CLINICAL CHEMISTRY 1

QUALITY CONTROL, LEVEY JENNINGS CHART, WESTGARD RULES

LECTURE | SIR EUGENE DAYAG| FIRST SEMESTER

QUALITY CONTROL

• QC, also called internal quality control or statistical

process control
• performs everyday
• a process to periodically examine a measurement
procedure to verify that it is performing according to
preestablished specifications
• The External Quality Assurance Services (EQAS) ▪ wider, shorter or increase in SD –
programs include specimen packs and documentation imprecision, random error
(reports); results are compared to other laboratories ▪ shift in mean to left/right – inaccuracy,
using the same methodologies, instruments, and systematic error
reagents ▪ Kurtosis – degree of flatness or sharpness
• part of the process management component of the on the peak of the set of values having
quality system that integrates good laboratory Gaussian distribution
practices to ensure correct patient results o in general, laboratories use the ±2SD criteria for
• Objectives: the limits of the acceptable range for a test
o to check the stability of the machine o when the QC measurement falls within that
o to check the quality of reagents range, there is 95.5% confidence that the
o to check technical errors measurement is correct
o only 4.5% of the time will a value fall outside
of that range due to change; more likely it will
be due to error
• Cumulative Sum (Cusum)
o calculates the difference between QC results
and target mean
o V – mask method: requires computerization
o identifies consistent bias problems
o this plot will give the earliest indication of
systematic errors (trend) and can be used with
13s rule
o very sensitive to small, persistent errors that
commonly occur in the modern, low – calibration
frequency analyzer
o results are out of control when the slope exceeds
45 degree or a decision (±2.7SD) is exceeded
• Youden Plot/Twin Plot
Quality Control Parameters o it is used to compare results obtained on high
and low control serum from different
• Accuracy – systematic error laboratories
• Precision – random error o display the results of the analyses by plotting the
• Diagnostic efficiency mean values for one specimen on ordinate (y –
• Practicability axis) and the other specimen on the abscissa (x –
• Reliability axis)
o points falling from a center but on the 45-degree
Quality Control Charts line suggest a proportional error
o points falling from a center but not on the 45-
• Gaussian Curve degree line suggest a constant error
o parametric • Shewhart Levey-Jennings Chart
o it occurs when data elements are centered o dot chart
around the mean with most elements close to the o most widely used system in the clinical
mean laboratory
o focuses on distributions of errors from the o allows the laboratorians to apply multiple rules
analytical method rather than the values from a without the aid of a computer
healthy patient population o a graphic representation of the acceptable limits
of variation in the results of an analytical method

[AUTHOR NAME] Trans by: Mel ♡

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 CLINICAL CHEMISTRY 1
QUALITY CONTROL, LEVEY JENNINGS CHART, WESTGARD RULES
LECTURE | SIR EUGENE DAYAG| FIRST SEMESTER

Practice

• shift – more than 6 consecutive values fall in one

side of the mean

Note: There is only one rule: All QC values should be

within ± 2SD to be considered accurate or precise

Westgard Rules

• it recognized that the use of simple upper and lower

control limits is not enough to identify analytical
problems
▪ causes of shift
• used the term control rule to indicate if the analytical
• improper calibration of the instrument
process is out of control
(main cause)
• error detection rates can increase w/o increase the
• change in reagent formulation/reagent
false rejection rate
lot
• major instrument maintenance
• failure in reagent dispense system
• trend – more than six consecutive values
steadily increase or decrease
▪ causes of trend
• deterioration of reagents (main cause)
• deterioration of light source/incubation
temp
• gradual accumulation of debris in the
sample/reagent tubing or on electrode
surfaces
• outlier – control values that are far from the main
set of values; caused by random or systematic
errors

[AUTHOR NAME] Trans by: Mel ♡

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 CLINICAL CHEMISTRY 1
QUALITY CONTROL, LEVEY JENNINGS CHART, WESTGARD RULES
LECTURE | SIR EUGENE DAYAG| FIRST SEMESTER

• 12s
o warning rule, can release result
o used as a rejection or warning when one control, • R4s rule
result exceeds the mean ± 2SD o if the difference between the 2 controls is equal
o for screening purpose to or greater than 4s
o random error o reject the run for probable random error

• 13s rule • 10x rule

o cannot release result o if 10 consecutive control values fall on 1 side of x
o if 1 control observation exceeds x ±3s (either + or -)
o reject run for probable random error o reject run for probable systematic error

• 22s rule • 6x rule

o systematic error o reject when 6 consecutive controls fall on one side
of the mean

• 41s rule
o if 4 consecutive clues exceed the same x+15 or • 8x
x-15 limit o reject when 8 consecutive controls fall on one side
o reject the run for probable systematic error of the mean

[AUTHOR NAME] Trans by: Mel ♡

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 CLINICAL CHEMISTRY 1
QUALITY CONTROL, LEVEY JENNINGS CHART, WESTGARD RULES
LECTURE | SIR EUGENE DAYAG| FIRST SEMESTER

• 9x
o reject when 9 consecutive controls fall on one side
of the mean

• 7T
o reject when 7 control measumenets trend in the
same direction
o get progressively higher or progressively lower

• 12x
o reject when 12 consecutive controls fall on one
side of the mean

Note: ALL RULES THAT ARE ODD NUMBER INCLUDING R

ARE ALL RANDOM ERRORS. ALL RULES THAT ARE EVEN
NUMBERS ARE SYSTEMATIC ERRORS

∑𝑥
1. compute for mean = 𝑥̅ =
𝑁−1
• 2of32s ∑ |𝑥−𝑥̅|2
o reject when 2 out of 3 control measurements 2. compute for SD = 𝑆𝐷 = √
𝑁−1
exceed the same mean +2s or mean -2s control 𝑠
3. compute for CV = 𝐶𝑉 = 𝑥100
limit 𝑥̅

12S RANDOM ERROR WARNING/REJECT

13S RANDOM ERROR REJECT

SYSTEMATIC
22S REJECT
ERROR
SYSTEMATIC
41S REJECT
ERROR
• 31s
o reject when 3 consecutive control measurements R4S RANDOM ERROR REJECT
exceed the same mean +1s or mean -1s control SYSTEMATIC
limit 10X REJECT
ERROR

[AUTHOR NAME] Trans by: Mel ♡

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

Carbohydrates

• Primary energy source

• Glycogen – storage form of glucose
• Contain C, H, and O
• Disease status involved Hyperglycemia (glucose in the
blood is increased) and Hypoglycemia (glucose is
decreased in blood)

General Description of Carbohydrates

• Carbohydrates may be classified based on following:

o Size of base carbon chain: triose (3 carbons),
o Stereochemistry of compound
tetrose (4), pentose (5), hexose (6)
▪ Different special arrangements around each
▪ Glyceraldehyde – contains 3 carbon atoms
asymmetric carbon, forming stereoisomers
▪ Ribose – pentose
▪ Two different series are possible
▪ Glucose, galactose, fructose – hexose
• D–configuration – all/most of the
o Location of CO function
hydroxyl group are on the right side
▪ Aldose: terminal carbonyl group (aldehyde
• L–glucose – hydroxyl group on the left
group)
o Important to be remembered
▪ Ketose: carbonyl group in middle, linked to 2
because some of the methodologies
other carbon atoms (ketone group)
and techniques that we will be using
in the determination and
identification of glucose, they can
only measure the D–glucose
o Number of sugar units

Classification Definition
Cannot be hydrolyzed to a
Monosaccharide or “simple simpler form
sugar” Ex. Glucose, Galactose,
Fructose
Formed by interaction of
two monosaccharides
• Maltose → 2 Glucose
▪ Carbohydrate Model
Disaccharides • Lactose → Glucose +
Galactose
• Fisher Projection – linear form
• Sucrose → Glucose +
• Haworth Projection – cyclic form Fructose
Linkage of many
monosaccharide units
Polysaccharides Greater than 10 sugar units
Ex. Starch, Glycogen
(polymers of CHO)

o Number of sugar units in chain

▪ Fructose – “Levulose” or “fruit sugar”

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

▪ Lactose – “Milk sugar” → found only in dairy ▪ Partially digested CHOs go from mouth to the
products stomach
▪ Sucrose – “Table sugar” → found in beets and ▪ No carbohydrate digestion occurs in stomach
sugar cane due to acidic pH
▪ Maltose – found in cereals, wheat, and malt ▪ Pancreatic digestion of CHO occurs in small
products intestine (duodenum and jejunum) in
▪ Glycogen – Storage form of glucose in the increased pH through pancreatic amylase
body → found in liver and skeletal muscles (amylopsin) followed by maltase, sucrase or
• Chemical Properties of Carbohydrates lactase (these enzymes hydrolyzes
o Reducing carbohydrates disaccharide to monosaccharides)
▪ To reduce, carbohydrate must have ketone ▪ Monosaccharides are absorbed by gut and
or aldehyde group → anomeric carbon or the transported to liver
carbon #1 ▪ Glucose is only carbohydrate directly used
▪ All monosaccharides & many disaccharides = for energy or stored as glycogen (because it
reducing agents is the simplest form); galactose & fructose
▪ Ex. glucose, maltose, fructose, lactose, must be converted to glucose
galactose ▪ Glucose is the only fuel in the pathway
▪ Benedict’s Test o Carbohydrate metabolism in the blood
o Nonreducing carbohydrates ▪ Goal of cell is to convert glucose to carbon
▪ Do not have ketone or aldehyde group & will dioxide and water for energy production
not reduce ▪ Storage as glycogen in the liver
▪ Sucrose ▪ Storage as triglycerides into adipose tissues
(abdominal area)
▪ Conversion to ketoacids, amino acids, or
protein
o Embden – Meyerhof Pathway
• Glucose/Dextrose ▪ Glycolytic Pathway
o Primary source of energy for humans; nervous ▪ Glucose is broken down into two- and three-
system totally depends on glucose from carbon molecules of pyruvic acid that can
extracellular fluid enter the tricarboxylic acid (TCA) cycle on
o The most important consumer: brain (50%) conversion to acetyl – coenzyme A (acetyl-
o It can be derived from: diet, body stores like CoA)
glycogen, endogenous synthesis from protein and ▪ Glucose → CO2 and H2O
triglyceride ▪ Pyruvate – product if aerobic
▪ Lactate – product if anaerobic
Glucose Metabolism
o Hexose monophosphate pathway (HMP)
• Fate of Glucose ▪ The oxidized product permits the formation
o Carbohydrate Digestion of ribose–5–phosphate and NADP in its
▪ Most ingested carbohydrates are polymers reduced form (NADPH)
(starch, glycogen) ▪ NADPH → important in neutralization of
▪ Glucose – hindi na kailangan idigest kasi peroxide to prevent the production of
simplest form na sya so magagamit siya ng methemoglobin and Heinz bodies
body agad agad ▪ HMP shunt also permits pentoses, such as
▪ CHO digestion starts in the mouth through ribose, to enter the glycolytic pathway
salivary amylase (ptyalin) but only partial ▪ Glycogen Synthase – important enzyme with
digestion converts Glucose–1–phosphate into
glycogen

