● Transports hemoglobin ESSENTIAL NON-ESSENTIAL
HEMOPEXIN ● Transports heme Arginine Alanine
● Transports two Fe3+ (ferric) Lysine Glutamic Acid
TRANSFERRIN Tryptophan Serine
● Ferritin – storage form of Fe3+ (ferric)
● Transports copper Histidine Asparagine
CERULOPLASMIN ● Wilson’s disease – deficiency of Methionine Glutamine
ceruloplasmin not copper Valine Tyrosine
● A.k.a transthyretin Isoleucine Aspartic Acid
PRE-ALBUMIN ● Transports retinol (vitamin A) and Phenylalanine Glycine
thyroid hormones Leucine Cysteine
● Most versatile protein carriers Threonine Proline
because it binds to almost everything
ALBUMIN
(iron, magnesium, calcium, bilirubin,
and drugs)

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10.2. Proteins and Non-Protein Nitrogen Compounds CLCHEM

D. AMINOACIDOPATHIES G. TYPES OF PROTEINS
● Refers to a class of inherited errors of metabolism in which ● Simple proteins – made up of only amino acids
there is an enzyme defect that inhibits the body’s ability to ● Conjugated proteins – amino acids + prosthetic groups
metabolize certain amino acids o Examples – lipids (lipoproteins), sugar (glycoprotein), and
hemoglobin (hemoproteins)
Table 3. Aminoacidopathies
DISORDERS DESCRIPTION H. HOW TO DIFFERENTIATE PROTEINS
● Absence of phenylalanine ● Differential solubility – based on pH, temperature, and ionic
hydroxylase strength
● Mousy or musty urine odor ● Molecular size
PHENYL-
● Part of the newborn screening act ● Molecular mass
KETONURIA
(RA 9928) ● Electrical charge – via electrophoresis (commonly used)
● Guthrie test – microbiological test ● Surface adsorption – same principle with chromatography
which uses Bacillus subtills
● Deficiency of enzyme branched 1. ELECTROPHORESIS
MAPLE SYRUP chain alpha-ketoacid ● Separates substances according to charge
URINE DISEASE decarboxylase o Kuryentehen mo si protein, tatakbo sila papunta sa
● Maple syrup urine odor support media
● Deficiency of homogentisate ● Support media – paper (barely used), gel (agarose, starch,
oxidase and polyacrylamide), and cellulose acetate
● Presence of alkapton or ● Positive electrode – anode
ALKAPTONURIA
homogentisic acid o Negative ions (anions) will move to the positive electrode
● Dark brown to black urine upon long (anode)
standing ● Negative electrode – cathode
● Deficiency of isovaleryl-CoA o Positive ions (cations) will move to the negative electrode
ISOVALERIC
dehydrogenase (cathode)
ACIDEMIA
● Sweaty feet odor ● A buffer is used because proteins are amphoteric
● Deficiency of cystathionine beta- o Proteins become positive if pH is acidic
HOMOCYSTINURIA
synthase o Proteins become negative if pH is alkaline
CYSTINURIA ● Inadequate reabsorption of cystine ● 8.6 – most common pH used in electrophoresis
● Excretion of tyrosine and tyrosine o Alkaline pH → proteins become negative (anions) → move
catabolites in urine caused by toward anode (positive pole)
deficiency of: ● Isoelectric point (pI) – pH at which substances will have net
o Type 1 – fumarylacetoacetate zero charge
TYROSINEMIA hydrolase ● Zones or regions are stained using Coomassie, brilliant blue,
o Type 2 – tyrosine amido black, and Ponceau S to visualize the bands
aminotransferase ● Bands are quantitated using densitometer
o Type 3 – 4-hydroxyphenyl ● Origin to anode (alkaline pH) – gamma → beta → alpha-2 →
pyruvate dioxygenase alpha-1 → albumin (fastest)
● Deficiency of argininosuccinic
acid synthetase (type 1) or a. ELECTROPHORETIC PATTERNS
CITRULLINEMIA
mutation of gene that makes the Table 4. Electrophoretic Patterns
protein citrin (type 2) CARRIERS DESCRIPTION
ARGININO- ● Deficiency of argininosuccinic ● Cirrhosis – tend to exhibit
SUCCINIC acid lyase BETA-GAMMA
elevated IgA which are a
ACIDURIA BRIDGING
member of the gamma region
● Monoclonal gammopathy
MONOCLONAL SHARP
E. ANALYSIS OF AMINO ACIDS ● Multiple myeloma – cancer of
SPIKE IN GAMMA
● Heparinized blood samples collected after at least 6 – 8 hours plasma cells which secretes a
REGION
of fasting massive amount of IgG
● Separate plasma promptly, do not aspirate the platelet and ● Acute inflammation
white cell layer ● Most acute phase reactants are
● Do not accept hemolyzed sample found in the alpha-1 and alpha-
INCREASED ALPHA-1
● Deproteinization should be performed within 30 minutes of 2
AND ALPHA-2
collection ● Acute phase reactants are
● Analysis should be performed immediately proteins that increase in acute
● If not analyzed immediately, sample must be frozen at -20°C – inflammation
-40°C DECREASED ALBUMIN ● Nephrotic syndrome
● Random urine specimen for screening urinary amino acids AND INCREASED
● 24-hour urine sample preserved with thymol or organic solvent ALPHA-2
for quantification of urinary amino acids DECREASED ALPHA-1 ● Emphysema
● Different specimen
F. STRUCTURES OF PROTEINS ● Use of plasma instead of serum
● Primary – refers to the sequence of amino acids INCREASED BETA ● Protein fibrinogen is only
● Secondary – folding or planar shape which include alpha- present in plasma and not in
helix, beta-sheet, beta-turn, or random structure of proteins serum
● Tertiary – folding of proteins into a three dimensional shape
● Quaternary – incorporation of two or more polypeptide chains
or subunits into a larger unit

