Clinical Chemistry Lecture Notes 2.

According to the location of the carbonyl functional group

December 1, 2021 ○ Two classification:
CARBOHYDRATES i. Aldose: carbonyl functional group located in the
● Introduction of Macromolecules (biochemistry) ALDEHYDE
Classifications ii. Ketose: carbonyl functional group located in the
● Pathways of Glucose Metabolism KETONE.
● Regulation of Glucose Metabolism 3. Based on the number of sugar units
● Disease states in Glucose Metabolism ○ Sugar units- pertains to the saccharide unit
○ Hyperglycemia- diabetes mellitus i. Monosaccharide
○ Hypoglycemia- decreased plasma glucose ii. Disaccharide
● Diagnosis of patients with Glucose Metabolic iii. Oligosaccharide
○ Using different methods iv. Polysaccharide
4. Based on the stereochemistry of the compound
INTRODUCTION ○ Pertains to the rotation of the sugar unit (structural rotation)
Carbohydrates- primary energy source, stored primarily in the form of i. L-type of sugar (levorotatory)- rotation of the sugar
glycogen in the muscle and liver is to the left (counter-clockwise)
● Provides energy for cell’s ATP production ii. D- type of sugar (dextrorotatory)- rotation of the
● Biochemically speaking, carbohydrates are the “Hydrates” of sugar to the right (clockwise)
aldehydes and ketones derived based on the location of the carbonyl
group Classification according to number of sugar units:
● Can be associated with several types of condition: 1. Monosaccharides/ Simple sugars-
○ Elevated: hyperglycemia ○ Sugar that cannot be hydrolyzed to a simpler form
○ Decreased plasma glucose: hypoglycemia ○ Sugar that contain 3, 4, 5, 6 carbon atoms (triose, tetrose,
- Disease states involved hyperglycemia and hypoglycemia pentose, hexose)
- Structure contains C, H, and O (Cx (H2O)y with C=O and -OH ○ Examples: fructose, glucose, galactose
functional groups) Glucose:
- the only carbohydrate directly used for energy
Classification of Carbohydrates: production of cells
1. Based on the size of the base carbon chain - The only carbohydrate stored as glycogen in the
○ Carbohydrate chain is classified on the number of carbon muscle and in the liver.
present: - The only sugar that is directly used by the brain as
i. Triose: 3 carbon (simplest carbohydrate) its energy source. Brain is completely dependent on
ii. Tetrose: 4 carbon blood glucose for energy production.
iii. Pentose: 5 carbon - ⅔ of glucose utilization among resting adults
iv. Hexose: 6 carbon occurs in the Central Nervous System
- Other monosaccharide must be converted to
glucose first before being utilized or consumed.
➔ Reducing Sugar- capable of converting an agent or substance using 3. Oligosaccharides- composed of more than 2 up to 10
reduction-oxidation reaction monosaccharides.
- The presence of the double bond and a negative charge in 4. Polysaccharides- complex sugar composed of more than 10
the inole anion group, makes a sugar an active reducing monosaccharide units.
agent. ○ Linkage of many monosaccharide units
- Example: glucose ○ Include starch (most common in the diet), glycogen, and
➔ Non-reducing sugar: cellulose
- Do not contain an active ketone/aldehyde group i. Glycogen- storage form of monosaccharides, spec.
- Example: sucrose Glucose in the liver or muscle. A reservoir
ii. Cellulose- derived from plants, not digested in the
- For the small intestine to absorb carbohydrates obtained from the GIT (resistant to digestion)
diet, the sugar must be in the form of monosaccharides (a simple - For oligosaccharide and polysaccharides, an important digestive
sugar). enzyme (AMYLASE) is necessary for breakdown of glycosidic bonds
- In the GIT, the polysaccharides are converted into simple sugars, or and for release of simple sugars.
else it won't be absorbed by the small intestine - Amylase is derived from:
1. Salivary gland (salivary amylase)
2. Disaccharides 2. Pancreas (pancreatic amylase)
- Formed by interaction of two monosaccharides (connected by - Amylase is responsible for breakdown/ hydrolysis of both
glycosidic bond) oligosaccharide and polysaccharides.
Examples:
i. Maltose = glucose + glucose CARBOHYDRATE MODELS
enzyme: Maltase Fischer projection
ii. Lactose = glucose + galactose ➔ Carbons are numbered
enzyme: Lactase ➔ it is in straight linear chain
iii. Sucrose = glucose + fructose ➔ linked to show a representation of the cyclic form
enzyme: Sucrase
- For disaccharides to be absorbed in the small intestine, it must be Haworth projection
hydrolyzed/ converted further into monosaccharides. ➔ carbohydrate compound is in
- In the small intestine, disaccharides are hydrolyzed in the the cyclic form
presence of an enzyme called disaccharidases (maltase, ➔ more representative of the
lactase, sucrase), which is responsible for hydrolysis of actual CHO structure
disaccharides into monosaccharides. ➔ formed when the functional
- Patients who are lactose intolerant cannot convert and absorb carbonyl group reacts with an
lactose, thus normal flora in the small intestine would try to break active alcohol group on the
down the lactose and be consumed by the bacteria causing diarrhea. sugar to form a ring
- Lactose intolerance is attributed to the deficiency of the
disaccharidase lactase- deficient Lactase enzyme = no
absorption of lactose
December 1, 2021 PART 2
Glucose Metabolism
Overview:
- In anatomy, whenever we consume starch-rich foods (being the
most common polysaccharide in our diet), INITIAL DIGESTION
happens in the mouth. Through the catalytic action of the salivary
amylase, starch conversion starts in the mouth.
- Mouth -> Intestine. In the small intestine, the monosaccharides are
absorbed. Absorption happens and the monosaccharides are now in
circulation.
- From circulation, sugars are consumed by the cells as source of
energy for ATP generation.
- Glucose is the monosaccharide that is directly consumed by the cell.
- Glucose directly fuels the brain.
- Unutilized glucose in the plasma will be stored in the liver and
muscle in the form of glycogen.
- Some of the excess glucose is converted into fatty acid
stored in the adipose tissue.
- Insulin is responsible for the normal reactivity of the body in
utilizing unused glucose.
After consumption of starch-rich foods, in the first 2 hours, it is normal to be
hyperglycemic. After 2 hours, the hyperglycemic state gradually normalizes
because of the action of the hormone INSULIN (hypoglycemic agent), which
is released from the pancreas when there is hyperglycemic state to promote
lowering of plasma glucose level.
Insulin (hypoglycemic agent)
➔ promotes the cell uptake of glucose to be used as source of energy
➔ promote conversion of excess glucose to glycogen in the muscle and
liver
➔ Promote conversion of excess glucose into fatty acids (triglycerides)
in the adipose
- Triglycerides: major storage form of fats in the
adipose.
A 2-hour postprandial test is practiced in phlebotomy because 2 hours after
eating is the reference to check if the plasma glucose has lowered down.
Glucose Metabolism
Kreb Cycle aka Tricarboxylic acid cycle-TCA
Glycolysis (Embden-meyerhof pathway)
● In the glycolysis these products are produced: lactate,
pyruvate, and ATP
○ This depends on the aerobic/anaerobic activity
■ Aerobic- pyruvate
■ Anaerobic- lactate
● Embden-Meyerhof Pathway or Hexose Monophosphate
pathway
● Glycolysis happens inside the cell. To be possible, the
glucose must be uptake by the cell
○ Cell uptake is promoted by the activity of the
INSULIN
Gluconeogenesis- formation of glucose from non- carbohydrate source (fats
or proteins)
➔ Formation of glucose-6-phosphate from non carbohydrate source
- Non carbohydrate source- fats and proteins.
- When fats undergo gluconeogenesis, it forms glucose-6-phosphate
and ketone bodies.
Ketone bodies- products of conversion/hydrolysis of fats
- For proteins, glucose and urea nitrogen are formed
Glycogenesis- conversion of glucose to glycogen (polysaccharide) for
storage
➔ Unconsumed/ unutilized glucose in the plasma is stored in the liver
and muscle as glycogen, thru process of glycogenesis
➔ Insulin also promotes this process
Glycogenolysis- breakdown of glycogen to glucose-6-phosphate (opposite of
glycogenesis)
“Lysis”- metabolism/ breakdown
➔ When glucose is needed, glycogen is used; regulated by glucagon.
➔ Glucagon is a hyperglycemic agent- when in need of glucose,
glycogen is the source.
 ➔ Glucagon and insulin have exact opposite functions:
- Glucagon: hyperglycemic agent
- Insulin: hypoglycemic agent
Lipogenesis -conversion of carbohydrates to fatty acids
➔ Fatty acids become triglycerides for storage in the adipose tissue.
(majority of fats)
➔ Excess carbs are converted to fats by insulin.
➔ Lypolysis: to generate ketone bodies and glucose-6-phosphate
The fat conversion and breakdown can also regulate glucose level in the
plasma.
Regulation of Glucose Metabolism
In the body, glucose is regulated by the hormones
Hormones- responsible for controlling the catabolism and anabolism and
breakdown of glucose.
2-7 hyperglycemic- promotes in elevating plasma level
Hormones:
1. Insulin- the only hypoglycemic agent
○ Primary hormone responsible for decreasing blood glucose
(hyperglycemic to normal level)
■ When impaired, laboratory-prepared insulin
(synthetic) is injected because it is the only
hypoglycemic agent.
○ Synthesized by the β-cells (major cell type in the pancreas)
of the islets of Langerhans (pancreas)
■ Beta cell- major cell type in the pancreas
○ Regulates blood glucose level by the process of
glycogenesis
■ ↑ glycogenesis, glycolysis and lipogenesis; and ↓
glycogenolysis.
○ Insulin is normally released in the plasma by the pancreas
when the glucose level is high.
■ Normal plasma glucose level- no insulin released
■ Normoglycemic- no insulin activity
○ THE ONLY HORMONE CAPABLE OF LOWERING GLUCOSE
LEVEL
○ Has reciprocal relationship with glucagon.
2. Glucagon- Primary hyperglycemic agent
○ Primary hormone responsible increasing blood glucose
○ Synthesized by the alpha cells of the islets of Langerhans
(pancreas)
■ Regulates blood glucose by ↑ glycogenolysis and
gluconeogenesis
○ Released during stress and fasting state
○ Glucagon enhances catabolic function of the body during
fasting period.
3. Epinephrine- hyperglycemic agent
○ released during times of physical and emotional stress
○ Aka the STRESS HORMONE
■ When stressed, the plasma glucose increases
because of hormonal change.
■ Overconsumption of carbohydrate due to stress
causes hyperglycemia (exogenous)
○ Associated with stress; promotes the inhibition and
secretion of insulin and promotes glycogenolysis and
lipolysis.
○ ↓ intestinal entry of glucose into the cell, ↑ gluconeogenesis,
glycogenolysis and lipolysis
4. Cortisol- hyperglycemic agent
○ Produced by the adrenal cortex, secreted in response to
ACTH (adrenocorticotropic hormone activity), ↑ plasma
glucose
○ Lowers interstitial entry of glucose into the cell; promotes
gluconeogenesis, glycogenolysis and lipolysis
5. Growth hormone - hyperglycemic agent
○ Produced by the anterior pituitary gland; ↑ plasma glucose
○ ↓ glucose entry to cells, ↑ glycolysis
 6. Thyroxine or T4 - Hyperglycemic agent
○ Has 4 iodine molecules. Important in the iodine related to
thyroid gland
○ Produced by the thyroid gland; ↑ plasma glucose Promotes
glycogenolysis, gluconeogenesis and glucose intestinal
absorption.
7. Somatostatin- hyperglycemic agent
○ Produced by the delta cells of the islets of Langerhans in the
pancreas and hypothalamus
○ Increases plasma glucose by inhibition of insulin, glucagon,
GH, etc
If glucagon is deficient/impaired, there are five other hormones that could
elevate plasma glucose level, but if insulin is impaired, there are no hormone
with similar actions
Synthetic insulin is needed.
DISEASE STATES IN GLUCOSE METABOLISM
Hyperglycemia
- When we consume starch-rich food we normally become hyperglycemic
and normalized by insulin 2 hours after consumption.
- But when insulin action is abnormal and deficient, hyperglycemia is
sustained. (even after 2hrs)
Coined pathologic as “hyperglycemic state”
● Defined as increase in plasma glucose level
○ “Hyper”- increase
○ “Gly”- carbohydrate or sugar
○ “Emia”- blood or plasma
● Causes:
○ Stress (overeating)
- Causes hormonal imbalance and releases
hyperglycemic agents in nature
○ Severe infection (metabolic stress in the proinflammatory
phase of the infection)
- ↑ Glycogenolysis, ↑ glucagon release, and insulin
resistance
■ Metabolic stress and hormonal changes occurs that
increases hormonal production
● Increased synthesis of Glucagon, ACTH release,
and insulin resistance
○ Dehydration (↓ plasma volume, ↑ solute level)
■ Glucose: major contributor of plasma osmolality
○ Major solutes in the plasma that contributes to the
plasma osmolality:
Major solutes in the plasma:
■ Glucose
■ Urea
■ Sodium
■ Chloride
■ Other major: urea, and electrolytes like Na and Cl
■ Level of solute present in the solvent/plasma
○ When there is dehydration, plasma volume
decreases and plasma solutes increase
(including glucose)
○ Pregnancy (hormonal surge)
○ Hormonal Imbalance result to hyperglycemia
○ Catabolic and anabolic changes causes
hyperglycemia
○ Pancreatectomy - due to infection or disease
○ Surgical removal of a portion of the pancreas.
○ Pancreas: Both endocrine and exocrine gland
■ site of major enzymes and hormones
synthesis including insulin
○ As Beta cell number decreases- insulin becomes
deficient.
○ Hemochromatosis- excessive iron accumulation or deposition in the
vital organs
- Exact opposite of IDA - Iron deficiency Anemia (low level of
Iron)
○ Excessive iron in the body accumulates in the pancreas and
can damage it.
 ■ When extra iron is deposited/stored in the pancreas, the iron can
Ketone bodies Tyrosine intake;
damage the pancreas and directly affect the insulin phosphatase IA-2 Thirst center of the
are increased
secretion/production. & IA-2B autoabs brain is activated and
because of the
direct person to take
active lipolysis
○ Insulin deficiency or abnormal insulin receptor. End result: no in fluid; increase
Increased in hypoglycemic osmolality
○ Can result in diabetes mellitus.
Ketone bodies, agent; plasma
○ Common cause of Hyperglycemia
plasma pH glucose stays Polyphagia
decreases high excessive eating;
FBS levels: =/> 126 mg/dL (considered hyperglycemia) associated with cells
Around 70-110mg/dL: reference range Signs and starvation; when
Absence of
○ 111-125: prediabetic stage (borderline diabetic symptoms are glucose in the plasma
Insulin with
stage) more severe than is not consumed,
excess in other types cells starved and
glucagon brain will direct the
Insulin injection, Idiopathic Type I person
HYPERGLYCEMIA DM- form of type To consume more
injected in the
Diabetes Mellitus- Metabolic disease characterized by hyperglycemia I DM that has no food; sign of DM
peritoneal area is
resulting from defects in insulin secretion, insulin action or both. ideology,
the med
Metabolic disorder- hormonal defects and abnormalities unknown cause Polyuria
interference.
-Strongly increase urine output;
Epidemiology Pathogenesis Characteristics inherited. direct effect of
and Pathophy -No beta cell polydipsia
autoantibodies.
Type 1 5-10% of all β-Cell destruction -Abrupt onset
cases of absolute insulin -Insulin dependence
formerly diabetes occurs deficiency linked Type 2 90% OF ALL Insulin resistance Non-insulin
known as in childhood and to autoimmune Ketosis tendency CASES OF w/ insulin dependent
adolescence; disorder caused prone to develop Stable type DIABETES secretory defect
“insulin-de
unstable by genetical ketoacidosis; plasma DM Ketosis tendency is
pendent defect becomes acidic Patients are Relative insulin seldom with greater
diabetes Absence of because ketone non-insulin resistant to deficiency tendency to develop
mellitus Insulin with Autoantibodies bodies are acidic in dependent diabetic hyperosmolar
(IDDM)” excess in (abnormal) nature DM type ketoacidosis Risk factors: nonketotic states
glucagon Islet cell Increased with (1,000 mg/dL of
Also known autoantibodies Thin patients & has Adult onset age glucose:
as - Brittle ketonemia Majority of cases Obesity >320mOsm/dL)
juvenile-on diabetes, Insulin are adults lack of exercise
set DM autoantibodies Sign and Symptoms Sedentary Milder symptoms
-Keto-acidosis
(3Ps) Strong lifestyle ● Polydipsia
(ketosis) prone Glutamic acid association with ● Polyphagia
diabetes decarboxylase Polydipsia genetics; Adipose cells are ● Polyuria
autoantibodies there is excessive inherited type resistant to
thirst; increase fluid
 Is it normal for the bodies to produce autoantibodies? No.
insulin Treatment: oral
medication
(metformin) / Diabetes Mellitus can be associated with secondary conditions, such
hypoglycemic drugs as:
● Genetic defects of β-cell function
Gestational Glucose Glucose Screening test is ● Pancreatic disease
type DM intolerance intolerance with performed among
● Endocrine disease
during onset during pregnant patients:
pregnancy pregnancy ● Drug or chemical induced
causing Oral Glucose ● Insulin receptor abnormalities – Insulin will not function
Due to metabolic hyperglycemia Tolerance Test without the receptors
and hormonal (OGTT) is performed ● Other genetic syndromes
changes Due to metabolic between the 24th and If the person has any of these conditions, they can also have DM as
and hormonal 28th week of their secondary condition.
High risk for changes gestation.
respiratory - To identify if
distress ↑ risk for the mother is Candidate type III DM
syndrome respiratory glucose ➔ Insulin resistance and insulin resistance in the CNS
hypocalcemia distress intolerant ➔ Proposed to connected with Alzheimer's disease (pathophysiology)
(low calcium in syndrome, ➔ Resistance to insulin in the brain causing hyperglycemia
the blood; baby hypocalcemia and ➔ Not yet published; still under research
has jaundice), hyperbilirubinemi
and a
Diagnostic Criteria for Diabetes Mellitus
hyperbilirubinem
ia 1. Random plasma glucose ≥200mg/dL, + symptoms of diabetes
○ Blood is collected anytime of the day. Preferably in the
Mothers with morning, because stress is increased in the afternoon; and
GDM after stress contributes to hyperglycemia
pregnancy can ○ No fasting required
develop type 2
○ Late Morning collection is preferred for lesser effects of
DM
hormones.
The mother must 2. Fasting plasma glucose >126 mg/dL FBG/FBS commonly
be evaluated ○ Average 8-hour fasting up to 10-hours
6-12weeks after 3. 2-h plasma glucose ≥200 mg/dL during an OGTT
giving birth to
monitor glucose Categories of fasting Plasma
tolerance and
➢ Normal fasting glucose: FBG <110 mg/dL
level
➢ Impaired fasting glucose (Pre-DM): FBG111-125 mg/dL
Immune response- how the body responds to foreign bodies. ➢ Provisional diabetes diagnosis: FPG >126 mg/dL
The body can identify the normal from abnormal cells.
The Immune system can discriminate cell from non-cell; normal from
abnormal
Categories of Oral Glucose Tolerance:
➢ Normal glucose tolerance: 2-h Plasma Glucose <140 mg/dL
➢ Impaired glucose tolerance: 2-h PG 140-199 mg/dL
➢ Provisional diabetes diagnosis: 2-h PG >200 mg/dL
Glucose should be regulated by insulin in under 2hrs after eating
OGTT Testing Procedure:
Patient Preparation: fasting (8 hours average)
In the laboratory, blood specimen is drawn; labelled as “fasting blood
specimen”
The patient will be given a “glucose load” (50g, 75g, 100g glucose)
Pregnant patient: 100 g
Male patient/ Non-pregnant: 75 g
Pediatric: computed glucose load: 1.75 g glucose per kg
body weight
Consumption of glucose load is needed within 5 minutes.
After an hour, blood specimen will be drawn again, labelled as “one
hour specimen”
After another hour, blood specimen will be drawn again, labelled as
“two hour specimen”
After another hour, blood specimen will be drawn again, labelled as
“three hour specimen”
The patient must not consume any food in the process
➔ Plasma glucose level is tested.
➔ 4 blood specimens will be tested for plasma glucose.
Diagnostic Criteria for Gestational Diabetes Mellitus
1. 1-h plasma glucose ≥140 mg/dL during an OGTT + symptoms of
gestational diabetes (100 grams glucose load)
2. 3-h plasma glucose: (75 g in practice)
■ Fasting (8-14 hrs): >95 mg/dL
■ 1 hr: ≥180 mg/dL
■ 2 hrs: ≥155 mg/dL
■ 3 hrs: ≥140 mg/dL)
Laboratory Findings
Hyperglycemia
1. Increased glucose plasma and urine
○ Glucosuria, mellituria, glycosuria (presence of glucose in
urine)
○ It is not normal to have glucose in the urine since kidneys
should reabsorb the essential molecules.
○ Kidneys have “renal threshold”, which is the maximum
concentration that the kidneys can reabsorb. When the level
exceeds the threshold, it is excreted.
■ Renal threshold of glucose: up to 180mg/dL
■ >180mg/dL = positive glucose in the urine.
2. Increased urine specific gravity
○ Because of presence of glucose
3. Increased Serum and urine osmolality
○ Since glucose is one of the major contributors of plasma
osmolality
4. Ketones in serum and urine is present (ketonemia and ketonuria)
○ Commonly seen in type I DM patients
→ acetoacetate (commonly measured in lab),
B-Hydroxybutyrate and acetoin
5. Decreased blood and urine pH (acidosis)
○ Due to ketone bodies
○ Increase ketone, decrease pH
6. Electrolyte imbalance: This affects the plasma level.
○ Low sodium in the plasma: hyponatremia
○ Increased potassium: hyperkalemia
- Inside the rbc, most of the potassium is found. Acidosis
causes hydrogen ion concentration in the plasma. Rbc will
buffer the pH, potassium inside will leave the cells to allow
the hydrogen to enter the cell, and sodium ions will also enter
the cell.
Diseases involved in Hyperglycemia:
Macrovascular problems: affects large blood vessels
Microvascular problems:
● Nephropathy- one of the most common complications
● Retinopathy- affecting the eyes
 ● Neuropathy- affecting the Central nervous system
● Increased risk for heart diseases
○ Patient blood also becomes viscous
○ Poor wound healing
Hypoglycemia
➔ Defined as low plasma glucose level
➔ Signs and symptoms are observable in the CNS
Causes:
● Result of Imbalance between glucose utilization and production
● When the plasma glucose level is between 65-70mg/dL, glucagon
and other hyperglycemic agents are released
● Observable signs and symptoms occur when the plasma glucose
level is between 50-55mg/dL
● 2 Classifications based on symptoms:
○ Neurogenic: composed of tremors, palpitations, anxiety,
diaphoresis
■ Diaphoresis- a condition characterized by excessive
sweating.
○ Neuroglycopenic: consists of dizziness, tingling sensation,
blurred vision, confusion, and behavioral changes
Patient appears healthy:
● No coexisting disease
○ Drugs
○ Insulinoma (tumor in the beta cell), Islet
hyperplasia/Nesidioblastosis (abnormal condition in the
islet cells)
○ Factitial hypoglycemia from insulin/sulfonylurea
○ Severe exercise, Ketotic hypoglycemia
● Compensated coexistent
○ Drugs/disease
Patient appear ill:
● Drugs
● Predisposing illness
● Hospitalized patient
Laboratory Findings:
➔ Decrease glucose in plasma
➔ Increased in patients with pancreatic βcells tumors (insulinoma) –
insulin levels are high; tumors promote over secretion of insulin
➔ Hyperinsulinemia because of tumor in βcells in the pancreas
Inborn errors of carbohydrate metabolism:
➔ Inherited conditions that manifested to the newborns/ infants
➔ The conditions under these are all tested in the newborns. This is a
type of inherited problem.
➔ Absence of essential enzymes that would promote carbohydrate
metabolism.
Galactosemia:
➔ It is a congenital deficiency of one of the three enzymes involved in
galactose metabolism
◆ galactose-1-phosphate uridyl transferase - the most
common cause of galactosemia
◆ Galactokinase (GALK)
◆ Uridine diphosphate galactose-4-epimerase (GALE)
➔ Laboratory feature: elevated blood and urine galactose
➔ Clinical features: jaundice, hepatomegaly, easy bruisability,
galactosuria, E.coli, sepsis, cataract, hypotonia, and sensorineural
deafness
➔ Diagnostic tests: erythrocyte galactose-1-phosphate uridyl
transferase activity
Essential fructosuria
➔ An autosomal recessive disorder characterized by fructokinase
deficiency.
➔ Fructose is present in the urine.
➔ Fructose catalyses the conversion of fructose to
glucose-1-phosphate
➔ Diagnostic indicator: the presence of fructose in urine.
Hereditary fructose intolerance
➔ A defect of fructose 1-6-bisphosphate aldolase B activity in the
liver, kidneys and intestine
 ➔ Inability to convert fructose-1-phosphate and fructose
IIIa Cori De Brancher (liver and Hepatomegaly, muscle
1-6-bisphosphate into dihydroxyacetone phosphate, Forbes muscle) weakness, retarded
glyceraldehydes-3-phosphate and glyceraldehydes. growth,
➔ Clinical features: irritability, lethargy, seizures, and hepatomegaly cardiomyopathy