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Regulation of Carbohydrate Metabolism o Produced from the alpha cells of the

▪ Processes Islets of Langerhans in the pancreas
▪ All connected using tricarboxylic acid o Primary hyperglycemic hormone
cycle/Krebs Cycle o Released during stress (fight or
▪ -lysis – break down flight response) and fasting states
▪ -genesis – beginning/formation of o Promotes glycogenolysis and
• Glycolysis: Glucose → Pyruvate → gluconeogenesis
Energy • Somatostatin
• Gluconeogenesis: Non–CHO sources (ex. o Produced by Delta cells &
triglycerides, proteins) → Glucose hypothalamus
(neophyte – bago) o ↑ plasma glucose
• Glycogenolysis: Glycogen → Glucose o Inhibits pituitary (growth hormone
• Glycogenesis: Glucose → Glycogen and thyrotropin) and pancreatic
• Lipogenesis: Glucose → Fatty acid (Beta– (insulin & glucagon) hormones
oxidation) • Epinephrine
• Lipolysis: Fat → Glucose o Produced by adrenal medulla
o Released during stress
o ↑ plasma glucose by inhibiting
insulin secretion
o ↑ glycogenolysis and lipolysis
• Glucocorticoid – Cortisol
o Released from the adrenal cortex on
stimulation by adrenocorticotropic
hormone
o Cortisol increases plasma glucose by
decreasing intestinal entry into the
cell
o ↑ gluconeogenesis, glycogenolysis,
▪ Hormones involve and lipolysis
• Insulin • Growth hormone
o The only and primary hypoglycemic o Increases plasma glucose by
agent/hormone decreasing the entry of glucose into
o Produced by beta cells of the Islets the cells
of Langerhans in the pancreas o Its release from the pituitary is
o Proinsulin is converted into insulin stimulated by decreased glucose
by the removal of C – Peptide levels and inhibited by increased
o Trigger/Stimulus: If patient is glucose
hyperglycemic, insulin is produced o ↓ glucose entry to cells, ↑
o It decreases plasma glucose levels glycolysis
by increasing the transport entry of o Peak during afternoon
glucose in muscle and adipose • Adrenocorticotropic hormone (ACTH)
tissue by way of nonspecific o ACTH is produced by the pituitary
receptors gland in response to low cortisol
o ↑ glycogenesis, glycolysis, levels
lipogenesis o Increases plasma glucose levels by
o ↓ glycogenolysis converting liver glycogen to glucose
• Glucagon and promoting gluconeogenesis

Trans by: Mel ♡

CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Thyroxine
o It increases plasma glucose levels
o ↑ glycogenolysis, gluconeogenesis,
and glucose intestinal absorption
o If too high glucose level in the body,
patients’ blood is viscous/thickened
• Incretins
o Incretin effect refers to the greater
and earlier insulin response to the
oral administration of glucose
compared with intravenous glucose
o The most important incretins GLP-1
and glucose-dependent
insulinotropic peptide (gastric
inhibitory polypeptide; GIP)
▪ GLP-1 rapidly stimulates insulin
secretion, suppresses glucagon
secretion, and slows gastric Anabolism, building up. Catabolism, breaking down.
emptying in response to a meal
▪ GLP-1 may also reduce appetite • Hyperglycemia
and promote weight loss o “Sugar rush”
▪ In vitro and animal studies o Increase in plasma glucose levels
indicate that GLP-1 can inhibit o In healthy patients, during a hyperglycemia state,
beta cell apoptosis, stimulate insulin is secreted
beta cell proliferation and o Hyperglycemia is caused by an imbalance of
neogenesis from precursor hormones
duct cells, and decreases alpha o Criteria for Testing for Prediabetes and Diabetes
cell mass ▪ All adults >45 years old should have fasting
▪ Plasma meal-stimulated GLP-1 blood glucose measured every 3 years,
levels are decreased in type 2 unless already diagnosed with diabetes
diabetes mellitus ▪ Testing should be earlier or more frequent
with these risk factors
• Overweight tendencies (BMI ≥25 kg/m2)
• Habitual physical inactivity
• Family history of diabetes in a first –
degree relative
• High – risk minority population (African
American, Latino)
• History of gestational diabetes or
delivering baby >9 lb
• Hypertension (≥140/90)
▪ Criteria for type 2 diabetes testing in
children, beginning at age 10 or at onset of
puberty & with follow – up testing every 2
years

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Family history (first- or second-degree) o Symptoms: increased hunger, sweating, nausea &
of type 2 diabetes vomiting, dizziness, nervousness & shaking,
• Race/ethnicity (African American, blurred speech & sight, mental confusion
Latino, Native American) o Relieved by giving carbohydrates or sugar load to
• Signs of insulin resistance patient
• Maternal history of diabetes or o Coke – reliever because it has high sugar content
gestational diabetes mellitus o Life threatening if not relieved
▪ Three methods of diagnosis (each must be o It results from an imbalance between glucose
confirmed by one of the others on a utilization and production
subsequent day) o Related to CNS → primary consumer of sugar is
• Diabetes symptoms + random glucose brain
level of ≥200 mg/dL o Diagnostic hypoglycemia value – ≤ 50 mg/dL (≤2.8
• A fasting plasma glucose of ≥126 mg/dL mmol/L)
• An oral glucose tolerance test (OGTT) w/ o Whipple’s triad – low blood glucose
2–hour post load (75g glucose level) concentration with typical symptoms alleviated
≥200 mg/dL by glucose administration
▪ Patients with following criteria have “pre – o Classification based on symptoms
diabetes” ▪ Neurogenic
• Fasting glucose of ≥100 mg/dL but <126 • Tremors, palpitations, anxiety, hunger
mg/dL • Triggered by Autonomic Nervous System
• OGTT 2 – hour level ≥140 mg/dL but ▪ Neuroglycopenic
<200 mg/dL • Dizziness, tingling, blurred vision,
▪ Criteria for testing and diagnosis of confusion, behavioral changes
Gestational Diabetes • Diminished glucose supply to the Central
• Age >25 years Nervous System
• Overweight Patients appear healthy
• Strong family history of diabetes • Insulinoma (Tumor in B–
• History of abnormal glucose metabolism cell, Islet hyperplasia
• History of poor obstetric outcome • Factitial hypoglycemia
No coexisting disease
• Presence of glycosuria (insulin/sulfonylurea)
• Diagnosis of polycystic ovarian • Severe exercise, Ketotic
syndrome hypoglycemia
• Ethnicity/race (African American, Latino, Compensated coexistent Drugs/disease
Native American) Patients appear ill
• Hypoglycemia Drugs, predisposing illness, hospitalized patient
o Life–threatening
o Involves decreased plasma glucose levels, and can
have many causes – some are transient (effect
can easily be relieved) and relatively insignificant, • Diabetes mellitus
but others can be life threatening if untreated o Group of diseases in which blood glucose levels
o Occurs in healthy – appearing and sick patients, are elevated
because of reaction to medication or of illness o Group of metabolic diseases characterized by
o Symptoms appear at glucose level of about 50–55 hyperglycemia resulting from defects in insulin
mg/dL secretion, insulin action, or both
o Critical value: <40 mg/dL o National Diabetes Data Group
▪ Type I
▪ Type II
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o ADA/World Health Organization (WHO) (HLA) DR3 and DR4 –