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10.2. Proteins and Non-Protein Nitrogen Compounds CLCHEM

I. DIFFERENT OF PROTEINS II. NON-PROTEIN NITROGEN COMPOUNDS (NPNs)
Table 5. Different of Proteins ● Not considered as proteins but contains nitrogen
PROTEINS ● Urea, amino acids, uric acid, creatinine, creatine, and ammonia
Pre-albumin Albumin Alpha-1 Antitrypsin o Most predominant is urea while the least predominant is
Alpha-1 Acid Alpha-1 Alpha-2 ammonia
Glycoprotein Fetoprotein Macroglobulin ● Most NPNs are waste products which can be used as part of
Alpha-Lipoprotein Haptoglobin Ceruloplasmin kidney function test, but some can be used for liver function
Pre-beta Beta-2 Beta-Lipoprotein assessment (particularly ammonia)
Lipoprotein Microglobulin o The job of the kidney is to throw away these waste
Complement Transferrin Fibrinogen products
Hemopexin C-Reactive Protein Immunoglobulins
SPECIAL TYPE PROTEINS A. UREA
HS-CRP Adiponectin Beta-Trace Protein ● End product of protein metabolism
B Type Natriuretic Cross-Linked C- Bence Jones ● Major NPN of the body
Peptide Telopeptidase Protein ● Produced by the liver and excreted by the kidneys but is
Cystatin C Fibronectin Troponin partially reabsorbed
Myoglobin ● Urea concentration vs BUN
o Common denominator is urea which is made up of carbon
J. PROTEINS IN OTHER SPECIMEN and oxygen in the middle with NH 2 on both sides
● CSF protein is less than 1% that of plasma protein ▪ Urea molecule = 𝑁𝐻2 − 𝐶𝑂 − 𝑁𝐻2
o CSF protein is expressed in mg/dL o Urea concentration – concentration of the whole urea
o Plasma protein is expressed in g/dL molecule
● Bence Jones proteins – monoclonal light chains excreted in o Blood urea nitrogen (BUN) – nitrogen content of urea
the urine of patients with multiple myeloma (only the N)
o Monoclonal light chains – only one kind of light chains
▪ Immunosero – two types of light chains (κ and λ) 𝐵𝑈𝑁 𝑡𝑜 𝑈𝑟𝑒𝑎 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝐵𝑈𝑁 𝑥 2.14
o Characteristically precipitates at 40°C – 60°C but 1
𝑈𝑟𝑒𝑎 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑜 𝐵𝑈𝑁 = 𝑈𝑟𝑒𝑎 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥
dissolves (solubilize) at 100°C 2.14
o Immunofixation – laboratory test used to identify (if κ and
λ) Bence Jones protein 1. METHODS FOR UREA
● Direct methods – diacetyl monoxime and o-phthaldehyde
K. METHODS FOR PROTEIN ANALYSIS ● Indirect methods:
1. KJELDAHL METHOD o Step 1 – urease
● Reference method o Step 2 – quantitation of ammonium ions using
● Measures the nitrogen content of proteins Nesslerization reaction, Berthelot reaction, coupled
● Assumes average nitrogen content of 16% enzymatic reaction, conductimetric method, and indicator
● Consists of acid digestion to release ammonium ions from dye method
nitrogen containing compounds
Table 6. Disorders Associated to Urea
● The ammonium can then be quantitated by Nesslerization or
DISORDERS DESCRIPTION
back titration
● Decreased protein intake
DECREASED ● Liver disease
2. BIURET REACTION
BUN ● Vomiting and diarrhea
● Barely used
● Pregnancy
● Quantifies protein by the number of its peptide bonds
● Pre-renal azotemia – reduced blood flow
● Positive result – violet color measured at 540nm
to the kidneys, diet, and increased protein
● Reagents (RANK) – Rochelle salt, alkaline copper sulfate,
catabolism
NaOH, and KI (potassium iodide) INCREASED
● Renal azotemia – decreased kidney
BUN
function
3. DYE-BINDING TECHNIQUES ● Post-renal azotemia – urinary tract
● Bromocresol purple – most sensitive and specific technique obstruction
● Bromocresol green – most commonly used
● Tetrabromophenol blue – protein pad of urine reagent strip
B. URIC ACID
● Methyl orange
● End product of purine metabolism in humans
● Hydroxyazobenzene benzoic acid (HABA)
● Strong reducing agent
● Ninhydrin – used for amino acids
● Produced in the liver and excreted through the kidneys
● Filtered but is reabsorbed in the kidneys
4. ACID PRECIPITATION TESTS
● Called acid precipitation test because the reagent used is acid
1. METHODS FOR URIC ACID
and the positive result is precipitation or turbidity
● Chemical method – Caraway (old method) or Henry method
● 3% sulfosalicylic acid (SSA) – confirmatory which detects all
● Enzymatic method
proteins (albumin, globulin, and Bence-Jones protein)
o Step 1 – uricase
● Trichloroacetic acid
o Step 2 – UV method, colorimetric method, and coupled
enzymatic reaction