Fructose -1-6-bisphosphate deficiency IIIb De Brancher (liver) Same as IIIa except

muscle weakness
➔ A defect in fructose-1-6-bisphosphate results in failure of hepatic
glucose generation by gluconeogenic precursors such as lactate IV Andersen Brancher Cirrhosis, esophageal
and glycerol varices, ascites
➔ Clinical features: hypoglycemia, lactic acidosis, convulsions and
coma V Mc Andle Muscle Myoglobinuria, muscle
Phosphorylase cramps
Glycogen storage Disease (GSD)
VI Hers Liver Phosphorylase Hepatomegaly,
➔ Deficiency of a specific enzyme involved in the metabolism of hypoglycemia
glycogen.
- Causing liver and muscle damage/defects VII Tarui Phosphofructokinase Pain and stiffness on
➔ It is an inherited autosomal recessive trait. exertion
➔ Serial blood specimens are collected
VIII Adenyl kinase Urinary excretion of
➔ Von-girke disease is the most common type of GSD
cathecolamines
Liver damage - types 1,2,3,4,6,9,0
Muscular defect - types 5 & 7 IXa Phosphorylase kinase Hepatomegaly,
➔ Other GSD’s: deficiencies of LDH, Pyruvate Kinase, phosphoglycerate (liver) hypoglycemia, delay in
kinase and mutase motor development
➔ Intravenous galactose tolerance test - test for type 1 GSD (low
IXb Phosphorylase (liver Hepatomegaly, retarded
glucose levels) and muscle) growth, muscle
hypotonia
Diagnostic Indicator:
X Cyclic Hepatomegaly
Types of Synonym Enzyme Deficiency Clinical Features AMP-dependent
GSD s (Tissue Affected)
XI Faconi- Glucose transporter-2 Hepatomegaly, rickets
Ia Von Glucose-6-Phosphata Hepatomegaly,retarded Bickel
Gierke se growth, seizures