the major locus is
▪ Type I Diabetes the major
▪ Type II Diabetes histocompatibility
▪ Other specific types of diabetes (MHC) on
chromosome 6
▪ Gestational Diabetes Mellitus (GDM) – • Microalbuminuria of
pregnant women due to hormonal 50 – 200 mg/24 hrs.
imbalances (Diabetic
nephropathy)
o Risk Factors • Idiopathic Type 1
▪ Physically inactive DM
▪ Family history & race (unknown/uncertain
cause)
▪ History of Impaired Fasting Glucose/Impaired
• Abrupt onset • Inhibited fatty
Glucose Tolerance (IFG/IGT) • Insulin dependent acid oxidation
▪ Hypertension (≥140/90 mmHg • Ketosis tendency (Fatty acid +
▪ Cardiovascular diseases • Signs and symptoms triglyceride
o Polydipsia, forming VLDL)
▪ Low HDL (less than 35 mg/dL) Polyphagia, • Untreated
▪ Elevated triglycerides (greater than 250 Polyuria Type 2 DM will
o Rapid weight result to
mg/dL) nonketotic
loss
▪ History of GDM o Possible loss hyperosmolar
▪ Polycystic ovarian syndrome (PCOS) Characteristics of coma – (>1,000
consciousness mg/dL)
Diabetes Mellitus o Severe
Type 1 Type 2 dehydration
Classification
• Adult Type or >500 mg/dL
• Insulin – dependent Maturity Onset o Electrolyte
Diabetes Mellitus DM imbalance
(IDDM) • Stable o Increase BUN
• Juvenile Onset Diabetes, and creatinine
Diabetes Mellitus easily o Elevated
Formerly known as osmolality
• Brittle Diabetes – controlled
DT1 is hard to • Ketosis >320 mOsm/dL
control, labile Resistant o autoAb – cells would attack our own cells
• Ketosis Prone Diabetes o ↑ glucose concentration – ↑ solute
Diabetes • Receptor –
Deficient DM
o Glucose threshold – 160–180 mg/dL
• 5–10% of all cases of • 90% of cases o Diabetic Ketoacidosis – It occurs when the body
Diabetes starts breaking down fat at a rate that is much too
• Occurs in childhood fast. The liver processes the fat into a fuel called
Epidemiology and
and adolescence
Pathophysiology ketones, which causes the blood to become acidic
• Absence of insulin
with excess o Hyperventilation – mechanism to lower down
glucagon
hydrogen concentration or increase pH, increase
• β–cell destruction –
β–cell produces release CO2
insulin o Normal blood pH – 7.35 to 7.45
• Islet cell of insulin
autoAb
• Glutamic acid
decarboxylase
Pathogenesis autoAb
• Tyrosine
phosphatase IA-2 &
IA-2B autoAb
• Genetic association
between type 1
diabetes and Human
Leukocyte Antigen

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Complications of DM
▪ Nephropathy – early stage DM is
Microalbuminuria
▪ Retinopathy
▪ Neuropathy
▪ Increase risk of heart disease
o Laboratory Findings
▪ Decreased blood and urine pH (acidic)
▪ Ketones (urine and blood) – increased
hydrogen ions making the urine and blood
acidic
▪ Increased specific gravity and osmolality –
presence of glucose in urine, Hyperosmolar
Coma
▪ Arterial Blood Gas (ABG) imbalance
(decreased bicarbonate and total CO2)
• Kussmaul–Kien respiration (Increased
respiratory or hyperventilation to
normalize pH)
▪ Electrolyte imbalance
• Decreased Na – polyuria and shift of
water from cells
• Increased K – displacement from cells in
o Cleave/Tatanggalin si C peptide para marelease si acidosis
Insulin • High K in blood circulation and
intracellular fluid would cause edema in
patients

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• 1 – hour – ≥180 mg/dL

• 2 – hour – ≥155 mg/dL
• 3 – hour – ≥140 mg/dL
o GDM converts to DM within 10 years in 30–40%
of cases
o Infants increase risk of:
▪ Respiratory distress syndrome
▪ Hypocalcemia
▪ Hyperbilirubinemia
• Fasting Plasma Glucose – should be 8-12 hours, NPO
(None per Orem/Nothing by mouth)
• 2–hour Plasma Glucose Level (Oral Glucose Tolerance
Test) – take the fasting glucose blood, give 75g of
glucose, after 1 hour collect ulit, after 1 hour collect
ulit for the 2nd hour → 3x magcocollect, this is to check
is px can metabolize 75g of glucose within 2 hours

Other types of Diabetes

• Pancreatic disorders
• Endocrine disorders – Cushing’s syndrome (abnormal
Increased of cortisol which leads to Hyperglycemia),
Pheochromocytoma (Increased epinephrine in body,
Hyperglycemia), Acromegaly (Increased Growth
hormone), and hyperthyroidism (Increased thyroxine)
• Drugs or chemical inducers of
o β – cell dysfunction (Dilantin and Pentamidine)
o Impaired insulin action (Thiazides, Diabetes Insipidus
Glucocorticoids
• Genetic syndromes – Down syndrome, Klinefelter’s • Pathophysiology
syndrome, Rabson–Mendengall syndrome, o Deficiency of ADH released by Neurohypophysis
Leprechaunism ▪ Severe polyuria (frequent urine) with low
specific gravity (glycosuria)
Gestational Diabetes Mellitus o Polydipsia
• Pathogenesis Inborn Errors of Carbohydrate Metabolism
o Glucose intolerance during pregnancy
o Due to metabolic and hormonal changes • Galactosemia
o Screening should be performed between 24 to 28 o Congenital deficiency of 1 of the 3 enzymes
weeks of gestation (all pregnant women) involved in galactose metabolism
▪ 1–hour Glucose Challenge Test – 50g Glucose o Nasa womb palang
Load o 3 enzymes
o A plasma glucose concentration of 140 mg/dL or ▪ Galactose–1–phosphate uridylyl transferase
greater (most common deficiency)
o Confirmatory: 3–hour GTT with 100g glucose ▪ Galactokinase (GALK)
o Oral Glucose Tolerance Test (OGTT) Results ▪ Uridine diphosphate galactose–4–epimerase
▪ Sample: Blood and Urine (GALE)
• FBS – ≥ 95 mg/dL o Laboratory features: elevated blood, and urine
galactose
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Clinical features: jaundice, hepatomegaly, easy

bruisability, galactosuria, E. coli sepsis, cataract,
and sensory neural deafness
o Diagnostic test: Erythrocyte galactose–1–PO4
uridylyl transferase activity
• Essential fructosuria
o Autosomal recessive disorder characterized by
fructokinase deficiency
o Fructokinase catalyzes the conversion of fructose
to fructose–1–phosphate
o Diagnostic indicator: the presence of fructose in
urine
• Hereditary Fructose Intolerance
o A defect of fructose 1–6 biphosphate aldolase B
activity in the liver, kidney, and intestine
o Clinical features: irritability, lethargy, seizures,
and hepatomegaly
• Fructose–1,6–biphosphate deficiency
o Results in failure of hepatic glucose generation by
gluconeogenic precursors such lactate, and
glycerol
o Clinical features: hypoglycemia, lactic acidosis,
convulsions, and coma

Glycogen Storage Disease

• Deficiency of enzyme for glycogen metabolism

• Inherited autosomal recessive trait
• Serial blood specimens are collected for 2 hours at 15
minutes intervals for testing
• Intravenous Glucose Tolerance Test (IVGTT) – test for
Type 1 GSD (decreased glucose levels)
• Glucose–6–phosphatase removes phosphate to
become glucose

Laboratory Diagnosis

• Specimen Consideration
o Specimen: Whole blood, serum, plasma, CSF,
serous fluid, urine
o Standard Clinical Specimen: Venous Plasma
Glucose
o Fasting Blood Sugar sample should be obtained
after 8 to 10/12 hours of fasting
o Venous blood is lower than arterial due to
delivery to cells
o Capillary blood glucose is higher than venous
blood (5 – 7%)

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Whole blood is 10 to 15% lower than serum or • Positive result: Arsenomolybdenum blue
plasma measured spectrophotometrically at
o Serum glucose is 5% higher than plasma 520 nm
o Serum should be separated from cells within 30 c) Neocuproine
to 60 minutes • Reagent: Neocuproine
o Glucose is metabolized at room temperature at a • Positive result: Orange–red or yellow to
rate of 7 mg/dL/hour – bumababa, undergoes orange
glycolysis ▪ Alkaline Ferric Reduction Methods
o Glucose is metabolized at 4C at a rate of 2 • AKA Hagedorn Jensen Method
mg/dL/hour • Negative/Inverse colorimetry
o Gray top tube is preferred because it has an • Used in Auto Analyzer (Technicon)
antiglycolytic agent Sodium fluoride meaning it • Reagent: Hot alkaline solution of K
preserves glucose in the sample (used if there are Ferricyanide
delay in testing in consideration of glycolysis rate • Principle: Reducing sugars reduce
or decline in glucose concentration) ferricyanide to ferrocyanide
▪ 2 mg of Na fluoride/mL of whole blood • Positive result: disappearance of yellow
prevents glycolysis for 48 to 72 hours color
▪ Fluoride binds with magnesium which causes
• Nonspecific for glucose
inhibition of enzyme enolase (mechanism of
• 320–340 nm
action of Sodium fluoride)
o Condensation Methods
o CSF glucose is 60 to 70% that of plasma
▪ O – toluidine or Dubowski Method
▪ Blood glucose should be collected 1 to 2
• Reagent: O–toluidine + GAC at 100C
hours before the spinal tap
• Positive result: Bluish green or green
▪ If CSF glucose analysis is delayed, although
measured at 620 to 630 nm
must be analyzed immediately, it must be
• Enzymatic Methods
refrigerated at 4C or frozen at -20C
o Glucose Oxidase
o Plasma glucose increases with age
▪ Measures B–D–glucose
▪ FBS – 2 mg/dL/decade
• Mutarotase: concerts a–D–glucose to B–
▪ Post prandial glucose – 4 mg/dL/decade
D–glucose
▪ GC – 8–13 mg/dL/decade
▪ Step 1
Methods used to measure Glucose ▪ Step 2
• Polarographic Method
• Chemical Method
o Measures O2 depletion through
o Oxidation – Reduction Methods
electrodes
▪ Alkaline Copper Reduction Methods
o 1 molecule Glucose = 1 O2 molecule
• Principle: Reduction of cupric ions consumed
forming cuprous oxide in hot alkaline o ↓ O2 rate = ↑ Concentration of
solution of glucose Glucose
a) Folin Wu o Sources of error: H2O2 left alone will
• Reagent: Phosphomolybdate cause erroneous results
• Positive result: Phosmolybdenum blue ▪ Remedy: Add Ethanol/Iodide
measured spectrophotometrically at • Colorimetric Method (Saifer
520 nm Gernstenfield)
b) Nelson Somogyi o Reagent: Glucose oxidase +
• Reagent: Arsenomolybdate peroxidase + chromogen