Table 7. Disorders Associated to Uric Acid

DISORDERS DESCRIPTION
DECREASED ● Liver disease
BLOOD URIC ● Defective reabsorption of uric acid by
ACID kidneys
● Increased production
● Enzyme defect
ICNREASED
● Uric acid excretion competition
BLOOD URIC
● Decreased uric acid excretion
ACID
● Diet rich in purine (innards and beans)
● Gout
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10.2. Proteins and Non-Protein Nitrogen Compounds CLCHEM

C. CREATININE
● End product of muscle metabolism
● Produced when creatine loses water or creatinine phosphate
loses phosphoric acid
● Levels are a function of muscle mass and remain
approximately the same in an individual from day-to-day unless
muscle mass or renal function changes
● Filtered but is not reabsorbed by the renal tubules in significant
amounts

1. METHODS FOR CREATININE

● Chemical methods – Jaffe reaction
o Reagent – alkaline picrate solution
o Positive – red orange color
o Endpoint (simplest type) and kinetic (preferred type)
● Enzymatic methods:
o Creatinine aminohydrolase – creatine kinase method
o Creatininase – hydrogen peroxide method

Table 8. Disorders Associated to Creatinine

DISORDERS DESCRIPTION
● Relative to muscle mass
INCREASED
● Renal function
CREATININE
● Rate of creatine turnover

D. AMMONIA
● Immediate product of amino acid deamination
o Deamination – refers to the removal of the amino terminal
end of amino acids
● Primary site of ammonia production is the small intestine
● Ammonia in the blood is derived from the breakdown of amino
acids obtained from dietary protein or hydrolysis of urea
● Toxic to the body when it accumulates inside the body which is
why the liver and kidneys will have to detoxify and excrete them

1. METHODS FOR AMMONIA

● Conway microdiffusion
● Ion exchange
● Coupled enzymatic reaction
● Ion selective electrode

Table 9. Disorders Associated to Ammonia

DISORDERS DESCRIPTION
● Impaired liver function
INCREASED
● Reye’s syndrome
AMMONIA
● Hepatic coma (hepatic encepaholopathy)

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Carbohydrates and Lipids CLCHEM