Ib Glucose-6-phosphate Same as Ib; recurrent 0 Glycogen synthase No Hepatomegaly;

translocase bacterial infections hypoglycemic
symptoms in morning;
II Pompe 1,4, Glucosidase Cardiomegaly, infantile mild growth delay
death
CSF GLUCOSE
➔ It is about 40-60% of the blood plasma glucose level
- The level of glucose in the plasma would directly affect the
glucose level of the CSF
➔ Any changes in blood sugar are reflected in the CSF glucose
equilibrium time
➔ For comparison, a blood glucose specimen should be collected
before the lumbar puncture. There is a lag period in the equilibrium.
- ↑ plasma glucose, ↑ CSF glucose
➔ A markedly decreased CSF glucose (<40 mg/dL) & increased WBC
count (neutrophils) can be associated with the infections in the
CNS. used for diff count for meningitis.
↑ wbc, ↓ csf glucose = infection meningitis
➔ Increased level: diabetes seen after an hour
➔ Decreased level: bacterial meningitis, tuberculosis, fungal and
amebic meningitis; subarachnoid hemorrhage, systemic
hypoglycemia
- Organisms responsible consumes the glucose “microbial
consumption of glucose”
Reference values: 40-70 mg/dL (adult)
60-80 mg/dL (child)
Normal CSF to glucose ratio: <0.5/less than 0.5
- CSF glucose can also be used for differential diagnosis of meningitis
- Patient with bacterial, tubercular, and amebic meningitis:
CSF glucose is decreased
- Viral meningitis: CSF glucose is unchanged.
- Because viral agents do not consume glucose.
Tests that involve glucose:
C-peptide test
➔ Formed during the conversion of proinsulin to insulin
- Proinsulin is the precursor of insulin in the Beta cell
- Whenever insulin is synthesized, the proinsulin is converted
to insulin and secreted by the beta cells, C-peptides are
formed.
➔ The amount of circulating C-peptide provides reliable indicators for
pancreatic and insulin secretion
- C-peptide is used as an indicator of the function of the
beta-cell.
➔ Can be used to monitor individual responses to pancreatic surgery.
➔ This test mainly evaluates hypoglycemia and continues assessment
of Beta cells
- Cause of hypoglycemia may be evaluated by the C-peptide
test.
➔ Specimen: Fasting blood
➔ Method of testing: immunometric assay
◆ Antibody reagent is used to measure C-peptide level
● C-peptide serves as the target antigen
● Reagent: Anti C-peptide
- Increase: insulinoma, type 2 DM, ingestion of hypoglycemia
drugs
- Decreased type 1 DM (Absolute insulin deficiency)
➔ Reference value: 0.90-4.3 ng/ml (Correction Factor: 0.333)
➔ Normal ratio of C-peptide: insulin = 5.1 to 15.1
➔ D-xylose absorption test - is a test to measure the level of D-xylose
in the blood and in the urine sample. (Dextrorotatory xylose: sugar)
- Test is used to determine possible pancreatic insuficiencies
or malabsorption syndrome.
- D-xylose is a sugar and is normally/ easily absorbed in the
small intestine.
- If D-xylose is given to a patient, if the D-xylose is high in the
plasma/blood/urine, there is absorption. If it is low, then
there is malabsorption syndrome in the small intestine.
Other methods in Diagnosing Glucose Metabolic Alterations:
DIAGNOSIS OF GLUCOSE METABOLIC ALTERATIONS
Specimen Considerations:
1. Whole blood glucose concentration is 11% lower than plasma
- The rbc in the whole blood can consume the glucose
present
2. Serum or plasma must be refrigerated and separated from the cell
within 1 hour to prevent cellular consumption of glucose.
- To prevent false decrease of measurement
 3. Sodium fluoride (gray top) can be used to inhibit glycolytic enzymes
- Gray top has anti-glycolytic activities that inhibits enzymes
4. FBG should be obtained in the morning after 6 to 8 hours fasting (10
hrs is allowed but not longer than 16)
- > 16 hours: over-fasting = hypoglycemia
- Average is 8 hours
- Up to 10 hours if glucose testing has other fasting test required
- Lipid profile: Triglyceride (10hr fasting is required)
- Chylomicrons: gone in the circulation in the 10th hour.
- These can falsely elevate lipid profile tests that is
why longer than 9hrs fasting is required.
Glucose (FBG) only: 8 hours average
Glucose and Lipid profile test: 10 hours required
Lipid profile test only: 12 hours required.
Methods:
FBS- Fasting Blood Sugar (FBS/Fasting Plasma Glucose)
Non enzymatic methods of glucose measurement: Chemical Method
➢ Nelson somogyi - copper reduction method. Uses copper
- Uses BaSO4 to remove saccharoids (interfering agents)
- Glucose is a reducing agent
○ Glucose + arsenomolybdic acid = arsenomolybdenum blue
- arsenomolybdenum blue is a colored product measured
colorimetrically/spectrophotometrically
- The intensity of the blue product is directly proportional to the
absorbance of glucose in the sample. (accdg to beer’s law)
↑ Intensity of color ↑ Absorbance
➢ Hagedorn Jensen- Ferric reduction method
○ Inverse colorimetry method is used.
○ Glucose and ferricyanide (yellow) becomes ferrocyanide
(colorless)
- Ferricyanide- colored reagent, when added to glucose (reducing
agent), becomes ferrocyanide (a colorless product)
↑ ferricyanide converted to ferrocyanide ↓ absorbance
➢ Ortho-toluidine (dubowski)
○ Condensation of carbohydrates with aromatic amines
producing Schiff bases (green)
↑ Intensity of color ↑ Absorbance
Enzymatic methods:
- More specific than chemical method
1. Glucose oxidase (saifer gernstenfield)
○ B-D-glucose + O2 + H2O - glucose oxidase → gluconic acid
+ H2O2
○ H2O2 + reduce chromogen - peroxidase → oxidized
chromogen + H2O
○ Couple reaction is known as Trinder’s reaction
- False low results due to ↑ uric acid, bilirubin (icteric) &
ascorbic acid (vitamin C presence)
- These agents can interfere with glucose oxidase
tests. Must be avoided.
- Oxidized chromogen has a colored characteristic
measured colorimetrically
○ O2 consumption electrode (polarographic glucose analyzer)
can also measure oxygen depletion
↓ oxygen level ↑ hydrogen peroxide
Negative interference: effect on result is LOW
Positive interference: effect on result is HIGH
2. Hexokinase (reference method for glucose measurement)
- “Hexo” means 6
- “kinase” transference enzyme that transfers phosphate
group from one substrate to another
- One phosphate group from ATP is transferred to the 6th
carbon of glucose
Glucose + ATP - hexokinase → glucose 6-PO4 + ADP
Glucose 6-PO4 + NADP+ -G-6-PD → NADPH + H+ + 6-phosphogluconate
- NADP+ serves as a coenzyme - a second substrate (specifically for
oxidoreductase enzyme)
- -dehydrogenase enzymes are called oxidoreductase
○ A transfer of phosphate groups.
 ○ Measures for the absorbance the change in the absorbance
of the second enzyme (NAD), which produces a colorless
product (either oxidized or reduced)
○ Increased absorbance is measured at 340 nm
■ Ultraviolet region - no visible color
○ False low results due to gross hemolysis and highly
increased bilirubin (hyperbilirubinemia)
➔ When the product is NAD/NADP, decreased absorbance is measured
at 340 nm
➔ When the product is NADH/NADPH, increased absorbance is
measured at 340 nm
3. Clinitest
○ Reducing substance + Cu+2 → Cu+1O
○ Used in urinalysis
○ Cupric is reduced to cuprous
POC- Point of Care Testing
- Utilizes a glucometer- a handheld device used to monitor plasma
glucose level
- Only useful for monitoring; but for quality control, large
machines in lab is more superior than a glucometer
- In the laboratory, CBG (Capillary Blood Glucose) test requires the
use of a glucometer. CBG aka HGT (hemo glucose test)
➢ Type 1 diabetes - 3 to 4 times/day to monitor blood sugar
➢ With a glucometer, you can measure a random blood glucose level
of a patient
2-hr postprandial sugar
OGTT- Oral Glucose Tolerance Test
➢ The patient fasting blood glucose is taken glucose load is
administered blood glucose is determined in 30 min, 1st, 2nd, and
3rd hour. (30 min is removed now)
➢ A screening test for GDM
- Only administered among ambulatory patient (walk-in patient)
- Because glucose is depleted on bedrest
Prior to OGT testing, patients must have an unrestricted diet of 150g of
carbohydrates per day for 3 days.
- done to stabilize the production of inducible glycolytic
enzymes.
- Should not smoke or drink alcoholic beverages prior to testing.
➢ 2 types of Glucose Tolerance Test:
based on administration of glucose load:
1. OGTT- Oral Glucose Tolerance Test:
○ Janney-isaacson method - also known as single dose method. Most
common type of OGTT
○ Exton rose method - also known as double dose method /divided
oral dose method
2. Intravenous Glucose Tolerance Test (IVGTT)- administered
intravenously; used for diabetics with gastrointestinal tract disorder.
- If the patient has GIT disorder and oral glucose
administration is not accurate, IVGTT is performed.
- 0.5 g of glucose per kg bodyweight of the patient is given
(administered) to the patient within 3 minutes
- Fasting is also required.
➢ First blood withdrawal is done 5 minutes after administration
intravenously
HbA1C- Glycated Hemoglobin A1C
➢ reliable method for monitoring long term DM
➢ Index for long term plasma glucose control (2-3 month period),
indicating compliance and efficacy of DM therapy
↑ HbA1C = indicates either patient not complying with the
scheduled therapies/ medications. Or medication is no longer
effective in controlling plasma glucose level
➢ The largest subtraction of normal HgA in both diabetic and
non-diabetic individuals.
➢ Formed by the attachment of glucose to hemoglobin to form a
ketoamine
- Dietary status of the patient will not affect the HbA1C level
➢ Specimen requirement is EDTA Whole Blood sample.
➢ Normal value is 4.5 to 8.0%
Methods of HbA1C measurement: based on structural differences
- Based on charged differences between glycosylated and
nonglycosylated hemoglobin
- Structural characteristics of glycogroups on hemoglobin
○ Immunoassays - polyclonal or monoclonal antibodies
toward the glycated N-terminal group of the beta-chain of
hemoglobin. Antibodies serve as reagent.
○ Affinity chromatography - separated based on chemical
structure using boronate group to bind glycosylated proteins
○ Cation-Exchange Chromatography- Positive-charge resin
bed attaches to negatively charged hemoglobin
○ Electrophoresis- Separation is based on differences in
charge.
Increased positive charge, further migration
○ Isoelectric focusing- Type of electrophoresis using
isoelectric point to separate different fractions of Hgb
○ HPLC (high performance liquid chromatography)-
■ A form of ion-exchange chromatography
■ Separates all forms of HbA1C (A1a, A2b, A2c)
Fructosamine Test
➢ Also known as glycated albumin / glycated protein
➢ A test for diabetes for that measures the average blood glucose
level over 2-3 weeks period
○ Used for short-term plasma glucose control
➢ Performed if patient has hemolytic disorders (hemolytic anemia)
○ Hb1Ac is not practical for patients with hemolytic anemia
Ketone
➢ Produced from catabolism of lipids
➢ Produced by the liver through metabolism of stored lipids
➢ 3 ketone bodies formed:
○ Acetone (2%)
○ Acetoacetic acid (20%) commonly measured in CC lab
○ 3-Beta-hydroxybutyric acid (78%)
The ketone body level in the plasma reflects the lipolytic activity in the patient
↑ Lypolysis ↑ Ketone Bodies
Ketonemia- accumulation of ketones in blood
Ketonuria- accumulation of ketones in urine
➢ Methods of ketone measurement:
○ Gerhardt’s test - specific only with acetoacetate
Acetoacetic acid + ferric chloride = red color
○ Nitroprusside test
- 10x more sensitive to acetoacetate than to acetone
Acetoacetic acid + nitroprusside -alkaline pH→ purple color
○ Enzymatic:
NADH + H + acetoacetic acid
beta-HBD → NAD + Beta hydroxybutyric acid
Microalbuminuria
➢ Presence of albumin in the urine
○ Present even before proteinuria develops
➢ Diagnosis at an early stage diabetic renal nephropathy and before
the development of proteinuria
➢ Persistent albuminuria in the range of 30-299 mg/24 hour or albumin
creatinine ratio of 30-300 micrograms/mg
○ Micral test- test performed to detect and measure the
microalbumin present in the urine sample
○ Specimen used is urine
➢ Diabetic nephropathy is the most common type
December 7, 2021 (quiz next week wednesday) MAJOR CLASSIFICATION OF LIPIDS/FATS:
Clin Chemistry Lecture 1. FATTY ACIDS
LIPIDS AND PROTEINS ➢ Composed of linear chains of C-H bonds that terminates with
Scope: -COOH (carboxyl group)
1. Lipid Chemistry ➢ Mostly found as constituents of phospholipids or triglycerides
2. Lipoprotein Structure - Mainly derived from the hydrolysis of triglycerides in the
3. Lipoprotein Physiology and Metabolism adipose tissue
4. Lipid Disorders ➢ Important source of energy/fuel for the cell.
5. Lipid and Lipoprotein Analyses ➢ Provides substance for the conversion of glucose in the process of
gluconeogenesis.
LIPID CHEMISTRY ➢ Classifications of Fatty Acids:
INTRODUCTION 1. Based on presence of ester bonds:
Lipids: Commonly referred to as fats ■ Unesterified → bound to albumin in the plasma
- Composed of mostly carbon-hydrogen (C-H) bonds (attached in the albumin)
➜ Primary source of fuel of the cell (just like the carbohydrates) ■ Esterified → constituent/part of the triglycerides or
➜ Provides stability to cell membrane and allow for transmembrane phospholipids (part of the TAG/phospholipid
transport structure)
- Cell membrane is chiefly made of lipid bilayers 2. Based on linked or number of fatty acid structure:
➜ Insoluble in blood or plasma but soluble in organic solvents (such ■ Structure: short: 4-6
as ether) ■ medium: 8-12
- Blood is mainly composed of water, lipids/fats are insoluble ■ long chain: >12
in water therefore also insoluble in blood/plasma 3. Based on the number of double bonds:
➜ Major Classifications of Lipids: ■ saturated: no double bonds
○ Fatty Acids - Butyric acid
○ Triglycerides ■ monounsaturated: one double bond
○ Cholesterol - Oleic acid
○ Phospholipids ■ polyunsaturated: 2 or more double bonds
○ Fat-soluble vitamins (Vitamin A, D, E, K) - Linoleic Acid
Glycerol (Triglyceride – sat)
- Since lipids are insoluble in the blood, it requires special transport - Triglycerides: composed of 1 glycerol
mechanisms, with the aid of lipoproteins. structure and 3 fatty acids
➜ Transported by lipoproteins: (VLDL, LDL, HDL) - Where fatty acids are mainly derived
■ Special transport mechanisms Other examples of fatty acids:
■ Needed to transfer lipids to the liver, cells, tissues Palmitic Acid
Stearic Acid
Arachidonic Acid
 2. TRIGLYCERIDE
- Also known as TRIACYLGLYCEROL or NEUTRAL FAT
- Composed of 3 molecules of fatty acids attached to 1 molecule of
glycerol
- Do not contain charged/hydrophilic group
➢ Contains saturated fatty acids or unsaturated fatty acids
➢ Very hydrophobic and Water Insoluble, Neutral lipid
➢ Main storage form of lipid in man; chiefly found in adipose tissues
➢ Constitutes 95% of the stored fat in the body is in this form
➢ In the plasma, the predominant form is GLYCERYL ESTER
Adipose tissue: Triglyceride
Plasma: Glyceryl Ester
➢ When triglyceride is broken down/ catabolized, fatty acids are
released to cells and converted into energy
➢ It is able to provide insulation (excellent insulator)
Facilitators of the breakdown of triglycerides:
1. Enzyme LPL: Lipoprotein Lipase
2. Hormone Epinephrine
3. Hormone Cortisol
3. PHOSPHOLIPIDS
- Also known as the CONJUGATED LIPIDS
- In the body, phospholipids are the most abundant lipids derived from
phosphatidic acid
➢ Contains:
Molecular tail: two fatty acids attached to one molecule of glycerol
Molecular head: On the third carbon position contains a
phospholipid head group (phosphorylated group is present)
➢ Phospholipids are Amphipathic: contain hydrophilic and
hydrophobic head groups
○ Hydrophilic is present on the outer part (directly exposed to
plasma)- makes phospholipids soluble in water POLAR
○ Hydrophobic is present on the inner part- water insoluble
portion NON-POLAR
➢ Most abundant lipid in the body
➢ Derived from phosphatidic acid
➢ Originates in the liver and intestines
- Called conjugated because it is a conjugation of two fatty acids and
a phosphorylated glycerol
➢ Not part of the Lipid Profile; not routinely measured in CC section
- The measurement would only provide little information in
the diagnosis/cases of abnormal lipid metabolism
➢ This is measured in Fetal Lung Maturity (serology and aubf)
○ During pregnancy, amniotic fluid is collected and tested for
phospholipids (spec. Sphingomyelin and lecithin ratio)
- To determine the fetal lung maturity of developing
fetus (on the third trimester)
○ Phospholipids can act as surfactants (could alter the fluid
surface tension (Surfactants: Sphingomyelin and lecithin
ratio)
➢ Also participates in cell metabolism and blood coagulation
Three forms of Phospholipids:
1. Lecithin or Phosphatidylcholine- Major form of
phospholipid (70%)
2. Sphingomyelin- Constitutes about 20% in plasma
3. Cephalin- Constitutes about 10% of phospholipids in
the plasma
4. Cholesterol
- Also known as 3-HYDROXY-5, 6 CHOLESTENE
● Unsaturated steroid alcohol contains four rings, component of
steroids
● Has a single carbon hydrogen sidechain tail (similar to fatty acid)
● Amphipathic: contain hydrophilic and hydrophobic head groups
● Found on the surface of lipid layers of the cell membrane
● An important constituent in the assembly of cell membrane and bile
acid. (Precursor to form the structures of the cell membrane and bile
salt/acids )
○ Bile acid contributes to the metabolism or digestion of fats
in the intestine
● Cholesterol is not catabolized by most cells
○ Does not serve as fuel or energy of the cells
○ The unmetabolized cholesterol in the cell, it is either
released in the blood or transported back to the liver
● Classifications of Cholesterol:
■ Unesterified → free cholesterol (amphipathic)
– 30% of the total cholesterol in the body
– Present in the plasma, serum, and in RBCs
– Polar form of Cholesterol
■ Esterified → cholesterol ester (neutral lipid)
– 70% of the total cholesterol in the body
– Also present in the plasma or serum
– Has hydrophobic form
– In the formation, an important enzyme is needed
➢ LCAT: Lecithin-Cholesterol Acyl Transferase
– Important for the esterification of
cholesterol
– It catalyzes the esterification of cholesterol
by promoting the transfer of fatty acids
from lecithin to cholesterol
– This is synthesized in the liver
○ APO A-I is the activator
- Both are present in the plasma or serum
- In the Total Cholesterol test, both esterified and
unesterified are measured.
● Converted to:
■ Bile salts: promote fat absorption in the intestine
- Cholesterol is a precursor for the formation of bile
salts
- Bile salts serve as emulsifying agent (emulsifier)
- Bile salt enhances fat digestion
- Produced in the liver and temporarily stored in the
gallbladder.
- Removal of gallbladder will be needed to
limit the fat consumption because there is
no storage of bile salt anymore
■ Steroid hormones: glucocorticoids, mineralocorticoid,
estrogen
■ Vitamin D and cell membrane synthesis
– Vit. D: important in absorption of calcium in the
intestine.
– Cholesterol is needed for the formation of lipid
bilayer
● Both unesterified and esterified is measured (total cholesterol in the
lipid profile test)
● Lipid profile test:
■ TRIGLYCERIDES (TAG): directly measured w reagent
■ LIPOPROTEINS (HDL, LDL, VLDL)
– HDL measured directly w reagent & machine
– LDL AND VLDL are not directly measured with
reagent, these are calculated/computed, but
separated in costs
■ TOTAL CHOLESTEROL: directly measured w reagent
● CHOLESTEROL: routinely measured
■ Cholesterol value is essential or important in the diagnosis
of lipoprotein diseases and management of lipoprotein
disorders and lipid abnormalities.
CHEMICAL STRUCTURE OF LIPIDS (amphipathic)
LIPOPROTEIN STRUCTURE
● Components:
○ composed of both lipids and proteins (apolipoproteins)
● Composition:
- The LIPID PORTION- inner and outer lipid
composed of amphipathic type
- Outer lipid (HYDROPHILIC)
- Free cholesterol, phospholipids are found on
the surface (hydrophilic)
- Inner lipid (HYDROPHILIC)
- Triglycerides and cholesteryl esters are found in the
core regions (hydrophobic)
- The PROTEIN PORTION- called apo portion/ apolipoprotein
 ● Functions of Apolipoproteins (protein part):
Apo E VLDL, HDL LDL receptor ligand
○ Maintain structural integrity (Highly stable)
- Protein is stable. WIth the stability of the protein Apo(a) Lp(a) Plasminogen inhibitor
portion of the lipoprotein, the overall structure is
maintained.
● MAJOR TYPES
○ Ligands for cell receptor
○ Chylomicrons- Biggest, Lightest: least dense
○ Activators and inhibitors of enzymes
○ VLDL - Very Low Density Lipoprotein
○ Amphipathic characteristic
○ LDL - Low Density Lipoprotein
○ HDL- Smallest; Heaviest: most dense
- Density is based on the protein constituents/components
○ More protein present= Heavier and more dense