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Chromogens [gives color]: O – ▪ GD is from Bacillus cereus

dianisidine (positive: blue), • Highly specific for glucose
Aminoantipyine (positive: rose – ▪ Sources of error: Maltose, Icodextrin,
pink) Galactose (these cause falsely increased
▪ Glucose oxidase is specific to B–D–glucose result)
• 2 forms: 35–36% a–D–glucose & 64– • Isotope Dilution mass Spectrophotometry or Gas
65% B Chromatography
▪ Source of error o Most accurate method for glucose measurement
• False positive: Oxidizing agents (ex. (Henry, 23rd edition)
bleach) • Selivanoff’s or Seliwanoff’s test for Fructose
• False Negative: Reducing agents (ex. o Reagent: Hot HCl + resorcinol
ascorbic acid, uric acid, bilirubin [enteric o Positive result: Red within 30 seconds
sample], hemoglobin) • Bial’s Test for Pentose
o Addition of K Ferrocyanide will decrease o Reagent: HCl, Orcinol, and FeCl3
interference with bilirubin o Positive result: Green
o Hexokinase Method • Laboratory Tests
▪ Reference Method o Random Blood Glucose (RBS)
▪ Step 1 ▪ Taken anytime of the day without fasting
▪ Step 2 ▪ Used for emergency cases (ex. hyperglycemic
▪ Absorbance is measured at 30 nm ketonic coma, insulin shock – nasobrahan sa
▪ NAD is required if G6PD is obtained from insulin intake, bagsak ang glucose
Leuconostoc mesenteroides concentration)
▪ NADP is required if G6PD is obtained from o Fasting Blood Sugar (FBS)
yeast ▪ Taken after at least 8 to 10/12 hours of
▪ Product: 6–phosphogluconate or 6– overnight fasting
phosphogluconolactone (anaerobic) ▪ Usually done during the morning to prevent
▪ G6PD makes the reaction specific for glucose the effects of diurnal variation (Cortisol)
in hexokinase method ▪ Categories:
▪ Modifications • Normal FBS – <100 mg/dL
• NADPH or NADH from 2nd step can be • Impaired FBS/Prediabetes – 100 to 125
reacted with chromogen such as mg/dL
Phenazine methosulfate with • Provisional DM diagnosis – ≥126 mg/dL
nitrophenyl tetrazolium derivative. o 2–hours Post–Prandial Glucose
Absorbance is measured at 520 nm ▪ Post–prandial – after meals
(kinetic analysis) ▪ FBS initially, the patient is given CHO load
▪ Sources of Error: Hemolyzed and icteric (usually 75g), and plasma glucose is
samples can cause falsely decreased results measured after 2 hours
o Glucose Dehydrogenase Method ▪ Categories:
▪ Variations • Normal – <140 mg/dL
• Spectrophotometric • Impaired glucose tolerance/Prediabetes
o Reagents: Mutarotase, GD, – 140–199 mg/dL
Diaphorase • Provisional DM diagnosis – ≥200 mg/dL
o Product: MTTH (Blue) + NAD o Glucose Challenge Test/Glucose Loading Test – no
• Through Electric Current fasting, administer 50g CHO, wait for 1 hr. then
o Reagents: Pyrroloquinoline extract blood
quinone, GD, Ferricyanide o 2 PPBS – after collecting FBS sample, instruct the
o Product: Ferricyanide + 2e patient to eat, then after 2 hrs. extract again
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Oral Glucose Tolerance Test (OGTT) ▪ Patient should drink the

▪ Series of glucose testing after 8–10 hours of glucose load within 5 minutes
testing ▪ If the patient vomits,
▪ Specimen: blood & urine discontinue the test
▪ Baseline sample: fasting (get sample with 1– • Exton Rose Method (Double Dose)
hour interval) o Uses 100g of dextrose powder and
▪ Patient cannot eat because it will alter result 650mL of water
▪ Categories: o First half is given after the FBS is
• Normal/Non–DM – <140 mg/dL taken
• Impaired GTT – 140 – 199 mg/dL o 2nd dose (the other half) is given
• DM – ≥200mg/dL after the collection of 30 min.
▪ Types: sample
• Janney – Isaacson Method (Single dose) o Intravenous GTT
o Most common ▪ Used for patients with GI tract disorders and
o Glucose load is given and series of for patients who cannot tolerate large CHO
blood sample for glucose assays are load
then collected 30 minutes, 1 hour, ▪ 0.5g of glucose/kg body weight (given within
and 2 hours after CHO load intake 3 minutes) given intravenously
o Guidelines for OGTT (Single Dose) ▪ Fasting sample is required
▪ Patient should go with normal ▪ First blood collection is after 5 minutes of
to high CHO diet (150g of administration
CHO/day) for 3 days prior the o HBA1c/Glycosylated Hemoglobin
test ▪ For long term glucose control for the
▪ If the patient is taking previous 2 to 3 months (18 to 20 weeks in
medications that affect glucose other books)
tolerance, it must be ▪ In every 1% increase in HBA1c = increase of
discontinued 35 mg/dL PG
▪ Avoid physical (excessive) ▪ Patient with shorter RBC lifespan = lower
activity; 8 to 10 hours of HBA1c (not suitable)
overnight fasting, but not ▪ Specimen: EDTA (sample: whole blood)
greater than 16 hours ▪ Methods: Affinity chromatography (based on
▪ Should be performed in the structural differences), Ion exchange (based
morning due to prevention of on electrical differences/charges),
the normal diurnal effect on Electrophoresis (interfere Hemoglobin F if
glucose >7%), Isoelectric Focusing or IEF (can
▪ Blood glucose is taken every 30 interfere A1c), and High Performance Liquid
minutes for 2 hours Chromatography or HPLC (separates
▪ Patient should be ambulatory, glycosylated hemoglobin)
may drink water, no smoking ▪ Ideal value: <7%
▪ FBS is measured before giving ▪ Estimated correlation between Mean PG and
the glucose load; if the test of A1c levels
FBS is >140 mg/dL, the test
should be discontinued
▪ Standard glucose load: 75g
(general), 100g (pregnant), and
1.75g of CHO/body weight
(children)

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: CARBOHYDRATES
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Fructosamine
▪ Also called glycosylated or glycated
albumin/plasma protein ketoamine
▪ Reflection of short–term glucose control (2– • Advanced Glycated Products (AGE)
3 weeks) o Patients with DM have more AGE than healthy
▪ Monitoring diabetic individuals w/ chronic subjects
hemolytic anemia and hemoglobin variants o Cannot be used if the serum albumin of patient is
(HbS or HbC) – decreased RBC lifespan ≤3mg/dL
▪ Not measured in cases of low plasma albumin
(<30g/L) – low fructosamine
▪ Reference values: 205 – 285 umol/L
o C–Peptide
▪ C–peptide is formed during the conversion of
proinsulin to insulin
▪ Indicators for pancreatic and insulin
secretions (B–cell function)
▪ Specimen: Fasting blood (serum)
▪ Decreased: Type 1 DM
o D–xylose Absorption Test
▪ Used to differentiate diagnosis of pancreatic
insufficiency from malabsorption
▪ Low blood or urine xylose
• Ketone
o Produced by the liver through metabolism of
stored lipids
o 3 ketone bodies
▪ Acetone (2%)
▪ Acetoacetic acid (20%)
▪ 3–B–hydroxybutyric acid (78%)
o Ketonemia – high ketone levels in blood
o Ketonuria – high ketone levels in urine
o Diagnosis of Glucose Metabolic Alterations

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

Lipids and Lipoproteins ▪ If there are only single bonds in the

chain (found in the carboxyl group –
Lipids
hydrophilic)
• “Fats”
• Lipids are soluble in nonpolar organic solvents,
such as chloroform and ether, but relatively
insoluble polar solvents such as water
• Composed of mostly carbon-hydrogen bonds.
• Water insoluble, non-polar, transported by
lipoproteins ▪ They have high melting points; they are
• Rich source of energy, efficient way to store solid in room temperature
excess calories (remember gluconeogenesis, ▪ Usually seen in animal sources though,
lipids converted to glucose and used as energy there are also plant–based saturated
sources) fatty acids like coconuts
• Breaking down of lipids will give away: fatty acids,
and glycerol – precursors to glucose for
gluconeogenesis
• Integral part of the cellular membrane
o Semi-permeable due to the phospholipid
bilayer
• Precursor to steroid hormones
o Corticoid hormones, estrogen, sex hormones

Forms of lipids o Unsaturated – good type

▪ If there are carbon–carbon double
1. Fatty acids
bonds in the chain, the fatty acid is
• A fatty acid has a carboxyl group (hydrophilic)
unsaturated (carboxyl group and at
at the polar end and a hydrocarbon chain
the chain)
(hydrophobic) at the non-polar tail
▪ The double bonds can have two
o Amphipathic – they have both the
configurations: cis and trans
hydrophilic and hydrophobic region.
▪ In unsaturated fatty acids, the
• A fatty acid that occurs in a living system
stereochemistry at the double bond
normally contains an even number of carbon
is usually cis rather than trans
atoms, and the hydrocarbon chain is usually
▪ Trans FA are not commonly found in
unbranched
nature; hydrogenation –
• Fatty acids are rarely found free in nature,
synthetically being produced
but they form parts of many commonly
through this process; addition of
occurring lipids
hydrogen atoms in the fatty acid
o Bound fatty acids are more common like
chain
glycerol
• Types:
o Saturated – named after their carbon
atoms