UERM CAHP 2023 First Semester LEC 10
Medical Technology Assessment Program 1 TOPIC 3
OUTLINE o If the body receives lactose after a long time, the body will not
I. Carbohydrates 1 be able to split and digest lactose which results in the
A. Carbohydrate Metabolism 1 accumulation in the intestine
o The intestine contains normal flora, particularly Escherichia coli,
B. Specimen Considerations 2
Klebsiella, and Enterobacter (EKE) which are known as rapid
C. Methods for Carbohydrate Analysis 2 lactose fermenters
D. Laboratory Tests for Carbohydrates 3 ▪ TSI – A/A (acid slant / acid butt)
E. Clinical Significance 3 o EKE ferments the lactose accumulated in the intestine which
II. Lipids 4 produces acid and gas which causes gastrointestinal discomfort
A. Triglycerides (TAG) 4 o After regular drinking of milk, the body will remember to produce
B. Cholesterol 4 lactase
▪ Calcium (milk) promotes nerve transmission
C. Phospholipids 5
D. Fatty Acids 5
E. Methods for Lipoproteins 5 A. CARBOHYDRATE METABOLISM
F. Specimen Considerations 5 1. Begins in the mouth via salivary amylase (a.k.a ptyalin)
G. Clinical Significance 5 o Chewing food well helps the stomach digest the food very
well which reduces belly fat
I. CARBOHYDRATES o Amylase is produced by the pancreas
o Trivia – according to studies, rice should be chewed
● Hydrates of carbon – carbon with water in them
approximately 30 times and meat should be chewed 40 –
● Water soluble
60 times
o Example – sugar (sucrose) dissolves in water
o Which goes down to the esophagus to the stomach
● Most are reducing sugars (capable of reduction)
2. Temporarily stopped at the stomach due to acidity
o Exception – sucrose (non-reducing sugar)
o No carbohydrate digestion because of the hydrochloric
● Used as source of energy
acid which has the pH of 1.0 – 3.0
o Example – glucose is the food of cells and is converted
o The acidity of the HCl will inhibit or inactivate the salivary
into ADP or ATP for biochemical processes
amylase
● A big family of different variety of sugars
o Most enzymes are sensitive to pH (denatured)
Table 1. Forms of Carbohydrates
o Responsible for more protein digestion (enzyme pepsin is
MONOSACCHARIDES not denatured in the stomach) than carbohydrate digestion
3. Resumes in the small intestines via pancreatic amylase (a.k.a
● A.k.a dextrose
amylopsin)
● Used by the cells as a source of energy
o The intestine is connected with the bile duct which is
GLUCOSE ● Readily converted by the body into
connected to the pancreas
energy
o Hydrolyzes polysaccharides into monosaccharides
o IV fluids usually contain glucose
(particularly in the form of glucose)
● A.k.a levulose or fruit sugar
o Small intestine – known as the organ of absorption
● Mainly found in fruits (mainly citrus)
4. Absorbed from the intestines into the blood leading to transient
● Detectable in semen or seminal fluid
hyperglycemia
FRUCTOSE produced by the seminal vesicles
o Monosaccharides are absorbed into the small intestine
o Serve as food for sperm cells
which is delivered to the blood and circulation
o Pineapple can increase the amount
o Transient hyperglycemia – temporary increase in blood
of fructose in seminal fluid
glucose levels after eating
GALACTOSE
▪ Often takes 30 minutes
DISACCHARIDES
▪ Indicates and ensures that the food was absorbed
● Glucose + galactose 5. Blood glucose returns back to normal via insulin
● A.k.a milk sugar o Pancreas (beta-cells of the islet of Langerhans) produces
LACTOSE o Primary sugar found in milk products the hormone insulin (hypoglycemic agent)
● Can cause gastrointestinal discomfort ▪ Insulin is not immediately produced
when accumulated in the intestine
▪ Initially produces proinsulin (inactive form)
● Glucose + fructose ▪ C-peptide fragment is removed in order to activate
● A.k.a table sugar proinsulin into insulin (active form)
SUCROSE ● Non-reducing sugar o Plan A – increases cellular uptake of glucose
● Not detected by the copper reduction ▪ Insulin pushes the glucose from the plasma into the
methods cell to be utilized as a source of energy
● Glucose + glucose ▪ Insulin serves as the key while the insulin receptor
MALTOSE
● A.k.a malt sugar (receiver) serves as the door
POLYSACCHARIDES ▪ Problem – increased glucose in the plasma
● Polysaccharide found in the cell wall of (hyperglycemia) because it cannot enter the cell
CHITIN
fungi either there is no insulin (key) or there is a problem in
● Storage form of glucose in the body the insulin receptor (door)
GLYCOGEN
● Stored or found in the liver and muscles → Type I diabetes mellitus – no insulin
● Main polysaccharide found in staple → Type II diabetes mellitus – defect in receptor
STARCH
foods (bread, rice, etc.) o Plan B – promote glycogenesis (glycogen synthesis)
▪ Excess glucose (monosaccharide) is combined to
NEED-TO-KNOW create glycogen (polysaccharide) which is stored in
● Glucose, fructose, and galactose are collectively referred to as the liver and muscles
hexoses (contains 6 carbon atoms)
o Reagent hexokinase – kinase (transfers PO4) that targets the
o Plan C – promotes lipogenesis (lipid or TAG synthesis)
hexoses ▪ Lipids are stored in the body in the form of adipose
● Lactose – can cause gastrointestinal discomfort when accumulated tissues which will eventually lead to weight gain
in the intestine ▪ Lipids are heat-stable and easily formed
o Under normal circumstances, the body can digest lactose by the
enzyme lactase which hydrolyzes glucose and galactose
o When the body does not receive lactose (not a frequent milk
drinker), the body temporarily stops the production of lactase

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10.3. Carbohydrates and Lipids CLCHEM