CHARACTERISTICS OF THE MAJOR HUMAN LIPOPROTEINS

Characteristics Chylomicrons VLDL LDL HDL

Density (g/mL) <0.93 0.93- 1.019- 1.063- 1.21

1.006 1.063

Diameter (nm) 80-1,200 30-80 18-30 5-12

CHARACTERISTIC OF THE MAJOR HUMAN APOLIPOPROTEIN
Total lipid (% by 98 89-96 77 50
Apolipoprotein Major LPP Location Function weight)

Apo A-I HDL LCAT activator, ABCA1 Triglyceride (% by 84 44-60 11 3

lipid acceptor weight

Apo A-II HDL Inactivates LCAT Total cholesterol (% 7 16-22 62 19

by weight)
Apo A-IV Chylomicrons, VLDL, HDL
Major Protein Apo B-48 Apo Apo-B100 Apo A-1
Apo B-100 Chylomicrons, VLDL, HDL LDL receptor ligand B-100

Apo B-48 Chylomicrons Remnant receptor

ligand ● Density is directly correlated with the protein constituent
○ Protein Constituent related to Total Lipid
Apo C-I Chylomicrons, VLDL, HDL ○ Eg. In Chylomicrons, there is 98% total lipid, therefore only
2% is protein
Apo C-II Chylomicrons, VLDL, HDL LPL cofactor
● Lipoproteins = LIPID + PROTEIN
Apo C-III Chylomicrons, VLDL, HDL LPL inhibitor
Clin Chemistry Lecture
MAJOR TYPES OF LIPOPROTEINS
Chylomicrons
➔ Largest and least dense
➔ Produced in the intestine
➔ Delivery of dietary lipids to hepatic and peripheral tissues
- When we consume fatty meals, chylomicrons increases in
the plasma
- Increased chylomicrons causes lipemic plasma; responsible
for fatty appearance of sample
- Released in the peripheral circulation as transporter
- Chylo is completely cleared within 6-9 hours after eating
- No more chylomicrons present which results in no false
results. If chylomicrons are still present, it can falsely
increase the lipid profile.
- Exogenous triglyceride- from the diet
➔ Major transporter of exogenous triglycerides
◆ Delivery of dietary lipids to hepatic and peripheral cells
➔ Completely cleared from the plasma or circulation within to 6-9
hours post-prandial
◆ That is why strict 12 hours fasting is required for lipid profile
testing
◆ False ↑ lipid profile, if chylos is still present in the blood
◆ Lipid profile is done to determine normal metabolism
➔ Lipid and Glucose Tests: 10 hours fasting
VLDL- very low density lipoprotein
➔ Aka pre-beta lipoprotein
➔ Produced in the liver
➔ When serum electrophoresis is performed, VLDL is found in the
pre-beta region in the electrophoresis
➔ Transfers triglycerides from liver to peripheral tissues
◆ Endogenous triglycerides- triglycerides formed in the liver
◆ If triglycerides come from the food we eat, it is called
exogenous triglycerides.
LDL- Low density lipoprotein
➔ Cholesterol-rich
➔ Also known as beta lipoprotein or bad cholesterol
➔ High LDL in plasma = higher risk to coronary heart disease/stroke
- Found in the beta region when serum is tested in
electrophoresis
- Considered as Bad cholesterol because it the most
cholesterol-rich lipoprotein
- Most atherogenic type of lipoprotein
● Contributes to the formation of Fatty plaque in the
arteries or blood vessels that can obstruct blood
flow.
➔ Formed from lipolysis of VLDL to IDL then to LDL
➔ Transfer dietary cholesterol to peripheral
➔ Product of Lipolysis of VLDL
◆ intermediate density lipoprotein (IDL) – further catabolized
to LDL
HDL- High Density Lipoprotein
- Alpha lipoprotein/ good cholesterol
- Present in the alpha region when serum is tested in electrophoresis
- Responsible for the transfer of excess cholesterol from cells to the
liver
- To recycle the cholesterol
- Clears the plasma of circulation of excess cholesterol (tagalinis ng
excess cholesterol)
- Decreased HDL = Increased risk of developing heart disease
- Nascent disc-shaped, discoidal shape
- Produced in the liver and known as Cardioprotective lipoprotein
- Excess cholesterol is given back to the liver
- reverse cholesterol transport mechanism
- If HDL is high, the risk for coronary heart disease is low.
- If LDL is high, the risk for CHD is high.
MINOR TYPES OF LIPOPROTEINS
Lipoprotein (a)
- LDL lipoprotein-like particle
- Increased Lipoprotein A increases risk for premature coronary heart
diseases and stroke
- Competed with plasminogen for fibrin
 ➔ In Hematology, there is fibrinolysis, the clot formed in the blood will Beta-VLDL (floating Beta lipoprotein)- minor and abnormal
prevent further blood loss called coagulation. When bleeding is ➔ Its migration mobility is similar to LDL however its density is the
controlled because of clotting, there is a healing process- repair same as VLDL
process. Once the damaged blood vessel is repaired, the clot should ➔ Found in the type 3 hyperlipoproteinemia or dysbetalipoproteinemia
be removed. ➔ Rich in cholesterol content than VLDL due to defective catabolism of
➔ To remove the clot, fibrinolysis follows once the clot is no longer VLDL, there is failure to convert VLDL to LDL causing IDL to also
needed. Plasminogen promotes fibrinolysis. accumulate.
◆ In the presence of LpA will inhibit/ prevent plasminogen
from lysing fibrin. And once inhibited by LpA, the fibrin clot Adult reference ranges for lipids:
will not be dissolved and will stay as is.
Analyte Reference Range Conversion Factor
◆ Fibrin clots will accumulate and will add up in the fatty (mmol/L)
plaque already found in the blood vessel -> premature
coronary heart disease manifestation will occur earlier. Total Cholesterol 140-200 mg/dL 0.026
➔ It is not normal to have clots in the blood vessels.
Characteristics: HDL Cholesterol 40-75 mg/dL
● Variable migrations LDL Cholesterol 50-130 mg/dL
○ LpA is found in the pre-beta region or sometimes
between LDL and albumin region. Triglyceride 60-150 mg/dL 0.011
● Also known as sinking pre-beta lipoprotein - Reference range may vary from one reference book to another
○ Called sinking because of its electrophoretic
mobility is same with VLDL, and density is similar to LIPOPROTEIN STRUCTURE:
LDL (kasing bigat) Lipoprotein Metabolism:
A. Lipid Absorption- happens in small intestine
IDL- Intermediate Density Lipoprotein ○ Conversion of dietary lipid into a more polar (amphipathic)
- When catabolized, it will first form VLDL-> IDL -> LDL subclass compound by the pancreatic lipase (enzyme)
- IDL is a subclass of LDL i. The triglycerides converted into monoglycerides
- Migration is seen in either pre-beta or beta region ii. Cholesterol esters become free cholesterol
iii. Phospholipids become lysophospholipids
○ All of this is due to the pancreatic lipase.
Lipoprotein X ○ The bile (produced in the liver but stored in the gallbladder)
- Abnormal lipoprotein can facilitate the emulsification of fats to allow better
- It is an abnormal lipoprotein found in obstructive jaundice and LCAT digestion, conversion and absorption.
deficiency ○ If fats are not absorbed, it will be defecated. There might be
- Specific and sensitive indicator of cholestasis. a possible problem in the lipid absorption or problem in the
- The lipid content is mostly phospholipids and free cholesterol intestine. This is called “phosphorea”
(unesterified) (90%) Involved lipoprotein: pancreatic lipase
- It contains Apo C and albumin
B. Exogenous Pathway
○ The ones involved in this are the chylomicrons. Arteriosclerosis
○ Chylomicrons are synthesized in the intestine, carrying
dietary lipids to the circulation
○ Lipoprotein lipase (LPL) hydrolyzes Triglyceride carried by
chylomicrons into fatty acid and glycerol or re-esterified for
long term storage in the hepatic cells (Re-esterification in
the liver)
○ Chylomicrons will be converted by LPL and turn into
chylomicron remnant particles.
Involved lipoprotein: LPL
Involved lipids: chylomicrons
C. Endogenous Pathway (within the body)
○ Triglycerides will now be synthesized in the liver and
packaged by VLDL
○ VLDL will transport triglyceride to the circulation
○ The TGL in the liver are packaged into VLDL, carrying lipids
to the circulation towards the different tissues
○ VLDL is converted into VLDL remnants by action of LDL and
taken up by the liver
○ Half of VLDL is transformed into LDL for delivery of
exogenous cholesterol to peripheral cells; half is taken up by Hyperlipoproteinemia
the liver (VLDL remnants)
- Involved lipoprotein: VLDL and LDL
- Involved lipids: endogenous triglycerides
D. Reverse Cholesterol Transport Pathway
○ HDL removes excess cholesterol transport pathway deliver
cholesterol to the liver
○ Cells do not use cholesterol as a source of energy.
Unconsumed cholesterol must be recycled and brought
back to the liver.
○ HDL is in charge of transporting excess cholesterol. HDL
can also be obtained from the diet:
i. Fish oil (omega-3)
○ Increased HDL, decreased risk for coronary disease
- Involved lipoprotein: HDL
- Involved lipids: cholesterol
LIPID DISORDERS:
➔ Deposition of esterified cholesterol in artery walls. If esterified
cholesterols are high, LDL is high
- Esterified cholesterol- hydrophobic type of cholesterol
- 70% is found in the circulation = deposited in the artery walls
- Indication of increased esterified cholesterol in the blood: increased
LDL (bad cholesterol)
➔ Increased smooth muscle cells, extracellular lipid, calcification,
fibrous tissues, macrophages, lymphocytes and platelets (plague)
- Fibrin clots that are not dissolved
➔ Rupture or erosion can cause thrombosis that block circulation that
could trigger diff diseases
Diseases that may manifest: CAD, PVD, CVD
- CAD - coronary artery disease. End result is angina and MI
- PVD - peripheral vascular disease. End result is arteries in
arms or legs
- CVD - cerebrovascular disease. End result is vessels of the
brain (stroke)
➔ Hypercholesterolemia
◆ Increased cholesterol
◆ Increased LDL. decreased receptors
➔ Hypertriglyceridemia
◆ Increased TGL
◆ Decreased LPL or Apo C-2
◆ VLDL becomes VLDL remnants
◆ Chylomicrons becomes chylomicron remnants
➔ Combined hyperlipidemia
◆ Increased TGL, cholesterol
◆ Increased VLDL and chylos remnants
◆ Presence of apo E2/2