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

▪ The notation used for fatty acids

indicates the number of carbon
atoms and the number of double
bonds – in this system, 18:0 denotes
an 18–carbon saturated fatty acid
with no double bonds, and 18:1
denotes an 18–carbon fatty acid with
1 double bond – oleic
▪ They spoil easily, to preserve,
hydrogenation is done.
▪ Lowers bad cholesterol and
increases good cholesterol.
▪ Trans fatty acid – opposite; lowers
good cholesterol – bad for the body
because they are synthetic since the
body does not have enzymes to
hydrolyze, digest these fatty acids.
▪ Why do we perform hydrogenation?
– unsaturated fats, albeit being
healthier than saturated fats, are
unstable and they spoil easily. To
preserve the quality of these oils and
fats, hydrogenation is performed.
Basically, hydrogenation is
performed to preserve the
unsaturated fatty acids.
o Madalas magbara o magkaroon ng
atherosclerosis in the arteries are
saturated and trans fats.
o Why unsaturated fat over saturated fat? –
this leads us back to their MELTING
POINTS. Due to a higher melting point,
saturated fats are solid in room
temperature. This also means that they
are in a solidified state when found inside
NOTE: Degree of unsaturation refers to the number of the body. Unsaturated fatty acids, on the
double bonds. The superscript indicates the position of other hand, are liquid. Therefore, those
double bonds. For example, 9 refers to a double bond at that usually cause blockages
the ninth carbon atom from the carboxyl end of the (atherosclerosis) in the body are the
molecule. The ratio included for example: 18:2 means the saturated fatty acids and the trans fats.
fatty acid has 18 carbon atoms and 2 double bonds. 2. Phosphoacylglycerol/Phospholipid
▪ The melting point is lower; liquid • In such lipid molecules, 2 fatty acids are also
form in room temperature (like oils). esterified to the glycerol molecule. The
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

resulting compound is called phosphatidic

acid (compound).
• One molecule of phosphoric acid can form
ester bonds both to glycerol and to some
other alcohol (phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl
serine, phosphatidyl glycerol and
phosphatidyl linocithol), creating a
phosphatidyl ester – they can combine to
essential compounds like cephalin, lecithin,
etc.
• Alcohols to which phosphoric acid can be
esterified include:
o Phosphatidylcholine
• Glycerolipids with other Head groups:
o Phosphatidylethanolamine
o Phosphatidylserine (PS) – very important
for RBCs in hematology
o Diphosphatidylglycerol (cardiolipin)
o Phosphatidylglycerol
• If the phosphate group of phosphatidic acid
o Phosphatidylinositol
will react to a second alcohol, it becomes a
phosphatidyl ester.
• Most abundant lipid in the body – our cells
contain phospholipid in their cell membrane.
• Serves as cell surfactant in the lungs.
• Forms:
o Lecithin/phosphatidyl choline (70%)
o Sphingomyelin (20%)
• Only phospholipid in membranes that is not
derived from glycerol but from an amino
alcohol called sphingosine
• Essential component of cell membranes
• It accumulates in the liver and spleen of
patients suffering from Niemann-Pick
disease.
• Niemann-Pick disease – disorder whereby
sphingomyelin is accumulated and not
metabolized. Sphingomyelin accumulates in
the liver and spleen
o Cephalin (10%)
3. Triglyceride (Triacylglycerol)
• AKA Neutral fat
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Possesses 3 molecules of fatty acids and a

molecule of glycerol which serves as a
backbone.
• Main storage of lipid in man (in adipose
tissue)
• Low calorie intake = low triglyceride level
• Function: When triglycerides are
metabolized, their fatty acids are released to
the cells and converted to energy – provides
excellent insulation.
• Fatty acids will enter the Kreb’s cycle as
acetyl coenzyme A and at the same time
glycerol enters as 3-phosphoglycerate in the
glycolytic pathway
• When an organism uses fatty acids, the ester
linkages of triacylglycerols are hydrolyzed by
enzymes called LIPASES
• The same hydrolysis reaction can take place
outside organisms, with acids or bases as
catalysts.

• Liberation of fatty acids to be used as energy

sources in the body
• When a base such as sodium hydroxide or
potassium hydroxide is used, the products of
the reaction, which is called saponification,
are glycerol and the sodium or potassium
salts of the fatty acids.
o These salts are soaps.
o When soaps are used with hard water,
the calcium and magnesium ions in the
water react with the fatty acids to form a
precipitate—the characteristic scum left
on the insides of sinks and bathtubs.
o The other product of saponification,
glycerol, is used in creams and lotions as
well as in the manufacture of
nitroglycerin.

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• The three ester groups are the polar parts of

the molecule
• The tails of the fatty acids are nonpolar
• The glycerol part is the hydrophilic part, and
the lower part is the hydrophobic part
• Fatty acids, phospholipids, and triglycerides
can all be used as energy sources In the example above, cholesterol can be converted to
testosterone, estradiol, and progesterone.
4. Cholesterol
• NOT a source of fuel because it is not Forms of Lipids (Cholesterol)
catabolized by animals – not a source of
energy. a. Cholesterol Ester (70%)
• Contains 4 rings and single C-H chain tail like • Most abundant
fatty acid • Composed of cholesterol ring and a fatty acid
• Precursor of five major classes of steroids: • It undergoes esterification by LCAT
o Progestins – sexual function o Lecithin-cholesterol acyl transferase
o Glucocorticoids – brings forth cortisol (LCAT)
o Mineralocorticoids – brings forth ▪ Catalyzes the esterification of
aldosterone cholesterol by promoting the
o Androgens – sexual function transfer of fatty acids from lecithin to
o Estrogens – sexual function cholesterol which results in the
• GLUCOCORTICOIDS and formation of lysolecithin and
MINERALOCORTICOIDS – very important in cholesterol ester.
the metabolism of carbohydrates and the • Hydrophilic in nature or water loving
regulation of carbohydrates and other very • Hydrophilic because of the fatty acids
important or essential electrolytes in the attached to them
body b. Free Cholesterol (30%)
• It is found on the surface of lipid layers; • Unesterified cholesterol
synthesized in the liver • Found on the surface of lipoproteins because
they are hydrophobic, they cannot be freely
transported in the plasma. They should be
transported by LIPOPROTEINS.
• Hydrophobic

Cholesterol from Acetyl-CoA

• A diversion from the Krebs cycle

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Synthesis from more than 25 enzymes (we will

not be talking about this)
• Principal steps:
o Conversion acetyl CoA is derived from Beta
oxidation of fatty acids to produce Acetyl CoA
o Oxidative decarboxylation of pyruvate to
beta-hydroxy beta-methyl glutaryl CoA
(HMG-CoA)
o HMG-CoA converted to mevalonic acid by
HMG-CoA reductase – the enzyme inhibited
by statins
• What are statins? Main component of the
maintenance drugs of those with cholesterol
problems. To lower cholesterol, we prevent its
production by inhibiting HMG-CoA reductase to
prevent the synthesis of mevalonic acid,
therefore inhibiting squalene formation, and
ultimately, cholesterol
• Mevalonic acid – Squalene and after cyclisation, o Highest amount of protein and phospholipid:
converted to Cholesterol HDL
o Highest amount of free cholesterol and
General Lipoprotein Structure cholesterol esters: LDL
• Lipoproteins o Highest amount of triglycerides:
o Carrier molecules of the hydrophobic lipids chylomicrons and VLDL
such as saturated fatty acids, free o Named according to densities because of
cholesterol, and triglycerides. their protein (apolipoprotein) component.
o Combination of lipids and proteins
o Large macromolecular complexes of lipids
with specialized protein, known as
apolipoprotein
o Main purpose: transport of TAG and
cholesterol to sites of energy storage and
utilization
o Composition:

o The entire structure is called LIPOPROTEIN

o Protein portion: APOLIPOPROTEIN
o Found inside or within the hydrophobic core
are the hydrophobic lipids: cholesterol ester,
free fatty acids, and triglycerides

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Cholesterol and Phospholipid are found on ▪ Normally found in the body

the surface ▪ Largest and the least dense among
lipoproteins; LPP with lowest density
(<0.95 kg/L)
▪ Transports exogenous TAG (triglyceride)
▪ Exogenous TAGs – TAGs from the diet
▪ Produced by the intestines; completely
cleared within 6 to 9 hours post prandial
▪ The reason why we have fasting hours for
lipids or lipid profile (10 hour fasting
time)
▪ When present at high levels, CM result in
“milky” plasma and accumulate as a
floating creamy layer when left
undisturbed for several hours.
o Densities are named with regards to their ▪ They will accumulate on top or float as a
buoyant density or electrophoretic mobility creamy layer
o Buoyant density – density of lipoproteins can ▪ Major composition: 90% of Exogenous
be seen based on ultracentrifugation TAG (80-95); 1-2% CHON
o Electrophoretic mobility – electrophoresis is ▪ The apolipoproteins in CM include apoB-
performed 48, apoA-I, apoA-IV, apoC-I, apoC-II,
o Chylomicrons being the least dense apoC-III, and apoE
o HDLs are the densest because it has the ▪ Take note of apoB-48 because it is unique
highest protein concentration to chylomicrons only.
o For electrophoretic mobility, the least dense ▪ In metabolism of CMs, apoC-II serves as
is the largest: chylomicrons are the largest. an activator of lipoprotein lipase
Therefore, the chylomicrons are left at the
point of origin
o B-lipoproteins are the LDL
o Pre-B-lipoproteins are the VLDL
o A-lipoproteins are the HDL