NEED-TO-KNOW C. METHODS FOR CARBOHYDRATE ANALYSIS
● It usually takes 2 hours until blood glucose returns to normal 1. CHEMICAL METHODS
o Principle behind the test 2-hour post-prandial blood glucose and
oral glucose tolerance test (OGTT) ● Old or traditional methods
o OGTT – collect FBS, give oral glucose load, and measure within
60 – 90 minutes with the end time being 120 minutes (2 hours) a. COPPER REDUCTION
Insulin ● Cupric form (Cu2+) – glucose can reduce the charge or oxidize
Receptor the cupric form of copper into the cuprous form (Cu1+)
Insulin ● Increased glucose = increased cuprous
o Glucose constantly converts cupric to cuprous
● Not specific for glucose because other reducing sugars can
also convert cupric
● Does not detect sucrose because it is a non-reducing sugar
↑ Glucose Glucose → Energy ● Not usually used for blood anymore (not specific), mostly used
for urine and other body fluids
● Cuprous form (Cu1+) is measured
Plasma ● Measures the appearance of the color
Figure 1. Insulin Plan A 𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑆𝑢𝑔𝑎𝑟
Step 1 𝐶𝑢 2+ (𝐶𝑢𝑝𝑟𝑖𝑐) − − − −−→ 𝐶𝑢1+ (𝐶𝑢𝑝𝑟𝑜𝑢𝑠)
B. SPECIMEN CONSIDERATIONS 𝐺𝑙𝑢𝑐𝑜𝑠𝑒
● Glucose can be detected in whole blood, serum, plasma, urine,
CSF, serous fluid, synovial fluid, etc. Step 2 Measure Cu1+ (cuprous form)
● Fasting venous plasma – standard clinical specimen
o Glucose analysis is usually collected and performed in the
morning Table 3. Copper Reduction Methods
o 8 – 10 hours of overnight fasting METHODS DESCRIPTION
▪ Over day fasting is theoretically not acceptable ● Reagent – arsenomolybdate
▪ NELSON
Reference ranges for glucose are standardized and ● Positive result – arsenomolybdenum
based with a morning specimen SOMOGYI
blue
● Reagent – phosphomolybdate
NICE-TO-KNOW FOLIN WU ● Positive result – phosphomolybdenum
● Glucose is affected by: blue
o Physical activities
o Mental activities – the brain consumes 25% of glucose intake ● Reagent – neocuproine or neocuprine
NEOCUPROINE
o Hormones (diurnal variation) – cortisol is the main ● Positive result – yellow to orange
glucocorticoid BENEDICT’S ● Mostly used for urine and other body
▪ Increases blood glucose levels, produced by the adrenal TEST AND fluids
cortex, and is a steroid or cholesterol based hormone CLINITEST ● Uses the principle of copper reduction
▪ Cortisol is elevated in the morning (6am – 8am) and
reduces in the afternoon (4pm – 8pm)
b. FERRIC REDUCTION OR HAFEDORN-JENSEN
Table 2. Specimen Considerations ● Other names – Hafedorn-Jensen
SPECIMEN DESCRIPTION ● Ferric form (Fe3+) – glucose can reduce the charge or oxidize
● Lower glucose level than arterial blood the ferric form of iron into the ferrous form (Fe2+)
● Venous blood usually contains waste ● Not specific for glucose because other reducing sugars can
VENOUS also convert ferric
products
BLOOD
● Arterial blood usually contains oxygen ● Does not detect sucrose because it is a non-reducing sugar
● Easier to collect ● Based on inverse colorimetry – measures the disappearance
● Gives 10% – 15% lower glucose levels than of color from yellow to colorless
serum or plasma
WHOLE
● Common mistakes – patients usually 𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑆𝑢𝑔𝑎𝑟
BLOOD
compare the glucometer (whole blood) results Step 1 𝐹𝑒 3+ (𝐹𝑒𝑟𝑟𝑖𝑐) − − − −−→ 𝐹𝑒 2+ (𝐹𝑒𝑟𝑟𝑜𝑢𝑠)
with laboratory (plasma) results 𝑌𝑒𝑙𝑙𝑜𝑤 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝐶𝑜𝑙𝑜𝑟𝑙𝑒𝑠𝑠
● May be used if separated from clot within 30
SERUM Step 2 Measure Fe2+ (ferrous form)
minutes – 60 minutes
● Use gray top tubes which contains sodium
fluoride (antiglycolytic agent)
o Prevents glycolysis (splitting of glucose) c. CONDENSATION METHOD
which will cause a false decreased result ● Dubowski – a method for glucose analysis
o NaF primarily targets and inhibits ● Ortho-toluidine method – uses the reagent ortho-toluidine
magnesium which subsequently inhibits (main) + acetic acid (HAC) + 100°C temperature (boiling)
PLASMA the enzyme enolase o Positive – bluish-green complex measured
o Antiglycolytic agent – 2mg/mL spectrophotometrically at 620nm – 630nm
o Anticoagulant – 6mg/mL – 10mg/mL ▪ Board exam question – choices were blue or green,
● At room temperature, glucose decreases at a choose green because “bluish” is an adjective that
rate of 7mg/dL per hour describes the green color
● At 4°C (refrigeration), glucose decreases at a
rate of 2mg/dL per hour
● 60 – 70% compared to plasma glucose
concentration
CSF
● Blood glucose is collected 1 – 2 hours
GLUCOSE
before the lumbar tap
o To allow equilibrium