Other lipid disorders:
1. Familial hypercholesterolemia (type 2a)
2. Familial dysbetalipoproteinemia (type 3
hyperlipoproteinemia)
3. Abetalipoproteinemia
4. Hypobetalipoproteinemia
5. niemann-Pick Disease
6. Tangier’s disease
7. Lipoprotein lipase deficiency
8. LCAT deficiency
9. Tay-sachs disease
LIPID AND LIPOPROTEIN ANALYSIS: (laboratory part)
- Plasma or serum can either be used
Cholesterol measurement
- There must be 2 weeks of preparation for the patient. The patient
must be on his usual diet.
- To assess the lipid profile much better.
- Fasting for patients with cholesterol is not strictly implemented.
- We can test the serum or plasma of non- fasting patients.
- Fasting has a little effect on the total cholesterol level
- In lipid profile test, there are other parameters tested so
fasting is required. But for cholesterol measurement, fasting
is not required.
- Fasting has little effect on the total cholesterol level in the blood.
Therefore fasting may be assayed.
- The presence of double-bond in the sterol group creates the
color in the coloremetric assay.
- The total cholesterol is measured
Precautions for cholesterol measurement:
- Hemolyzed blood must be avoided. Because the free Hgb
can falsely increase the cholesterol level
- Icteric specimens must be avoided.
- Avoid water contamination of the plasma/serum. Because
in cholesterol measurement, there is a dehydration process.
Presence of excess water can cause error in cholesterol
assay.
- Precise and accurate timing for the color development.
- Incubation period and reading
Components of Color developer mixture for cholesterol
measurement:
- Glacial acetic acid
- Acetic anhydride
- Concentrated sulfuric acid
NON-ENZYMATIC METHOD: (Abell Kendall)
Principle: dehydration and oxidation of cholesterol to form a colored
compound.
- Colored compounds are measured colorimetrically.
4 general methods for chemical testing:
1. One step method: only involves colorimetry (mix reagent and
sample, incubate, read)
2. Two step method: involves extraction and colorimetry
- Extraction of lipids/ target fats before the actual
measurement (colorimetry)
3. Three step method: Saponification with Extraction, and Colorimetry
4. Four step method: Precipitation + Saponification + Extraction +
Colorimetry
➔ Cholesteryl esters are hydrolyzed with alcoholic KOH
➔ Unesterified cholesterol is extracted with hexane
Measured using liebermann-burchard reaction ( color green result)
Cholesterol + sulfuric acid + acetic anhydride → Green solution called
(CholestadienylMonosulfonic Acid) (visible light region)
In Salkowski reaction = red solution
- Absorbance of the dye of the solution is directly
proportional to the concentration
ENZYMATIC METHOD: (cholesterol oxidase)
➔ I. Total cholesterol (cholesteryl ester and cholesterol) + H2O
-Cholesteryl esterase → Cholesterol + Fatty Acid
➔ ii. Cholesterol + O2 -Cholesterol oxidase → 4-cholestenone + H2O2
➔ iii. H2O2 + dye -peroxidase → Color (Quinoneimine dye)
- The result will be quinoneimine dye
 - Absorbance of the quinoneimine dye is directly proportional to the b. Glycerol - Phosphate Oxidase
concentration of the sample Glycerophosphate + O2 - GO -> Dihydroxyacetone + H2O2
H2O2 + Dye - Peroxidase -> Color
CENTER’S FOR DISEASE CONTROL of America (CDC)
➔ CDC reference method for cholesterol measurement is: Abell, Levey Lipoprotein measurement:
and Brodie method ➔ Preferred sample must be placed in an SST (serum separator tube)
- Hexane is used for extraction of cholesterol following hydrolysis routinely done in the hospital
using alcoholic potassium hydroxide. ➔ Sample of choice for lipoprotein studies is EDTA plasma (for
- Add the color development. End product: green solution research studies)
➔ Fasting is not necessarily required.
Triglyceride measurement - Non-fasting sample (lipemic sample): we can measure HDL
➔ Requires strict fasting of 12 hours. (for triglyceride only) and Total Cholesterol measurement .
◆ Triglyceride + FBS = 10 hours is accepted. - If fasting specimens, you can measure other lipoproteins as
➔ Fasting has a significant effect in the TGL level in the serum well.
➔ We can use plasma or serum for measurement ➔ Lipemic sample of patients who underwent fasting indicates
diseases
Non enzymatic method: ➔ Lipemic sample must be treated thru: to remove unnecessary lipids
◆ CDC reference method for Triglyceride measurement is the ◆ Enzymatic cleavage
modified van handel and zilversmith ◆ Ultracentrifugation
- In triglyceride measurement, we measure the glycerol content and
not the fatty acid. Methods in measuring lipoproteins:
- The blue/ yellow is measured through the absorbance ◆ General Methods:
➔ Triglyceride - Alcoholic KOH -> Glycerol + Fatty Acid Ultracentrifugation- reference method for lipoprotein quantitation
➔ Glycerol + Periodic Acid -> Formaldehyde ● Based on molecular density
● Uses specific type of solution: potassium bromide reagent
- Formaldehyde formed in the first reaction will now react to either with specific gravity of 1.063
Colorimetric or Fluorometric Method. ○ Plasma (centrifuged for 24hrs) with the reagent is
a. Van Handel and Zilversmith (Colorimetric Method) used in this procedure
Formaldehyde + Chromotropic Acid -> Blue Solution ● Order from lightest to heaviest: Chylomicrons -> VLDL -> LDL
b. Hantzch Condensation (Fluorometric Method) -> HDL
Formaldehyde + Diacetyl acetone + NH3 -> Yellow Solution Electrophoresis - based on the protein constituents
● Based on migration in an electric field
Enzymatic method: Glycerol Kinase ● Lipid stains (OIl red O, Fat Red 7B, and
➔ Triglyceride - Lipase -> Glycerol + Fatty Acid Sudan Black)
➔ Glycerol + ATP - Glycerokinase -> Glycerophosphate + ADP ● 4 bands with fat stain:
a. Pyruvate Kinase ○ Alpha- lipoprotein (HDL)-
ADP + PEP - Pyruvate Kinase -> ATP + Pyruvate fastest
Pyruvate + NADH + H + -LD -> Lactate + NAD+ (340nm)
 ● The most anodic is the HDL (alpha), followed by pre-beta
(VDL, and LpA), followed by beta (LDL), then chylomicrons
in the origin
- Supporting medium: AGAROSE medium (most sensitive supporting
medium for lipoprotein fraction determination)
◆ HDL method - High Density Lipoprotein Methods
● Polyanion precipitation- two step process for measurement
of HDL
● 1. Precipitation- removes non-HDL lipoprotein
○ Supernate will be used as a sample to measure the
HDL using the Abell-Kendall method. (a cholesterol
assay)
- Lipoproteins (LDL, VLDL) are precipitated with polyanions (e.g.
Heparin, dextran sulfate or Na phosphotungstate) in the presence of
divalent cations (e.g. Mg or Mn), which are sediment by
centrifugation (10-30mins for 10,000 g or 3 mins for 15,000g)
- HDL is quantified in the supernatant by Abell-Kendal
◆ LDL method
● Derived values
● LDL= Total Cholesterol - (HDL + VLDL)
Friedewald:
VLDL = Triglyceride /5 = mg/dL
VLDL = Triglyceride /2.175 = mmol/L
● Ultracentrifugation of serum at native density gradient of
1.006 g/L to float VLDL
Lipid profile test:
➢ Total cholesterol
➢ Triglyceride
➢ HDL
➢ LDL
➢ VLDL
- Fatty acids and phospholipids are not routinely measured in the
laboratory
◆ Apolipoprotein method- protein portion
➔ Measured to determine the level of Apo B - for det of LDL and VLDL
- Increased concentration of of Apo B,
increased concentration of LDL & VLDL
Apo A-1 - for det of HDL concentration
- Increased concentration of of Apo A-1,
increased concentration of HDL
Immunoassay - antibody is involved in the test
- Immunonephelometric assay
- Immunoturbidimetric assay
- Immunochemical assay
- Used for measure target apolipoprotein
Target= Apo A-1
Reagent used is: Anti Apo A-1
POSITIVE RISK FACTORS:
➢ Age: ≥45 years for men; ≥55 years or premature menopause for
women
➢ Family history of premature CHD
➢ Current cigarette smoking
➢ Hypertension: BP ≥140/90 mmHg or taking antihypertensive
medication
➢ LDL cholesterol concentration: ≥160 mg/dL, with ≤ 1 risk factor
➢ LDL cholesterol concentration: ≥130 mg/dL, with ≥ 2 risk factor
➢ LDL cholesterol concentration: ≥100 mg/dL, with CHD or risk
equivalent
➢ HDL cholesterol concentration: <40 mg/dL
➢ Diabetes mellitus = CHD risk equivalent
➢ Metabolic syndrome (multiple risk factor)
Negative Risk Factor:
➢ HDL cholesterol concentration ≥60 mg/dL (maintain high HDL thru
diet)
○ To recycle excess cholesterol
➢ LDL cholesterol <100 mg/dL (lower LDL)
○ To delay fatty plaque formation
December 14, 2021
Clin Chemistry Lecture
PROTEINS
CHARACTERISTICS
PROTEINS-these are macromolecules composed of polymers of covalently
linked amino acids.
Amino acid- building block of proteins
- The chemical properties of amino acids determine the
biologic activity of proteins
- Since amino acids are composed of proteins, the chemical
property of amino acids can directly influence the biologic
activity of proteins.
● Peptide bond makes the protein highly stable
● Considered as Amphoteric
○ Can bear both positive and negative charges
○ due to the presence of acid and basic type of amino
acid present in the structure
● Based on structure: Proteins contain the following atoms:
- CHONS (carbon, hydrogen, oxygen, nitrogen, sulfur)
○ nitrogen content of protein differentiates proteins
from other macromolecule such as carbohydrates
and lipids
○ Nitrogen level in the body is determined by protein
catabolism or anabolism
○ Proteins are the macromolecules that carry the
nitrogen in the blood
● Proteins have high molecular weight: making them a good
immunogen
○ Immunogen- agents/substance that could
trigger/stimulate the immune system to produce
immune response
○ Immune response can be in the form of antibody
production
■ ex. Vaccination: where protein is an integral
part;
■ In vaccination, protein makes the
vaccination immunogenic/makes the
circulation life longer.
■ Usual protein content: ALBUMIN
■ The aim of the vaccine is to mimic the
actual infection to trigger the immune
system to produce antibodies.
● Proteins are considered as good immunogens because of
high MW
● “Most” proteins are synthesized in the liver specifically in the
hepatocytes where it is secreted to the circulation
○ Most proteins are proteins are produced in the
hepatocyte
○ Low protein levels indicate infection, cirrhosis,
hepatitis; protein level is affected
○ IMMUNOGLOBULINS/ANTIBODIES: Group of
proteins that are extra-hepatically produced
(outside the liver);
■ Produced outside the liver: produced in the
plasma cells
■ Plasma cell: terminally cell-differentiated
b-lymphocytes
● Side notes: Liver is responsible for producing many plasma
constituents
Other important characteristics of Proteins:
1. Biochemical reactions are catalyzed by enzymes (protein)
a. Can speed up reaction (enzyme)
b. Enzyme is an example of protein
2. Structure of cells and the extracellular matrix that surrounds the
cells (e.g. collagen)
a. Serve as structural support (collagen)
3. Transport of materials (eg transferrin for transfer of iron), receptors
for hormone, transcription factors
4. Makeup of antibodies (formed outside the liver)
Synthesis and Catabolism
● The information encoded in the genes, specifically the nucleotide
sequence, it can specifically provide each protein with its own
unique amino acid sequence
- All information are encoded in the genes
- When there is abnormal protein formation/occurrence, these
are all caused when there is an abnormal gene (level)
● Determined by the sequences of bases
● The amino acid sequence of protein (polypeptide chain) is
determined by the corresponding sequence of the DNA bases:
(guanine, adenine, cytosine, and thymine)
○ Found in the DNA
● Synthesis of protein occurs in cell, specifically in the Ribosomes:
○ Site of protein synthesis in the cells: Ribosomes
○ Essential for protein synthesis
○ ANABOLIC PROCES
Catabolism:
Disintegration of protein to amino acids
- Aka protein catabolism
● The following is where catabolism of proteins take place:
○ Digestive Tract/GIT
○ Kidneys
○ Liver (major site for protein catabolism/disintegration)
● In the disintegration process, there are two pathways:
1. Lysosomal pathway – degrades extracellular (proteins in
plasma/interstitial space) and some intracellular proteins
2. Cytosolic pathway – degrade intracellular proteins
(cytoplasm)
Transamination
- It involves the removal of nitrogen from the free amino acids
ultimately producing two products: ammonia and keto acid
1. Central reaction that removes amino acid nitrogen
2. This is catalyzed by intracellular enzyme called transaminases
3. It is a reversible reaction
DESTINATION OF THE BI-PRODUCTS:
■ When Keto acid is produced by transamination, it is further
oxidized by means of citric acid cycle. After which, it is
converted to glucose or fat.
■ When Ammonia is produced by transamination, it is further
converted to urea in the liver (in the hepatocyte) by the
so-called urea cycle, and then excreted as part of the urine.
‒ Ammonia: a toxic gas. This is detoxified and
converted in the liver. If not converted, It can
accumulate and severely damage cells irreversibly
and it is neurotoxic (CNS).
‒ Urea: major component of urine
Nitrogen Balance
- Aka nitrogen equilibrium
- Occurs when the nitrogen input/uptake equals the nitrogen
output/excretion
- Nitrogen input: protein intake of the person (nitrogen rich
foods). Nitrogen taken or is part of protein during anabolism
- Nitrogen ouput: level of nitrogen excreted in the body, found
in the urea. Released from proteins during catabolism
(mainly in the kidneys)
● The balance of nitrogen output subtracted from the nitrogen input
● Average nitrogen content of serum protein is approximately 16%
- Affected when there is imbalance in nitrogen balance in
blood:
Two Types:
A. Negative Nitrogen Balance
■ Protein excretion exceeds protein uptake (Excretion
> protein uptake)
■ There is greater protein catabolism than protein
anabolism (catabolism > anabolism)
■ Commonly found in Pathologic Conditions
○ Excessive tissue destruction (Burns,
wasting disease, high fever, and starvation
 B. Positive Nitrogen Balance secondary structures:
■ Protein uptake exceeds protein excretion ○ Alpha helix
■ Greater protein anabolism than protein catabolism ○ Beta-pleated sheets
(anabolism > catabolism) TERTIARY:
■ There is greater nitrogen uptake than excretion ● Overall conformation (fold) of the protein molecule
■ Associated with physiologic conditions ● Responsible for the function and physical and chemical properties
○ Growth of a protein
○ Pregnancy ● Due to interaction of side chains (ex. hydrophobic)
○ Repair Process
STRUCTURE OF PROTEINS QUATERNARY
➢ Levels of protein structure ● Interaction of more than one protein molecule or subunits (ex.
○ Primary Hemoglobin)
○ Secondary ● Most complex
○ Tertiary - Proteins are highly stable due to the peptide bond (peptide
○ Quaternary linkage) that is responsible for connecting the structures of
proteins
- This is why proteins are considered as good
immunogens; long circulation life because of stable
peptide linkage = more effective in stimulating
immune response; they are not affected by
proteolytic enzymes
- Proteolytic enzymes are responsible for degrading
proteins in the liver, intestines etc. targeting the
peptide bonds.
PRIMARY: amino acids in a specific sequence
-It represents the number and types of amino acids in the specific amino Protein Denaturation
acid sequence ● defined as the significant change in protein structure with resulting
➔ amino acids sequence must be in the correct sequence for proteins loss of the functional and chemical characteristics
to function properly - Tertiary structure is mostly affected
● Defective amino acid pattern = gene level occurrence
● Amino acids are in a specific sequence Factors that affect denaturation:
● Structure is somewhat linear A. Changes in temperature
● If there is error in amino acid sequence in the protein structure, it can - higher the temperature = more prone for
cause defects and will not function according to its role denaturation to occur
SECONDARY: repeating structures - temperature is at 56 degC, protein denaturation can
● Regularly repeating structures stabilized by hydrogen bonds occur
between the amino acids within the proteins - Serum inactivation - one way to inactivate
● Responsible for the strength and flexibility of proteins unnecessary protein is by heating the serum sample
 at 56degC because it promotes denaturation and it
3. TRANSPORT Proteins that transport movement of ions, small
will avoid interference in the test measurements PROTEINS molecules or macromolecules (eg. hormones,
- Complement proteins: can be denatured lipids) across a biologic membrane; it can also
@56degC transport insoluble substance in the blood
B. Hydrolysis by strong acid or alkali - Iron, transferrin, lipoproteins
C. Enzymatic action - proteolytic enzymes/protease changes ● Albumin: most common protein transporter
the structure in the plasma
○ major transporter/
D. Exposure to urea or other substances
○ Most abundant protein in plasma
E. UV light exposure
4. Produced by B-cells lymphocytes (plasma cells)
CLASSIFICATION BY PROTEIN FUNCTIONS IMMUNOGLOBULI that mediates immune response (immunoglobulins)
NS (antibodies) Ig= immunoglobulin
CLASSIFICATION FUNCTION G,A,M,E,D= heavy chains
○ IgM = Mu
1. ENZYMES Proteins that catalyze chemical reactions ○ IgG = Gamma
● It is not consumed nor altered in the during ○ IgA = Alpha
the chemical reaction ○ IgD = Delta
○ It only speeds up the ○ IgE = Epsilon
process/velocity; thus ● Produced extra-hepatically
accelerating/hastening the ● Immunogen vs Antigen
chemical reaction ○ Has Anti- (antibodies)
● Enzymes catalyze biologic chemical ○ Remove anti- (immunogen/antigen)
reactions (organic reactants) ● Antibodies are produced to eliminate
● Different metabolic functions or activities in immunogens/ foreign substances/
the cell becomes evident and the reaction is pathogens
a lot faster
● Heat generation is low if there is no enzyme 5. STRUCTURAL Fibrous proteins are the structure of cells and
● Cannot digest food readily if there is no PROTEINS tissues (e.g. collagen, elastin and keratin) o
enzyme ○ Collagen: skin & blood vessels
○ Keratin: hair, skin, nails (epidermal
2. HORMONES Proteins that are considered as chemical derivatives)
messengers that control/regulates the actions of ○ Elastin: skin & arteries (blood
specific cells or organs vessels)
● Can influence/ affect growth and ● Important structures in the body
development, metabolism,and other
important biologic processes in the body. 6. STORAGE Proteins that serve as reserves of metal ions and
● Peptide hormones: protein PROTEINS amino acids
classification/types of hormones ● If for transport: transferrin
○ Not all hormones are protein in ● If storage: ferritin (for iron)
nature; some are lipid in nature
● Produced by glands 7. ENERGY ● Plasma proteins serve as a reserve source
● Liver is considered as a gland that can SOURCE of energy for tissues and muscles
produce hormones
 ● Macromolecules provide energy and can
also provide glucose (gluconeogenesis)
8. OSMOTIC ● Plasma proteins function in the distribution
FORCE of water throughout the compartments of
the body.
○ Albumin is responsible for the
distribution of water throughout the
compartment of the body; regulates
osmotic pressure
● Plasma protein like Albumin can regulate
the water movement in and out the blood
vessels and in the compartments of the
body
● Low albumin (in the blood): (water is not
regulated in and out); it will result to:
Edema: High tissue fluid retention due to
down regulation of water movement
■ Poor osmotic force
■ Ex: patient with renal
disease/dialysis =
hypoproteinemia/hypoalbuminemia
- Albumin (essential) is no longer
reabsorbed, instead, it is secreted in
the urine (Kidney is
destroyed/damaged)
- Manifestation: edema in the lower
extremities
- The essential molecules are
excreted (Proteinuria)
- Since albumin is low, water is not
regulated, therefore there is water
retention
9. HEMOSTASIS ● Mechanism on controlling/preventing
further blood loss by promoting
coagulation/blood clot formation
● Coagulation factors are protein in nature
● Proteins participate in coagulation in blood
● Also produced in the liver
○ Patient with liver disease may also
develop hemorrhagic conditions
(bleeding) because of affected
coagulation factors (also proteins)
in the liver
○ Low coagulation factors in plasma=
longer clot formation = no bleeding
control
10. ACID-BASE ● Proteins may also act as buffers
BALANCE ● Participation as buffers to maintain pH in
the plasma
○ Buffers resists/ prevents sudden
change in the pH of plasma
● May act as buffer: Hemoglobin, Albumin,
Plasma Proteins
● Participates in buffering plasma pH is
7.35-7.45 is maintained.
○ Average plasma pH= 7.4
CLASSIFICATION OF PROTEIN STRUCTURE
1. Simple Proteins- structure containing peptide
chains
○ Contain peptide chains composed of only
amino acids
○ May be globular or fibrous:
Globular: Albumin and immunoglobulins
- seen in the plasma or circulation
Fibrous: connective tissues, tendons,
bone and muscle
- seen in the structural support of
tissues
2. Conjugated Proteins- amino acids and
nonprotein prosthetic group
○ Consist of a protein and a nonprotein
prosthetic group
○ Name of the conjugated proteins
depends on the attached prosthetic
group present
1. Metalloprotein
● Metal ions attached - ferritin (iron), Ceruloplasmin
(copper)
 ● Complex metal – hemoglobin and flavoproteins Side note:
(flavin) (from anode to cathode)
2. Lipoproteins 1. HDL - a1 (alpha)
● Lipids attached – HDL, LDL, VLDL 2. VLDL - between beta and a2
● Protein component: Apolipoprotein 3. LDL - beta
3. Mucoproteins or proteoglycans 4. Chylomicrons - in the cathode
● With higher carbohydrate – mucin
4. Glycoproteins Different types of plasma proteins:
● Smaller concentration of carbohydrates attached to Prealbumin - Not visible in the typical serum/plasma electrophoresis (pic)
CHONS ● Indicator of nutrition
● 10%-40% carbohydrate – haptoglobin, and ○ Malnourished individual = decreased level of prealbumin
α1-antitrypsin ● Binds thyroid hormones (T3,T4)
● Binds retinol-binding protein (vitamin A)
5. Nucleoproteins
● Also known as transthyretin
● Combined with nucleic acids – Chromatin