▪ Chylomicrons are broken down by lipases

to produce chylomicron remnants.
o Very Low-Density Lipoproteins (VLDL)
▪ Aka pre-B LPP
▪ VLDL particles are produced by the liver
• Normal Lipoproteins ▪ Supply the tissues of the body with
o Chylomicrons triglycerides of endogenous, primarily
hepatic, origin, and cholesterol.
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

▪ Contains approximately 50% triglyceride, peripheral tissues or in the plasma are

40% cholesterol and phospholipid, and collected and returned to the liver to
10% protein (apolipoproteins) store or process further
▪ Density: 0.95-1.006 kg/L ▪ Major Apolipoprotein: ApoA-I
▪ Apolipoproteins: mostly apoB-100, apoC- ▪ In an electrophoretic set-up, the support
I, apoC-II, and apoCIII, but also apoE medium used is a porous material. The
o Low-Density Lipoproteins (LDL) bigger the molecule, the harder it is to
▪ Aka B-lipoproteins pass through the pores of the medium.
▪ BAD CHOLESTEROL The smaller the molecule, the faster the
▪ LDL is produced through the metabolism molecule passes through the medium
of VLDL in circulation and constitutes and progresses faster through the
about 50% of the total lipoprotein mass support medium.
in human plasma
▪ They deliver cholesterol to the peripheral
tissues. The more cholesterol, the more
LDL there is in the body
▪ VLDL to IDL to LDL • Which lipoprotein is the least dense?
▪ Transports cholesterol from the liver to Chylomicrons
the peripheral tissue • Which lipoproteins is the densest? HDL – Alpha
▪ Remember that cholesterol is also lipoproteins
important in the adrenal glands, the • What lipoprotein carries exogenous TAG?
testes, and the ovaries because Chylomicrons
cholesterol is a precursor of steroid • What lipoprotein carries endogenous TAG? VLDL
hormones • Good cholesterol: HDL
▪ Constitutes about 50% of total LPP in • Bad cholesterol: LDL
plasma
▪ Density: 1.019-1.063 kg/L • Minor Lipoproteins
▪ Apolipoprotein: apoB-100 with traces of o Intermediate density lipoprotein (IDL)
apoC ▪ Also known as VLDL remnants
o High-Density Lipoproteins ▪ Upon digestion of VLDL through lipase,
▪ Aka A-lipoprotein VLDL is catabolized into IDL before it
▪ GOOD CHOLESTEROL becomes LDL (carries cholesterol)
▪ Smallest and heaviest LPP ▪ Type III hyperlipoproteinemia
▪ Produced by the liver and intestine (dysbetalipoproteinemia or broad beta
▪ Can exist either as disk-shaped or disease)
spherical particles ▪ IDL is increased
• DISK-SHAPED – HDLs capable of ▪ Concentrations of IDL also contribute to
collecting excess cholesterol the development of CHD
• SPHERICAL – HDLs that have already o Lp(a)
collected cholesterol, kumbaga may ▪ Sinking pre-B lipoprotein (VLDL)
laman na ▪ During electrophoresis, it will migrate to
▪ HDL is involved in reverse cholesterol the pre-beta region but during
transport – excess cholesterol from centrifugation, it does not have the same
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

buoyancy as VLDL. It sinks during o Free fatty acids and glycerol from hydrolysis
ultracentrifugation of triglycerides by lipoprotein lipase can then
▪ LDL lipoprotein-like particle activity be taken up by the liver
▪ Increased Lp(a) confers increased risk for o Deliver exogenous TAG that has been
premature coronary heart disease and absorbed to be carried by chylomicrons into
stroke the liver for processing
o LpX Lipoprotein • Endogenous Pathway
▪ Obstructive biliary disease o VLDL loses core lipids, causing dissociation
▪ LCAT deficiency and transfer of apolipoproteins and
▪ LpX formation associates with a high phospholipids to other lipoprotein particles
level of hepatic cholesterol synthesis o During this, VLDL will be converted to VLDL
▪ Observable in disease states remnants, which can be further transformed
o Beta-lipoproteins (B-VLDL) by lipolysis into LDL
▪ B-VLDL (floating beta-lipoprotein) ▪ Enzyme: lipoprotein lipase
▪ VLDL will migrate in the pre-beta region o Half of the VLDL is converted to LDL – carrying
but due to certain conditions, VLDL is cholesterol from liver to peripheral tissues;
observed to be floating on the beta- remainder is taken up as VLDL remnants by
region. liver remnant receptors
▪ Accumulates in type 3 • Reverse cholesterol transport pathway
hyperlipoproteinemia. o HDL removes excess cholesterol from cells
▪ Uptake of cholesterol ester-rich B-VLDL o Spherical HDL
by macrophages induces foam cell
formation.
▪ Collection: EDTA. If heparin, it affects the
electrophoretic mobility of the
lipoprotein.

Lipoprotein Metabolism

• Lipid Absorption Diagram of Major Lipoprotein Metabolism Pathways

o Within intestinal lumen
o In digestion, dietary lipids are converted to
amphipathic lipids – hydrophobic and
hydrophilic
o Amphipathic lipids form micelles in intestinal
lumen
o Micelles encounter microvillus membranes
of intestinal mucosal cells and are absorbed
• Exogenous Pathway
o Which is the main transporter? Chylomicrons
o Chylomicrons interact with proteoglycans on
surface of capillaries in various tissues in
circulation

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• HDL will cleanse or harbor excess cholesterol in

the peripheral tissues and even in the
bloodstream
• HDL removes it by interaction with LDL by
transferring cholesterol from LDL directly to LDL
with the help of CETP and HDL brings it back to
the liver
• HDL can remove it by interacting with cells
through the apc-A1 receptor of the cell and will
start to collect cholesterol with LCAT
• HDL from discoid shape will become spherical
HDL and delivered back cholesterol to the liver

Diagram of Major Lipoprotein Metabolism Pathways and

Associated Diseases

• Dietary lipids will become amphipathic lipids and

it will be emulsified by bile
• Bile is made up of cholesterol (cholesterol is
precursor to bile)
• will enter the intestine
• Exogenous tags will be absorbed by the body and
carried by chylomicrons
• Exogenous tags are being broken down into its
primary components: 3 fatty acids + glycerol with
the help of LPL as well
• Chylomicrons broken down to VLDL remnants
with the help of LPL
• Delivery to the liver • Chylomicron retention – hindi nadidigest si
• Stored in the liver or adipose tissues for storage chylomicron
• If needed by the body, the liver will begin to
synthesize triglycerides
• Liver synthesizes endogenous tags
• VLDL carries the endogenous tags
• LPL will break down VLDL to IDL and then to LDL.
50% of VLDL becomes LDL and the other 50% will
just become remnants
• LDL will begin to deliver cholesterol to different
parts of the body: in the liver or the cells

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• How cells take up cholesterol:

• LDL encounters its receptor. Engulfed by endocytosis

where the LDL only is taken in.
• Cholesterol will be used as components of cell
membrane, steroid hormones, and bile acids.
• Over supply of cholesterol inhibits DNA
replication and disrupts HMG CoA reductase
• Stopped because cholesterol is oversupplied

Lipoprotein Physiology and Metabolism

• Women have, on average, HIGHER HDL

CHOLESTEROL levels but LOWER TOTAL
CHOLESTEROL and TRIGLYCERIDE LEVELS than
men

Trans by: Mel ♡

 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

THE MECHANISM OF ATHEROSCLEROSIS • Why? Women have estrogen, higher HDL, lower
cholesterol
Atherosclerosis
• After menopause, no difference in total
Deposition of lipids in the blood vessels cholesterol
specially the arteries • Why? After menopause, lower estrogen, lowered
LIPID DEPOSITED IN ARTERY – arterial HDL, leveled cholesterol with men
function of oxygen delivery is disrupted. It • Life expectancy in male and women are different,
will now result to myocardial infarction increased cholesterol is one of the leading causes
LDL and cholesterol in peripheral tissue is in men, particularly atherosclerosis.
deposited. INCREASED LDL = INCREASED • Total & LDL cholesterol & triglyceride levels all
CHOLESTEROL = LOW HDL increase with age, in both men & women.
LDL becomes oxidized and induces an • Total & LDL cholesterol & triglycerides are much
immune response. The macrophages will lower in young children than adults.
be summoned into the site. The • At puberty, boys’ HDL cholesterol drops 20% to
macrophages will attempt to phagocytize adult male levels, but girls’ does not change.
the excess LDL. HDL cannot function to
perform this because they are low.
• Lower rates of LDL cholesterol & heart disease in
Asians
When macrophages have engulfed LDL,
they will try to digest and destroy it. Diagnosis and Treatment of Lipid Disorders
However, macrophages cannot digest LDLs,
so they will attempt to engulf more LDLs • Dyslipidemias – another term for lipid disorders
which will cause them to transform into • Arteriosclerosis or Atherosclerosis
what is known as the FOAM CELLS o Single leading cause of death & disability in
FOAM CELLS – macrophages with increased U.S.
lipids in the cytoplasm o Caused by lipids, in form of esterified
cholesterol, being deposited in artery walls,
The foam cells will accumulate in the lining
of the arteries. But it will build up overtime resulting in fatty streaks
and in time it will cause a disrupted blood o Fatty streaks develop into plaques that can
flow in the blood vessels. block blood flow
When there is total blockage of the blood o Tanger’s Disease: HDL cannot collect
vessels by the plaque, there occurs the cholesterol
myocardial infarction.
WHAT HAPPENS TO HDL AFTER DEPOSITION OF
Worst case scenario is the rupture of the CHOLESTEROL IN THE LIVER?
artery
LDL can transverse tissues.
FOAM CELLS do not undergo apoptosis There are three types of cholesterol: HDL 1, HDL 2, HDL
because the coding gene for apoptosis has 3
also been compromised due to an excess in They unload cholesterol and return to their discoid
cholesterol shapes and return to the liver as well