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10.3. Carbohydrates and Lipids CLCHEM

2. ENZYMATIC METHODS NICE-TO-KNOW
● Uses enzymes which are known to be quick and able to ● Patient blank or sample blank is also used for xanthochromic (pink,
red, and orange) CSF samples to correct for the color contributed by
accelerate the reaction
the specimen
● Automated instruments utilize enzymatic methods most of the
time
D. LABORATORY TESTS FOR CARBOHYDRATES
● Common tests used to diagnose diabetes mellitus – FBS,
a. GLUCOSE OXIDASE METHOD OGTT, and HbA1c
● Specific for glucose only
● Oxidizes glucose
● Glucose in the presence of oxygen and glucose oxidase will
1. RANDOM BLOOD GLUCOSE (RBS)
convert glucose into gluconic acid ● Collected randomly or at any point or time of the day without
regard to fasting
● Oxygen is converted into hydrogen peroxide (H2O2)
● Polarographic method – measures oxygen depletion ● Nice to use for emergency cases to monitor insulin shock
o Increased glucose = oxygen depletion o Signs and symptoms of hypoglycemia is similar to
hyperglycemia which is a problem for emergency cases
o Oxygen is consumed by glucose and is converted into
hydrogen peroxide o Common protocol is to know why the patient fainted
o Rarely used because oxygen is a gas and is measured (patient is diabetic with high sugar or because of low sugar
due to insulin overdose)
using electrodes (high maintenance)
● Colorimetric – glucose oxidase (first enzyme) is coupled with
peroxidase (second enzyme) + chromogen (color reagent) 2. FASTING BLOOD GLUCOSE (FBS)
o Double sequential enzyme ● 8 – 10 hours of overnight fasting
o Peroxidase – targets the peroxide (H2O2) ● Used for short term monitoring of glucose control
o Peroxidase gets one oxygen from hydrogen peroxide
which becomes water (H2O) and is combined with the 3. GLYCOSYLATED HEMOGLOBIN (HbA1c)
chromogen ● Long term monitoring of glucose control over the past 2 – 3
o Chromogen transforms into oxidized chromogen months
o Positive – change in color
4. FRUCTOSAMINE
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑂𝑥𝑖𝑑𝑎𝑠𝑒 ● Also used for medium term of glucose control over the previous
Step 1
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 − −→ 𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝐴𝑐𝑖𝑑 + 𝐻2 𝑂2 2 – 3 weeks
● Rarely used
Step 2 Polarographic or colorimetric method
5. 2-HOUR POST PRANDIAL GLUCOSE
● First specimen – fasting
Colorimetric Method
● Second specimen – collected 2 hours after eating
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒 o Glucose levels should go back to normal after 2 hours
𝐻2 𝑂2 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → 𝐻2 𝑂 + 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛
𝐶ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝐶𝑜𝑙𝑜𝑟 6. ORAL GLUCOSE TOLERANCE TEST (OGTT)
● Can be used for pregnant women and diagnosing diabetes
NEED-TO-KNOW mellitus
● Glucose oxidase is not the reference method because: ● First specimen – fasting
o Too specific – only detects the beta-glucose
▪ Alpha-glucose comprises 35% of the total glucose
● Second specimen – collected 30 minutes after giving oral
▪ Beta-glucose comprises 65% of the total glucose glucose load
▪ Remedy – add mutarotase which will convert the alpha- o Average glucose load is usually 75g and should be
glucose into the beta-glucose drank within 5 minutes
o Affected by interferences – oxidizing agents and reducing o If the patient vomits the oral glucose load, the test is
agents discontinued
▪ Oxidizing agents (detergents) – promotes oxidation ● Third specimen (optional) – collected after 60 – 90 minutes
which will cause a false positive
▪ Reducing agents (ascorbic acid and uric acid) –
● Last specimen – collected after 120 minutes
promotes the process of reduction which will cancel out
the glucose oxidation reagent causing a false negative 7. ORAL GLUCOSE CHALLENGE TEST (OGCT)
● Used to screen patients with gestational diabetes
b. HEXOKINASE + GLUCOSE-6-PHOSPHATE o Rarely encountered because it only screens gestational
DEHYDROGENASE (G6PD) diabetes so physicians directly request for OGTT
● Reference method – not as affected by interferences ● Same with OGTT except the glucose load used is only 50g and
o Affected by hemolyzed specimens and icteric specimens is only measured after 1 hour
o Icteric specimens can be remedied by patient blank or ● First specimen – fasting
sample blank which will correct or adjust for color ● Second specimen – collected 30 minutes after giving oral
contributed by the patient’s specimen glucose load
● Hexokinase – transfers phosphate to hexoses o Glucose load given is 50g and should be drank within 5
(monosaccharides) minutes
o One phosphate molecule from ATP (three phosphates) is ● Last specimen – collected after 60 minutes
transferred to hexose becoming hexose-6-phosphate and
the ATP then becomes ADP (two phosphates) E. CLINICAL SIGNIFICANCE
▪ Hexose-6-phosphate pertains to the location of the ● Hypoglycemia – decreased blood glucose level
phosphate which is attached at the 6th carbon ● Hyperglycemia – increased blood glucose level
o Hexokinase alone does not target glucose only, it targets
other hexoses
● Glucose-6-dehydrogenase (G6PD) – only targets glucose-6-
phosphate