PLASMA PROTEINS
- When we perform serum or plasma protein electrophoresis, we
have 2 major fractions:
● Albumin
● Globulin
○ Globulin subfractions:
- Alpha 1 globulin (α1-Globulins)
- Alpha 2 globulin (α2-Globulins)
- Beta globulin (β-Globulins) Albumin- most anodic type of protein
- Gamma globulins (γ-Globulins) - Highest peak in the electrophoresis
- When we perform electrophoresis, the pH is always set in an - Highest peak because it has the highest concentration in the
alkaline level. healthy human serum/plasma
- Basic pH of 8.6, proteins are negatively charged - Smallest in terms of size (maliit pero pinaka madami)
- Most anodic type of protein
- Binds bilirubin (specifically bilirubin 1; b1), steroids, fatty acids
Electrophoresis: (+) anode ; (-) cathode
- Major contributor to oncotic/osmotic pressure to regulate
dissemination of fluid in and out of the vessels.
- Dietary protein: mostly composed of Albumin
Globulins:
Alpha 1 globulin
- α1 - antitrypsin
● Acute phase reactant
○ ACP- proteins increased when there is acute
inflammation
● Protease inhibitor
 - α1 - fetoprotein
● Principal fetal protein
○ Normally high in fetus
○ Low/decreased in adults
- Elevated: spina bifida, neural tube defects, fetal
distress
- Decreased level: down syndrome (trisomy 21),
trisomy 18 (aka Edward’s syndrome)
● High in a fetal specimen
● In adults, ↑ AFP is found among people with
malignancies
○ it is used as a tumor marker
○ The malignancy associated is hepatoma/
hepatocellular carcinoma
- α1 - acid glycoprotein
● Acute phase reactant present in alpha 1 fraction
- α1 - lipoprotein
● Transports lipids (HDL) - specifically cholesterol
from the tissues
● Pathway: Reverse Cholesterol Transport System
● Cardioprotective; Good Cholesterol
- α1 - antichymotrypsin and Inter-α-trypsin inhibitor
● Inhibits serine proteinases
- Gc-globulin: group-specific component
● Transports Vitamin D and binds to protein actin
○ Vitamin D needs a transporter because it
is derived from cholesterol
(lipid;insoluble)
● GC means Group specific component globulin
Alpha 2 globulins
Protein subfractions:
Haptoglobin
● Acute phase reactant
○ Substances that are increased in the plasma/serum
whenever there is acute inflammation
● Binds with free form hemoglobin – hemoglobin that is
released from BRCs during hemolytic conditions
○ When Red cells are lysed, free Hgbs will be released.
Haptoglobins bind with these free Hgb.
- Hemolytic anemia: ↓ haptoglobin level in plasma
- More lysis of rbcs, more free Hgbs are produced. Thus, more
haptoglobins are needed to bind with the free Hgb
Ceruloplasmin
● Acute phase reactant, contains copper
● ↓ Ceruloplasmin in plasma: Wilson’s disease, Menkes
syndrome, (kinkey hair disease) Kayser Fleischer rings in
cornea
● ↓ Ceruloplasmin in plasma: ↑ free copper level in plasma
Alpha 2 macroglobulin
● Inhibits protease enzyme
Beta globulins
1. Pre Beta lipoprotein
➔ Transports lipids (VLDL triglycerides) endogenous
triglycerides
➔ Found between Alpha 2 and Beta region
2. Transferrin
➔ Transports iron
➔ increased in IDA; decreased in hemochromatosis (high iron
level)
◆ The opposite of IDA is hemochromatosis
◆ Transferrin transports iron to bone marrow and liver
to be recycled
◆ IDA: free iron is low;
◆ Hemochromatosis: free iron is high
➔ Transports iron from plasma to the liver or the bone marrow
- Where iron is recycled/utilized
- Used in erythropoiesis- integration of iron in Hgb
Iron deficiency anemia: ↑ Transferrin level in plasma
➔ transferrin is responsible for transport of iron in plasma to liver
➔ IDA patient: low level of iron in plasma; more transferrin unused
= Higher level of transferrin in plasma
Hemochromatosis: ↓ Transferrin level in plasma
● High levels of iron in plasma/blood will utilize more transferrin for
transporting iron to liver/ bone marrow
➔ If there is free iron in the plasma, it is transported to the liver
3. Hemopexin
➔ Acute phase reactant, binds to heme portion of hemoglobin
 4. Beta lipoprotein 2. Immunoglobulin Alpha (IgA)
➔ Transports lipids (LDL bad cholesterol) ● Antibodies present in the secretion and in the plasma
➔ Transports dietary cholesterol ● Provides mucosal immunity
➔ Endogenous pathway (with VLDL) ○ Because it is high and predominant in secretion
5. Beta 2 microglobulin ● Also present in the colostrum along with IgG.
➔ Component of HLA molecules ● Can also be transferred through breast milk
◆ HLA - Human Leukocyte Antigen
● Part of immune response Immunoglobulin Mu (IgM) - biggest antibody
● Antigens in the WBCs and tissues
● Antibodies in early immune response
6. Complement proteins: C4, C3, C1q complement
● Predominant antibody in primary immune response/early
➔ Immune response
immune response
➔ Non-specific serum proteins that enhances activity of
antibody against antigens
➔ They are called complement because their presence would Immunoglobulin Epsilon (IgE)
enhance the activity of antibodies in eliminating pathogens ● Otherwise known as Reaginic Antibody
7. Fibrinogen ● Antibodies increased in allergic reaction and parasitic
➔ Precursor of fibrin clot infections particularly in helminthic infections (reagen,
➔ Factor I not Factor 1 allergy) worm infections
➔ forms stable fibrin clot that stop and clog to prevent further ● Present or increased in allergy or hypersensitivity reactions
blood loss/bleeding
8. C-reactive protein (CRP) Immunoglobulin Delta (IgD)
➔ First acute phase reactant to increase/elevate during acute ● Surface antibody (surface of lymphocytes)
stage inflammation. (fastest and first to increase) ● Present on the surface of B-cells
➔ Motivates phagocytosis in inflammation (cell engulfment) ● Not yet fully established in terms of the function
➔ Part of the immune system ● Produced in the plasma
➔ Protein found in the beta region
Arrangement of Immunoglobulins based on Concentration in the normal
Y Gamma globulins - other term for antibodies (rarely used term now) healthy serum:
- Closest to cathode (-) and farthest from anode (+) G, A, M, D, E