• Hyperlipoproteinemia
o Diseases associated with elevated lipoprotein
levels

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Includes hypercholesterolemia, o Low levels of lipoproteins

hypertriglyceridemia, & combined o Two forms: hypoalphalipoproteinemia &
hyperlipidemia hypobetalipoproteinemia
• Hypercholesterolemia • Hypoalphalipoproteinemia
o Lipid abnormality most closely linked to heart o Isolated decrease in circulating HDL
disease (concentration <40 mg/dL), without
o Why heart disease? Because cholesterol is presence of hypertriglyceridemia
not burnt up so where it is found, it can only o Alpha denotes region in which HDL migrates
be stored and not used up. on agarose electrophoresis.
o Familial hypercholesterolemia (FH): genetic o Associated with several defects, often
abnormality predisposing people to elevated genetic, most of which are linked to
cholesterol levels increased risk of premature CHD
▪ Homozygotes: rare (1:1 million); first o HDL – indirect marker for atherosclerosis
heart attack in teens (because as HDL increases, lower chance of
▪ Heterozygotes: more common (1:500) CVD, lower HDL, higher chances of CVD)
• Hypertriglyceridemia o LDL – direct marker for atherosclerosis
o Elevated triglyceride levels: high, 200–500
mg/dL; very high, >500 mg/dL
o Due to either genetic abnormalities or Lipid and Lipoprotein Analyses
hormonal abnormalities
o Hypertriglyceridemia hormonal • Lipid Measurement
abnormalities. Cholesterol increases tags, o Lipids and lipoproteins are important
tags induce activation of cortisone measures of CHD risk
transforming to cortisol. o Standardized decision cut-points for CHD risk
o So elevated cholesterol results to elevated developed by NCEP
tags • Cholesterol Measurement
• Combined hyperlipoproteinemia (CH) o Hexane extraction after hydrolysis with
o Elevated levels of serum total cholesterol & alcoholic KOH, followed by reaction with
triglycerides Liebermann-Burchard color reagent
o Increased risk for coronary heart disease • Triglyceride Measurement
(CHD) o Useful in detecting metabolic disorders and
o Genetic forms CVD risk
o Familial CH: some in family have only • Lipoprotein Methods
elevated cholesterol, others only elevated o Measure physical properties: density, size,
triglycerides, others both charge, apolipoprotein
o Familial dysbetalipoproteinemia: very rare o Methods: ultracentrifugation,
• Lp(a) Elevation electrophoretic separation, chemical
o Increased risk of CHD and cerebrovascular precipitation, chromatographic,
disease immunochemical
o Cerebrovascular disease means STROKE – sa • HDL Methods
brain kasi kapapg sa HEART – myocardial o In past, 2-step separation by chemical
infarction precipitation was used.
• Hypolipoproteinemia
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o Now, 3-step process: ultracentrifugation to Direct measurements denote that there are reagents
remove VLDL, heparin manganese available for the measurement. Indirect measurements
precipitation to remove LDL, & analysis of do not have reagents that is why they are calculated.
supernatant cholesterol by Abell-Kendall However, as of today, there are already reagents
assay developed to directly measure LDL. This is not the case
• Lipoprotein Methods for all laboratories.
o Beta-quantification: most common;
combines ultracentrifugation & chemical Computation: FRIDEWALD EQUATION
precipitation TC = HDL + LDL + VLDL
o Friedwald calculation: bypasses LDL = TC - (HDL+VLDL)
centrifugation; commonly used in routine & VLDL = TAG/5 (mg/dL)
sometimes research labs VLDL = TAG/2.175 (mmol/L)
• Compact Analyzers
o Mobile point-of-care testing systems Blood Sampling and Storage
o Can measure cholesterol, triglycerides, HDL
• Biologic Variations
cholesterol, & glucose from a finger stick o Cholesterol varies from individual to
sample individual
• Apolipoprotein Methods o Cholesterol, the coefficient of physiologic
o Apo B is measured directly in serum by within an individual averages about 6.5%, but
immunoassay. it can be higher in certain individuals
o Apo A-I is measured by separation & analysis o When measured in serial samples from the
of HDL cholesterol. same person, cholesterol levels in 95% of the
o Lp(a) is commonly measured by various sample will vary by about 13% above or
immunoassays. below that person’s mean level
o Cholesterol levels rise with age, starting in
• Phospholipid Measurement early adulthood, in both sexes. Women have
o Can be measured by an enzymatic reaction lower levels than men, except in childhood
sequence and after the early fifties.
o Fatty Acid Measurement o Estrogen in women specifically, estradiol,
• Commonly analyzed by gas-liquid cause cholesterol in women to be lower.
chromatography o Estradiol is the potent estrogen in women.
Laboratory Diagnosis for Lipids o This also explain why women have longer life
expectancies due to males being more
• Cholesterol, triglycerides, and lipoproteins – predisposed to cardiovascular diseases.
tested in the library and collectively called LIPID o At early fifties, variations can be seen
PROFILE because of menopause. With menopause,
• Lipid Profile estrogen levels will diminish as well causing
o Measures cholesterol to elevate.
▪ Cholesterol – directly
▪ TAG – directly
▪ HDL – directly
▪ LDL – indirectly; compute
▪ VLDL – indirectly; compute
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Fasting
o Ideally, patients should fast for 12 hours
before venipuncture
o Generally, TC and HDL-C levels can be
measured in non-fasting individuals, greatly
facilitating screening and monitoring
o When TG and LDL-C are being measured,
fasting becomes a requirement
o The NCEP Adult Treatment Panel III (ATP III)
(NCEP, 2002) has recommended that
patients fast for at least 6 – 9 hours before
blood specimens are taken for lipid and
lipoprotein analysis (BASED ON THE
CLEARANCE TIME OF CHYLOMICRONS)
o CMs are almost completely cleared within 6
to 9 hours, and their presence after a 12-hour
fast is considered abnormal
o In average, 10 – 12 hours
o TRIGLYCERIDES AND LDL are the ones that
require fasting.

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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

Types TAG CHOLE LDL VLDL CM Feature

Low cardiac risk; eruptive
Type 1 – hyperchylomicronemia.
↑ N N N ↑ xanthomia, recurrent
LPL Deficiency
pancreatitis
High cardiac risk; tendon
Type 2a – familial xanthomas; corneal
N ↑ ↑ N N
hypercholesterolemia arches; hypothyroidism;
nephrotic syndrome
Type 2b – mixed defect – familial
↑ ↑ ↑ ↑ N High cardiac risk
combined hyperlipidemia
Type 3 – familial Eruptive and palmar
↑ ↑ N ↑ N
dysbetalipoproteinemia xanthomas
Type 4 – familial
↑ N N ↑ N Low cardiac risk
hypertriglyceridemia
Low cardiac risk; eruptive
Type 5 ↑ ↑ N ↑ ↑
xanthomas, pancreatitis

☛ VLDL also carries 40% of cholesterol

☛ LDL increased, high risk
suggesting that factors other than simply
hemodilution may also operate
o Why? Remember that chylomicrons
o Current NCEP guidelines recommend that
are the ones that carry exogenous
patients be seated for 5 minutes before
triglycerides in the diet, and they are
sampling to prevent hemoconcentration
cleared after 6 hours. You need to fast
to remove any chylomicrons in the
circulation that is carrying triglycerides.
Practically, to remove the chylomicrons
• Venous Vs. Capillary Samples
in the blood.
o In general, measurements in capillary blood
o TAGS being measured: ENDOGENOUS
samples seem to be a little lower than in
o Why? Fasting is performed to take out
venous samples
chylomicrons which carry the
o Measurements in fingerstick samples tend
EXOGENOUS TAGS
to be more variable than in venous samples
o YOU NEVER FAST FOR CHOLESTEROL
obtained at the same time, probably as the
o NOTICE THAT CHYLOMICRONS ARE
result of pre-analytic sources of error BUT
NOT BEING MEASURED IN THE LIPID
it is good to keep in mind first that the
PROFILE BECAUSE IT IS MEANT TO BE
epidemiologic data from which risk levels
REMOVED BEFORE MEASUREMENT OF
for lipids and lipoproteins are derived are
LIPID PROFILE
based on measurements in venous samples
• Posture
• Plasma Vs. Serum
o Standing to reclined position = Decreases of
o Both plasma and serum can be used in the
as much as 10% in the concentrations of TC,
measurement of lipid profile
LDL-C, HDL-C, apoA-I, and apoB (Miller et
o Plasma or serum can be used when only
al, 1992) have been observed after a 20-
cholesterol, TG, and HDL-C are measured,
minute period of recumbence. The
and LDL-C is calculated from these three
decrease in TG is about 50% greater,
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