𝐻𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒
𝐻𝑒𝑥𝑜𝑠𝑒 + 𝐴𝑇𝑃 − −→ 𝐻𝑒𝑥𝑜𝑠𝑒 − 6 − 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐴𝐷𝑃

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10.3. Carbohydrates and Lipids CLCHEM

1. HYPERGLYCEMIA II. LIPIDS
● Increased blood glucose ● Contains C, H, and O
● Positive urine glucose – apparent when the glucose exceeds ● Water insoluble
the renal threshold value (160mg/dL – 180mg/dL) ● Transported by lipoproteins (chylomicrons, VLDL, LDL, and
o Renal threshold value varies depending on exposure HDL) so they can travel to the bloodstream
● Increased specific gravity – glucose adds to the specific
gravity A. TRIGLYCERIDES (TAG)
● Increased serum and urine osmolality ● TAG = triacylglycerol
o Osmolality – affected by sodium, glucose, and urea o Glycerol backbone + 3 fatty acids
(BUN) which are all directly proportional to osmolality ● Main storage form of lipids in humans
o Hyperglycemia leads to hyponatremia – if there is an ● Found in adipose tissues
increased in glucose, the osmolality increases, but the ● Used as an insulator and shock absorber
body tries to return osmolality to normal by lowering the
sodium content which will lower the osmolality 1. METHODS FOR TRYGLYCERIDES
▪ Sodium is the principal contributor to osmolality a. STANDING PLASMA TEST
which is more efficient compared to BUN
● Allow the plasma to stand overnight in the refrigerator
▪ BUN is a waste product of protein metabolism which ● Presence of chylomicrons – floating creamy layer in plasma
should be thrown out of the body instead of after overnight standing in refrigerator
accumulating it inside the body o Chylomicrons – transports exogenous TAG
● Presence of VLDL – turbid plasma in plasma after overnight
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝐵𝑈𝑁
𝑂𝑠𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 1.86𝑁𝑎 + + +9 standing in refrigerator
18 2.8 o VLDL – transports endogenous TAG
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝐵𝑈𝑁
𝑀𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝑂𝑠𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 2𝑁𝑎 + + b. CHEMICAL METHODS
20 3 ● Old or traditional method
● Positive ketones – products of increased fat metabolism ● Van Handel Zilversmit and Hantzsch
o Glucose accumulates in the plasma causing ● Goal is to quantify TAG
hyperglycemia
o Plan A of insulin is not applicable so the glucose cannot STEPS
enter the cell which will result to the cell not receiving 1. Extraction – separating TAG with protein carriers
glucose for energy causing the cells to starve and die (chylomicrons and VLDL)
o The cell finds another source of energy, which are the 2. Saponification (hydrolysis) – splits TAG into glycerol and
triglycerides fatty acids
o The body converts the TAG indirectly to energy through o Most methods target the measurement of glycerol
acetyl CoA o Measuring fatty acid will not be accurate because the free
o Increased TAG metabolism will happen to compensate for fatty acids present in the serum will falsely increase the
the energy needed result
o Some acetyl CoA are subsequently converted to ketones 3. Oxidation – oxidizes glycerol
o Reason why some diabetic patients experience sudden o Oxidized alcohols (-ol) transforms into aldehyde
weight loss o Glycerol transforms into formaldehyde (measurable and
● Acidic pH – connected with ketones because 98% of ketones visible with color reagents)
are acidic 4. Colorimetry – addition of color reagent
o Beta-hydroxybutyric acid – 78% o Positive result – change in color
o Acetoacetic acid or diacetic acid – 20%
o Acetone – 2% c. ENZYMATIC METHOD
o One of the complications of diabetes is diabetic ● All enzymatic methods start with lipase which splits TAG into
ketoacidosis glycerol and fatty acids
● Glycerol kinase – targets the glycerol
NICE-TO-KNOW
● Case 1 – the blood glucose of patient A is 210mg/dL, is this
considered as hyperglycemia? Will the urine glucose be positive?
DIFFERENT MANUFACTURERS
o Yes, because FBS > 126mg/dL is already provisional for ● Lipase + glycerol kinase + glycerophosphate dehydrogenase +
diabetes mellitus. A single FBS alone is not sufficient, it has to diaphorase
be 2 FBS or 1 FBS and 1 OGTT ● Lipase + glycerol kinase + glycerophosphate oxidase +
o Yes, because 210mg/dL already exceeds the renal threshold peroxidase
● Case 2 – the FBS of patient B is 140mg/dL, is this considered as
hyperglycemia? Will the urine glucose be positive?
● Lipase + glycerol kinase + pyruvate kinase + lactate
o Yes, because FBS > 126mg/dL is already provisional for dehydrogenase
diabetes mellitus
o No, because 140mg/dL does not exceed the renal threshold B. CHOLESTEROL
● Case 3 – patient C has an increased renal threshold value of ● Integral part of the hormones (steroid)
250mg/dL with blood glucose levels of 210mg/dL, will the urine
● Found in vitamins (particularly calciferol)
glucose be positive?
o No, because 210mg/dL does not exceed the patient’s renal
● Found in cell membranes of RBCs which provide tensile
threshold value strength
● Esterified cholesterol (cholesterol ester) – accounts to 70%
CASE 1 CASE 2 CASE 3 of the total cholesterol
210mg/dL 140mg/dL 210mg/dL ● Free cholesterol – accounts to 40% of total cholesterol
BLOOD Hyperglycemic Hyperglycemic Hyperglycemic
RTV = 160–180 RTV = 160–180 Increased RTV
KIDNEYS ↓ ≠ 250mg/dL
URINE
Positive Negative Negative
GLUCOSE
FA
Figure 2. Cholesterol Ester (Left) and Free Cholesterol (Right)

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1. METHODS FOR CHOLESTEROL F. SPECIMEN CONSIDERATIONS
a. CHEMICAL METHODS ● At least 12 hours of fasting
● Rarely encountered due to its tedious process ● Use dry equipment to avoid water contamination (water-
insoluble)
Table 4. Chemical Methods for Cholesterol
METHODS DESCRIPTION G. CLINICAL SIGNIFICANCE
ZLATKIS ● One step procedure ● Factors – age, BMI, waist and hip ratio, etc.
ZAK AND BOYLE (colorimetry only) ● Atherosclerosis
LIBERMAN BURCHARD ● Coronary heart disease
● Two step procedure
CARR AND DREKTER
(extraction and colorimetry)
● Three step procedure
ABELL (extraction, saponification,
and colorimetry)
● Four or complete step
SCHOENHEIMER OR procedure (extraction,
SPERRY saponification, purification,
and colorimetry)

b. ENZYMATIC METHOD
● Cholesterol esterase + cholesterol oxidase + peroxidase
● First enzyme used should always be cholesterol esterase
o Cholesterol esterase – converts cholesterol ester (70%)
to free cholesterol
● Measuring the cholesterol oxidase first will not be accurate
because it only detects the free cholesterol (only 30%)

NICE-TO-KNOW
● Usually when there is oxidase, it is then followed by peroxidase
o Glucose oxidase + peroxidase
o Glycerophosphate oxidase + peroxidase
o Cholesterol oxidase + peroxidase

C. PHOSPHOLIPIDS
● Looks similarly like TAG
o Glycerol backbone + 2 fatty acids + phosphate
● Found in the cell membrane (phospholipid bilayer) which is
responsible for permeability
● Found in amniotic fluid for assessing fetal lung maturity
o Act as surfactant (lubricant) for the lungs
o Allows the lungs to expand during inhalation and prevents
them from sticking to each other during exhalation
o Mature lungs = more phospholipids
o Lecithin-sphingomyelin ratio (L:S) – lecithin is a
phospholipid
o Important for premature birth to make sure that the baby’s
lungs are mature enough for breathing

D. FATTY ACIDS
● Building blocks of lipids

E. METHODS FOR LIPOPROTEINS

● Proteins carriers of lipids

Table 5. Methods for Lipoproteins

METHODS DESCRIPTION
● Separates lipoproteins based on
charge
ELECTRO-
● Origin to anode – Chylomicrons →
PHORESIS
beta-LPP → pre-beta LPP → alpha-
LPP (fastest)
● Separates lipoproteins based on
density
● Chylomicrons (top layer) → VLDL →
LDL → HDL (bottom layer)
o Chylomicrons are the largest
but lightest lipoproteins
ULTRA-
because they contain 80% –
CENTRIFUGATION
95% TAG by weight
o HDL are the smallest
(compact) but heaviest
lipoproteins because they
contain 45% – 55% protein by
weight

CLCHEM (10.3) Paculan, Coleen Danielle 5 of 5

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