measurements (di kasama si LDL-C, o In most lipid classes, the effect of storage
icalculate lang siya) temperature over 1 week is minimal
o Plasma is preferred when the lipoproteins between 4C, -20C, and -80 C (Zivkovic et.al.
are measured by ultracentrifugation or 2009)
electrophoretic methods
Triglyceride Measurement Methods
▪ Why? Because lipoproteins are labile,
and they require to be preservation • Chemical Methods
with the use of an anticoagulant. o Van Handel and Zilversmith (Colorimetric)
▪ K2 EDTA is the preferred anticoagulant o Hantzsch Condensation (Fluorometric)
even though cholesterol and o Modified Van Handel and Zilversmith
triglyceride concentrations in EDTA ▪ All have the same steps except for
plasma are about 3% lower than in measurement
serum • Generalized Steps for Chemical Methods
▪ Why not others? o Extraction
▪ Purpose: To remove lipids from
• Citrate affects osmotic pressures
proteins (VLDL)
disrupting lipoproteins whose
▪ Reagent: Organic solvents
integrity are not preserved and are (Chloroform, isopropanol,
destroyed even before diethylether)
measurement, result in falsely low
plasma lipid and lipoprotein
concentrations. ▪ Additional step: adsorption
• Heparin cannot be used as well • Purpose: To remove non-TAG
because it causes changes in the glycerol (phospholipid,
electrophoretic mobility of the monoglycerides, diglycerides, and
lipoproteins other interfering substances such
o Because the samples can be cooled to 4° C as glucose and bilirubin)
immediately to retard changes that can • Reagent: Alumina adsorbent
occur in the lipoproteins at room mixture, Zeolite, Florisil, Silisic acid
temperature. o Hydrolysis or Saponification
▪ Purpose: To cleave TAG into fatty acid
o When plasma is to be used, blood is cooled
(3 parts) and glycerol (1 part)
in an ice bath as soon as it is drawn, and the
▪ Reagent: Alcoholic potassium
cells are removed as soon as possible, hydroxide or Sodium methoxide
generally within 3 hours. The plasma is then o Oxidation
stored at 4° C until it is analyzed. ▪ Purpose: To convert glycerol to a
• Storage measurable compound
o Serum and plasma can be stored for 2 ▪ Reagent: Oxidizing agent (Periodic acid,
months under –70C Na periodate)
o Short term: 1–2 months -20C but they ▪ Glycerol is being measured instead of
should not be stored in a self-defrosting the TAG itself
freezer o Colorimetry (Van Handel Zilversmith)
o Storage at 4C, no changes in the ▪ Purpose: Aids in measurement by
imparting color to the product of
lipoproteins will be observed if stored for a
oxidation
shorter period
▪ Reagent: Color reagent
o Shorter period is more than 2 hours but less
• Chromotropic acid and sulfuric
than 1 week or 1 month acid: Pink (500 – 600 nm)
• Diphenylhydrazone
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

• Diacetyl Acetone and Ammonia in • Hyperlipoproteinemia types I, IIb,

(HANTZSCH METHOD), III, IV, V
Fluorometric method: Yellow • Alcoholism
o Spectro – 412 nm • Nephrotic syndrome,
o Fluoro ExL – 400 nm • hypothyroidism,
o EmL – 485 nm • pancreatitis
▪ MODIFIED: The one with the addition ▪ Decreased in
of the ADSORPTION stage • Malabsorption syndrome,
• Enzymatic Methods • hyperthyroidism (increased
o Initial Enzymatic Reaction metabolism fats are burnt
▪ AKA Lipase (enzyme lipase is used for immediately),
anything that has ester bonds) and • malnutrition,
Glycerokinase/Glycerokinase Method • brain infarction
▪ Product: Glycerophosphate + ADP o Values to remember
▪ TAG → Lipase → 3FA + Glycerol ▪ ATP Classification
▪ Glycerol ATP-ADP → Glycerokinase → • <150 mg/dL Normal
Glycerophosphate • 150 – 199 Below high
o Second Enzymatic Reaction
• 200 – 499 High
▪ Lipase – Glycerokinase coupled with
• ≥ 500 Very High
Glycerophosphate Dehydrogenase and
• Conversion factor: 0.0113
Diaphorase
• Reference range: 10 – 90 mg/dL
• Measurement of absorbance of
• Reference range: (0.11 – 2.15
NADH at 340 nm (colorless) after
mmol/L)
the addition of glycerophosphate
dehydrogenase or absorbance of Total Cholesterol Measurement Methods
Formazan dye at 500 – 600 nm
• Chemical Methods
▪ Lipase – Glycerokinase coupled with o Principle: Dehydration and oxidation of
Glycerophosphate–oxidase and Cholesterol to form a colored compound
Peroxidase o Methods:
• Product: Quinoniemine dye @ 500
– 505 nm
• Interference: Hgb = falsely high
• Ascorbic acid = falsely low Method Procedure
• INTERFERENCE OF BILIRUBIN may Pearson Stern, Mac
be minimized by the addition of K One step - Colorimetry
Gavack
ferricyanide Two step – Colorimetry,
▪ Lipase – Glycerokinase coupled with Bloors
Extraction
Pyruvate kinase and LDH Three step –
• Product: Lactate and NAD Abell - Kendall Colorimetry, Extraction,
• NAD measured Saponification
spectrophotometrically at 340 nm Four step –
or fluorometrically ExL 355 EmL Schoenheimer Sperry, Colorimetry, Extraction,
460 nm Parekh and Jung Saponification,
• CDC Reference Method: Modified Van Handel Precipitation
and Zilversmith • Generalized Steps for Chemical Methods
o Pink color at 500 – 600 nm o Extraction
o Emerging method to replace this: IDMS ▪ Purpose: Cholesterol is separated from
o Clinical Correlation protein
▪ Increased (TAG) in
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 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

▪ Reagent: • Enzymatic Method

• Bloor’s reagent (ethanol – ether at o Spectrophotometric
3:1) ▪ Reagent: cholesterol ester, cholesterol
• Chloroform oxidase, peroxidase, phenol, 4-
• Hexane aminoantipyrine
▪ Additional step – adsorption ▪ Product: Quinoneimine dye measure at
• Purpose: To extract all cholesterol 500 nm
o Saponification/Hydrolysis ▪ Most used method
▪ Purpose: Cholesterol esters are o Oxygen Consumption
hydrolyzed into free cholesterol and ▪ Reagent: Cholesterol ester hydrolase,
fatty acids cholesterol oxidase
▪ Reagent: Alcoholic Potassium ▪ Product: H2O2 (electrode measures the
hydroxide O2 released from H2O2 in the final step)
o Purification deletion of oxygen is directly
▪ Purpose: To precipitate free proportional to cholesterol level
cholesterol; removes nonspecific ▪ Disadvantage: Requires a lot of
interferences cholesterol oxidase
▪ Reagent: Digitonin (measurement of • Reference Method for Total Cholesterol
cholesterol before and after digitonin Measurement
treatment to determine cholesterol o CDC REFERENCE METHOD: Abell, Levy, and
fraction Brodie
o Colorimetry ▪ Extraction with hexane
▪ Purpose: Color development, measure ▪ Hydrolysis with alcoholic Potassium
spectrophotometrically hydroxide
▪ Reagent: Color reagent ▪ Extract dried in vacuum
▪ Color developer: ▪ Extract treated with Liebermann
• Glacial acetic acid Burchardt reagent (Acetic Anhydride +
• Acetic anhydride Sulfuric Acid)
• Concentrated sulfuric acid ▪ Read at 620 nm after 30 mins
▪ Clinical Correlation
Liebermann
Salkowski • Increased levels:
Burchardt o Hyperlipoproteinemia (types
Acetic Anhydride Sulfuric Acid +
Reagent II, III, V),
+ Sulfuric Acid Fe3+
o biliary cirrhosis,
Cholestadienyl
Product monosulfonic
Cholestadienyl o nephrotic syndrome,
disulfonic acid o poorly controlled diabetes
acid
Green (unstable; mellitus,
Na sulfate to o alcoholism,
End Color stabilize) Red (stable) o primary hypothyroidism
measured @ 410 • Decreased levels:
nm or 620 nm o Severe hepatocellular disease
o Precautions for the chemical methods: o Malnutrition
▪ Avoid hemolyzed sample: falsely high o Severe burns
result (RBCs have cholesterol in the o Hyperthyroidism
membrane) o Malabsorption syndrome
▪ Avoid icteric specimen: 5 -6 mg%
increase in cholesterol per mg of
bilirubin above normal
▪ Avoid water contamination ▪ Values to Remember
▪ Precise and accurate timing • ATP Classification:
Trans by: Mel ♡
 CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

o <200 mg/dL – Desirable

o 200 – 239 – Below High
o ≥240 – High
• Reference range: 150 – 250 mg/dL
(3.88 – 6.47 mmol/L)
• Conversion factor: 0.026
Phospholipid Measurement Methods
• Most of the phosphorous in the body is in the
form of phosphate
• Enzymatic method makes use of coupled
enzymes
• Chemical Methods
o Extraction
o Oxidation
▪ Purpose: Converts phospholipid
phosphorous to inorganic phosphorous
▪ Reagent: Concentrated acid (wet-ash
oxidation)
o Colorimetry
▪ Reagent: Molybdate
▪ Product: Molybdenum blue
o Calculation
▪ Total Phosphorous x 25 = Phospholipid
mass
• Enzymatic Method
o Reagent: Phospholipase D, Choline oxidase,
Peroxidase
o Product: Quinoneimine dye @ 500 nm
• Values to Remember
o Reference range: 150 – 380 mg/dL (1.50 –
3.80 g/L)
o Conversion factor: 0.01
Fatty Acid Measurement Methods
• Chemical Method
o Extraction
o Purification with Dilute Sulfuric Acid
o Titration
▪ Reagent: Dilute NaOH
▪ Indicator: Thymolphthalein
• Values to Remember
o Reference range: 9 -15 m

Trans by: Mel ♡

CLINICAL CHEMISTRY LECTURE
MIDTERM: LIPIDS AND LIPOPROTEINS
LECTURE | SIR. EUGENE DAYAG | 3MT | FIRST SEMESTER | SOURCE: BISHOP

Trans by: Mel ♡