1. Immunoglobulin Gamma (IgG)- smallest antibody OTHER PLASMA PROTEINS

● Considered as the smallest antibody
● It is the only antibody that can cross the placenta
● Can provide neonatal immunity
○ Because it can travel from the mother to the
developing fetus through placenta
● Antibody with the highest concentration in the healthy
human plasma
● The predominant antibody in the secondary/ memory/
anamnestic response.
● Smallest but the most abundant
● Myoglobin
○ Protein that carries oxygen in the muscles
○ Earliest cardiac marker to increase in case of Acute M.I
(myocardial infarction)
- In Acute Myocardial Infarction, myoglobin increases
- Fastest and first to elevate when there is Acute MI
Protein first to increase in AMI: Myoglobin
Enzyme first to increase in AMI (specific): CK-MB (creatine kinase MB
subfraction/subtype)
○ Increases 2-3 hours of onset, peak at 8-12 hours
○ Increased in crushing injury and muscle dystrophy
 ● Troponin - Rough measurement of total protein whether it is albumin or globulin
○ Cardiac marker for acute coronary syndrome - Hypoproteinemia and Hyperproteinemia
○ Also increased in patients with AMI
In the laboratory, Total Protein Measurement is done.
● Brain Natriuretic Peptide Total protein measurement- measures both the albumin and
○ Marker for congestive heart failure globulin contents of the plasma
○ A Neurohormone that affect body fluid homeostasis and - Aside from TP, Albumin and Globulin ratio is also measured
➔ TPAG- Total Protein Albumin Globulin Ratio
blood pressure level
Total protein measurement can reflect the ff:
● Fibronectin Fetal fibronectin (fFN) ➔ Nutritional status of the individual
○ Cellular interaction (ex. Cell adhesion) ➔ Kidney disease
○ Adherence of the placenta to the uterus ➔ Liver disease
↑ fFN = preterm labor and delivery ➔ And many other conditions
○ Increased in preterm labor and delivery ● Total protein is the sum of albumin and globulin measurement
● Cross-linked C telopeptides
○ Proteolytic fragment of collagen 1 Hypoproteinemia
○ Biochemical marker of bone resorption
- It occurs when there is an abnormal nitrogen balance
■ Bone resorption- destruction of bone and minerals
such as calcium is released from the bones - Can be due to the following:
● B-trace protein (Beta-trace protein) ➢ Excessive loss of protein
○ Synonym: prostaglandin D synthase ○ due to increased excretion of protein in cases of renal
○ Found in the CSF disease.
○ Marker for CSF leakage ○ Leakage into the GIT
↑ B-trace protein in plasma = leakage of CSF ○ Loss of blood or hemorrhage
■ (ex. Head injuries, trauma, damage in BBB - blood ○ Extensive burns
brain barrier) ➢ Decrease or low intake of protein
● Cystatin C ○ Due to malnutrition
○ Cysteine proteinase inhibitor ○ Intestinal malabsorption syndrome
○ Serum marker for glomerular filtration rate (GFR)
Total Protein Albumin Globulin Disease
- kidney function
● Amyloid
Normal, ↓ ↓ ↑ Hepatic Damage
○ Fibrous protein aggregates
● Cirrhosis β-γ bridging
○ Formed from alteration of Beta pleated sheaths in ● Hepatitis; ↑ γ-globulins
secondary structure of proteins ● Obstructive jaundice; ↑α2
○ Increased/elevated in amyloidosis Burns, trauma
○ Differential diagnosis tool for alzheimers Infections
- Used in alzheimer's diagnosis ● Acute α1 and α2 globulins
○ Low A-Beta-42 with high Tau proteins ● Chronic ↑ α1, α2, γ-globulins

TOTAL PROTEIN ABNORMALITIES ↓ ↓ Normal Malabsorption

- Measurement of albumin and globulin content of the plasma as well Inadequate diet
as the albumin and globulin ratio
 How electrophoresis is used in Protein diseases:
Nephrotic syndrome
● ↑ α2-globulins (alpha) ● M spike
● ↑ β-globulins (beta) ○ Monoclonal gammopathy
● ↓ γ-globulins (gamma) ○ associated with multiple myeloma
● Nephrotic syndrome
↓ Normal ↓ Immunodeficiency syndrome (i.e ● Acute inflammation
γ-globulins AIDS)
● Hypogammaglobulinemia
↓ ↓ ↓ Salt retention syndrome ○ Severe
● Alpha 1 antitrypsin deficiency
● Cirrhosis
1. Normal; ↓; ↑ ○ Albumin is low
➔ When albumin is slightly decreased, the increased globulin can ○ Alpha 1 is slightly decreased
compensate for the slightly decreased albumin = Normalized TP ○ Alpha 2 is slightly decreased
➔ When albumin is markedly decreased, the increased globulin cannot ○ Beta and gamma is joined together - beta-gamma bridging
compensate for the markedly decreased albumin = Low TP ● Chronic inflammation
2. ↓; ↓; Normal ○ Albumin is not affected but everything is affected/increased
➔ Total protein is low because albumin is low (majority of the protein) Reference interval
3. ↓; Normal; ↓
● 6.5-8.3 g/dL (65-83 g/L)
➔ Because gamma globulin is significantly decreased
● 6.0-7.8 g/dL (60-78 g/dL) in recumbent positions
- References by books vary, reference value used in practice is the one
Hyperproteinemia - is not an actual disease but rather a complication of provided by the manufacturer of reagent
other disease
➔ Result of an underlying cause TOTAL PROTEIN METHODS:
Summary of methods on total proteins:
Total Albumin Globulin Disease Kjeldahl - REFERENCE METHOD FOR TP
Protein
➔ Digestion of protein; measurement of nitrogen content
↑ ↑ ↑ Dehydration ➔ Reference method.
- When plasma volume is low, all ➔ Assumption: average nitrogen content of 16%
analytes (glucose, urea, sodium, Refractometry
potassium) in plasma is high ➔ Measurement of refractive index due to solutes in serum
➔ Refractometry of serum has the same principle with
↑ Normal ↑ Multiple Myeloma Refractometry in the urine
- Malignancy of plasma cells Biuret method
Monoclonal & Polyclonal gammopathies ● Formation of violet-colored chelate between Cu2+
ions and peptide bonds
Plasma cells: antibody-producing cells. Dye binding
- Malignancy in the plasma cells makes it hyperactive ● Protein binds to dye and causes a spectral shift in the
(excessive production of abnormal antibodies)
absorbance maximum of dye
- Hypergammaglobulinemia
● Interaction or reaction of protein with specific dye to
↑ antibodies ↑ gamma globulin
cause spectral shift in the absorbance of the dye
1. Kjeldahl 3. Biuret method
● Total nitrogen is measured ● Formation of violet-colored chelate between Cu2+
● Acid preparation (TCA or tungstic acid) protein with ions and peptide bonds (measured at 540 NM)
measurement of total nitrogen ● 2 or more peptide bonds must be present to
○ TCA can be used as the acid precipitating agent produce a violet reaction/solution
A. Kjeldahlization - conversion of nitrogen to ammonia with the ● Composition of the Biuret reagent:
addition of sulfuric acid as a digestive agent at 340-360 degC and
○ Cupric ions - Are used to break the peptide
cupric sulfate as a catalyst
● Sulfuric acid is used as the digestive agent. bonds
● Sulfuric acid will digest proteins at 340-360 deg C ○ Tartrate salt - used to keep the copper in the
● Cupric sulfate is used as the catalyst to speed up the solution
reaction H2SO4 ○ Potassium iodide - used to stabilize the
○ Nitrogen -H2SO4 → NH3 cupric ions
● Aside from cupric sulfate, you may also add potassium
sulfate 4. Dye binding
● To increase the boiling point of the solution and to improve ● Proteins bind to dye and cause a spectral shift in
● the efficiency of digestion of proteins
the absorbance maximum of dye.
B. Ammonia measurement:
● Colored product is directly proportional to the
1. Nessler’s reaction (double iodide of
Hg and K) concentration of the protein present
● Yellow solution is the end product (Ammonium ● More intense colored product, more intense
dimetric iodide) concentration
○ Measured colorimetrically ● Examples of protein binding dyes:
● Gum ghatti ○ Bromphenol blue
● Ammonia + Nessler's reagent -Gum ghatti → ○ Ponceau S
yellow solution (Ammonium dimercuric iodide) ○ Amido black 10B
2. Berthelot reaction ○ Lissamine green
● Indophenol blue is the end product.
○ Coomassie brilliant blue
○ Measured colorimetrically.
● Na nitroprusside
● Alkaline hypochlorite If specific to albumin, there is a separate set of method and dyes
● Ammonia + alkaline hypochlorite -Na Set of dyes & method for Albumin:
Nitroprusside → Indophenol blue 1. Salt preparation
2. Refractometry ● Globulins are precipitated in high salt concentration
● Same principle with the specific gravity measurement in the ● Albumin in supernatant is quantitated by biuret
urine reaction
○ In urine sample, there is correction when protein is ● Sodium sulfate is the common precipitating reagent
present in urine; unlike the one used in blood for albumin
● Measurement of ○ Globulins precipitate in the high salt
refractive index (velocity concentration of reagent leaving albumin in
of light in air and water) the supernatant
due to solutes in serum. 2. Dye binding:
A. Methyl orange
● Nonspecific for albumin
 B. HABA (2,4 HYDROXYBENZENE BENZOIC ACID) - Intense blue: Albumin (most abundant in conc.)
● Used but has many interferences such as drugs, - High concentration = high dye binding capacity
salicylates, bilirubin)
C. BCG (Bromcresol green)
● Sensitive
● Overestimates low albumin levels
● Most commonly used dye
D. BCP (Bromcresol purple)
● Specific, sensitive and precise

Electrophoresis
PRINCIPLE: Proteins separated based on electric charge
densities
- Protein separation based on electric charge densities
➔ Higher charge at pH 8.6 ; farther migration (closer to anode +) of
protein fraction
● Give overview in relative changes in different protein fractions
➔ The more and negatively charged particles it has, has a farther
migration from the point of application.
➔ Albumin is the most anodic and gamma globulin is the least anodic

Reference/normal values for electrophoresis:

➢ Albumin, 53-65% (3.5-5.0 g/dL) highest concentration
➢ Alpha 1 globulin, 2.5-5% (0.1-0.3 g/dL)
➢ Alpha 2 globulin, 7-13% (0.6-1.0 g/dL)
➢ Beta globulin, 8-14% (0.7-1.1 g/dL)
➢ Y-globulin, 12-22% (0.8-.1.6 g/dL)

Electrophoresis procedure:
Cellulose acetate/agarose gel (support media) Monoclonal increase
1. Protein fractions are separated
2. After separation, protein fractions are immersed in acid solution A: Normal
then stained by dyes (ex. Coomassie blue) C: Normal (represents A)
- Protein bands are stained using dye (Coomasie dye) to
demonstrate fractions
B: Monoclonal component
3. After adding coloring dye, the medium is placed in scanning
D: monoclonal spike (M spike) (represents B)
densitometer (to determine absorbance of each fractions) the which
compute the area under the absorbance - Gamma globulin has almost same
- To determine the quantity of protein fraction concentration with albumin
- Seen in patients with Hyperproteinemia (specifically those with
increased monoclonal antibodies)
- Associated with Multiple Myeloma (increased gamma
globulins); monoclonal gammopathy
1. Monoclonal Gammopathy/ Multiple Myeloma
- M-spike: increased gamma globulins
- Normal Albumin, alpha1, alpha2 |
2. Nephrotic syndrome
- ↓ Albumin | ↓sightly alpha1 and beta | ↑ alpha2
3. Acute inflammation
- Slightly ↓ albumin | slightly ↑ alpha1 | ↑ alpha2
- Increased acute phase reactants
4. Hypogammaglobulinemia (pt with immunodeficiency)
- Opposite of Monoclonal Gammopathy
- Normal Albumin, alpha1, alpha2 | markedly decreased
gamma
- Severe immunodeficiency syndrome (AIDS)
5. Alpha-1 antitrypsin deficiency
- Normal Albumin, alpha2, and gamma globulins | very low
alpha1
6. Cirrhosis
- Beta-gamma bridging
- Characteristic of patient with liver cirrhosis
- ↓ Albumin | slightly ↓alpha 1 and alpha 2 | beta and gamma
fusion (beta-gamma bridging)
- Beta-gamma bridging is seen in px with liver cirrhosis
7. Chronic inflammation
- Normal Albumin; ↑ alpha1, alpha2, beta, gamma
Acute phase reactants- increased in acute stage inflammation
- Only sensitive to inflammation
- Non-specific
● Fibrinogen
● Haptoglobin
● Ceruloplasmin
● Serum amyloid
● CRP
ACP is caused mainly by 2 conditions:
1. Tissue Damage
2. Infection
- Acute phase reactants increases
- The first/fastest one to increase is CRP
- ACP cannot determine the site of inflammation
- Certain of inflammation but not specific to site
High resolution protein Electrophoresis:
● Uses higher voltage couple with a cooling system and more
concentrated buffer
- End result: instead of just 5 fractions, more protein bands
are separated and more fraction bands are demonstrated.
● In this procedure, it can demonstrate:
○ prealbumin
○ Specific proteins (antibodies, transporter proteins,
lipoproteins)
● Modification: Higher electric current, higher voltage, cooling system,
buffer is more concentrated
● Advantage: more protein fraction is separated and demonstrated in
electrophoresis
● HRPE is performed to demonstrate other specific proteins in the
globulin fractions and outside the albumin fraction
Proteins present in other body fluids: used in urine protein determination autoantibodies
● Urine is the second bodily fluid; this method can also be used for other ○ oligoclonal banding are present in the gamma
body fluids (i.e CSF, transudates, exudates) globulins
● Multiple myeloma vs Multiple sclerosis:
1. Turbidimetric methods (SSA,TCA, benzethonium chloride) ○ Multiple myeloma - involves plasma cells; sample:
- Sulfosalicylic acid, trichloroacetic acid, benzethonium chloride plasma or serum
PRINCIPLE: Proteins are precipitated as particles, turbidimetry is
○ Multiple sclerosis - involves anti-myelin; sample:
measured spectrophotometrically.
CSF
Rapid test and easy to use/perform but the sensitivity is
unequal for individual protein
● Proteins are precipitated and turbidimetry is applied
- Turbidimetry principle: Light blocking capacity of
particles/precipitate

2. Biuret
Accurate method for urine protein testing
● Same with serum/plasma protein determination
● Violet-colored chelate is measured
● More than one peptide should be present

3. Follin-lowry
Very sensitive test for urine protein
● Initial biuret reaction, oxidation of AA (tyrosine, etc
residues by folin phenol reagent;
● measurement of resultant blue color (blue-colored
product)

4. Dye binding (coomassie blue, Ponceau S)

There is limited linearity of the reaction
Unequal sensitivity for individual proteins
● Proteins bind to bye, causes shift in absorption maximum

5. CSF protein electrophoresis

● Presence of oligoclonal banding demonstrated in the
electrophoresis using CSF as the sample, it is indicative or
diagnostic for multiple sclerosis
● In multiple sclerosis, px has autoantibodies against the
myelin sheath of the nerve cells (axon); autoantibodies are
concentrated in the CSF
● Oligloconal banding signifies the presence of
 Urea 45-50% 86.0%
January 4, 2022
Clin Chemistry Lecture Amino Acids 25% Not excreted in urine;
NON-PROTEIN NITROGEN COMPOUNDS (NPN) essential = reabsorbed;
- Proteins are macromolecules located in liver/muscles
- Nitrogen are mainly regulated by proteins not plasma
- Aside from Protein macromolecules, there are other molecules that
Uric Acid 10% 1.7%
contain Nitrogens. (NPNs)
Historical Perspective: Creatinine 5% 4.5%
- Originated in the early days of clinical chemistry
- Analytic methods require deproteinization process (not practiced Creatine 1-2% Not normally present in
urine; used as reservoir of
anymore; tedious and time-consuming) of specimen before analysis
energy; normally found in
- Since protein in specimen could interfere with the NPN
muscle and not excreted in
measurement urine (normally)
- Deproteinized specimen produces PFF: protein-free filtrate
- PFF is then analyzed Ammonia (trace) 0.2% 2.8%
- The concentration of the nitrogen-containing
- Constituents of plasma can also be seen in urine
compounds are measured/quantified
- Waste products and excess are normally excreted/removed
spectrophotometrically.
- Urea is highest in plasma and urine
- NPN determination could provide information in diagnosis of patient
- One of the major contributors of plasma osmolality
Condition commonly assessed: Kidney function - Three other major contributors: Chloride, Glucose, Sodium
- Since NPNs are normally excreted by the kidneys - When solvent is decreased, there is increased number in
- NPN measurement in the plasma/serum is useful in assessing solute
kidney functions and liver functions. - Amino acids are not normally present in urine since it should be
- Since some NPNs are produced in the liver. reabsorbed.
- Liver function would affect the formation of NPNs in plasma - Amino acids are important for formation of proteins
- Kidney function would affect the concentration of NPNs in (building blocks)
the plasma - Almost all creatinine are removed in the plasma
➔ Deproteinization process is not performed anymore because it is a - Creatine is not normally seen in urine because it serves as reservoir
tedious and time-consuming procedure of energy in the form of creatine phosphate.
◆ Instead of deproteinization process, methods used nowadays - Normally found in muscle not in urine
are specific and sensitive
UREA
- Protein contents do not interfere that much anymore
Physiology
- NPNs can be measured even with protein contents
● Major waste product of dietary protein catabolism
NPNs: Urea, Amino Acid, Creatinine, Creatine, Ammonia
○ Also a product of dietary protein
Compound Approx. Approx. ■ The level of urea in plasma is influenced by the
Plasma Concentration Urine Concentration intake of meat/ protein-rich diet
(% of total NPN) (% of excreted nitrogen) ● Excreted by the kidneys
● NPN compound with highest concentration in blood and urine
 ● Urea is formed in the liver from the amino group and free ammonia, ○ Kidneys are the major organ for the removal of urea
generated during protein catabolism ■ Renal function altered/abnormal = all that are not
excreted will be brought back and reabsorbed.
● plasma levels increases when they are not
excreted
● BUN – term used to refer to blood urea determination
● urea N – more appropriate term

Clinical Application of UREA

● Used to evaluate renal function
- First metabolite to increase in renal disease
- Also used to diagnose renal disease
- Through the process of Deamination (in the Liver)
○ Urea N (mg/dL) ↔ urea (mg/dL)
- Free amino group is converted to Ammonia -> Ammonium
○ 1 urea N → 2.14 urea
(less toxic form) -> In the urea cycle -> Urea
○ 0.467 urea → urea N
- Catalyzed by enzyme: Deaminase
○ 0.357 mg/dL (conventional unit) → mmol/L (SI Unit)
● From liver, carried in the blood then to the kidneys
● Assess hydration status
○ Where it is readily filtered from the plasma by renal tubules/
- Major contributor of plasma osmolality
renal glomerulus
- Dehydration: Decreased plasma volume; increased urea
■ Most of urea in plasma is filtered, and excreted in
- Normal kidney function, but increased urea in
the urine
plasma = due to dehydration.
● Not all urea is completely excreted; some of the urea in the
● Determine Nitrogen Balance
glomerular filtrate are:
- Good indicator of nitrogen intake
○ passively reabsorbed back to the circulation in the renal
- Increased urea in plasma= high protein intake
tubules through passive diffusion (small amount)
- Abnormally high urea in plasma is correlated (good
● The amount of urea reabsorbed depends on the ff factors:
indicator) to possible abnormal condition: negative nitrogen
1. Urine flow rate
balance
2. Extent of hydration
- Excreted as waste product
● Aside from the kidneys, small quantities of urea about <10% of total
● Diagnosis of renal disease
urea are excreted through:
● Verify adequacy of dialysis
○ the GIT (stool/feces) and skin (sweat and perspiration)
- Urea is easily removed by dialysis
○ Some patients with renal failure (dialysis patients) have
- Dialysis is the process of separating macro from
sweat with urine-like scent.
micro molecules.
- Since urea is micromolecule while protein is the
The concentration of urea in the plasma is determined by the ff factors: macromolecule.
● Protein content in the diet - Urea level in the plasma before and after dialysis would
○ Red meat increases the urea content ensure the effectiveness of the procedure.
● Rate of protein catabolism - Before dialysis, NPNs should be high, after dialysis
○ Fast catabolism = higher urea is formed (effective process) NPNs should be decreased
● Renal function and perfusion (rate of blood delivered to the tissues)
Methods of Analysis (Lab topic) for UREA
1. Enzymatic method: INDIRECT METHOD
- Urease catalyzes the reaction
- Measurement of urea is always an INDIRECT METHOD
- Urea -> ammonium carbonate
- Ammonium level is directly proportional to the urea
level
- After
➔ First step: conversion of urea to ammonium carbonate
Urea + 2H2O → urease → NH4 + CO3 2-
After conversion, Ammonium ions generated is measured in three ways:
1. GLDH (Glutamate Dehydrogenase) coupled enzymatic method
- disappearance of absorption of NADH is measured at 340 NM
I. Used on many automated instruments and best
performed as a kinetic measurement
NH4 +2-oxoglutarate + NADH + H ← GLDH → glutamate + NADH + H2O
- Reduced NAD is oxidized
- Decreased NADH is measured at 340nm
Ammonium ions generated would react to dye:
2. indicator dye: ammonium ions generated would react with a pH
indicator to produce a color change
- When ammonium is produced, the pH is changed and
becomes basic, the change in the pH would produce a color
change.
- Indicator dye is also used in:
- automated system
- multilayer film reagents
- dry reagent strips
- NH4 + pH indicator → color change:
- change in pH would produce color change (becomes basic)
1. Nessler’s reaction (HgI2/KI) = yellow product
formation
a. NH4 + nessler’s salt → Gum Ghatti →
yellow color
2. Berthelot reaction = indophenol blue
a. NH4 + alkaline hypochlorite → Na
nitroprusside → indophenol blue
- Products are measured colorimetrically
- The absorbance of the yellow and blue product is directly
proportional to the ammonium level and therefore in urea
concentration in the sample.
3. Conductimetric
- Ammonium generated from urea can increase the
conductivity of the sample
- It is highly specific and rapid method
- Conversion of unionized urea to NH4 and CO3 results in
increased conductivity
- It is indirect. Urea must first be converted to ammonium
and carbonate before it can be measured
2. Non-enzymatic/chemical method: DIRECT METHOD
- Direct measurement of urea is performed.
- No need for conversion and urea as it is.
- Non-specific, interfering agents may affect the quantifying
urea.
- No longer used because it uses toxic reagents.
➔ Fearon’s Reaction
I. Urea + DAM (Diacetyl Monoxime Mtd.) → yellow solution
(Diazine derivative) measured colorimetrically
II. Non-specific, uses toxic reagents
- Interfering substance may affect the measurement
III. DAM reagent is toxic and could harm MTs.
IDMS (Isotope Dilution Mass Spectrometry)- reference method for UREA.
- Pertains to the detection of characteristic fragments following
ionization procedure.
- After ionization procedure, it is followed by quantification using
isotopically labeled compounds.
Reference intervals
● Adults:
○ Plasma/serum - 6-20 mg/dL | 2.1 - 7.1 mmol/day
○ Urine/24-hour 12-20 g.day | 11.5 - 4.4 mmol urea/day
Specimen requirements for UREA (Lab topic)
● Use fasting blood sample since high protein diet affects urea
- If the person is taking a single-containing protein meal
(minimal/regular serving), fasting may not be required since
it has a minimal effect on the urea level.
- BUN (blood urea nitrogen)) requested along with FBS, urea
will be under fasting because of the FBS fasting requirement
- BUN test alone:
- High protein diet: fasting required
- Mininal protein diet: no fasting
● Avoid fluoride or citrate anticoagulants since they inhibit urease
- Affects the enzymatic method
- Urease that converts urea to ammonium is inhibited.
- Avoid gray or light blue top
- Plain tube is accepted.
● Refrigerate samples to avoid bacterial decomposition
- Examine the urine specimen as soon as possible. If delayed,
refrigerate the specimen.
- Especially urine sample in delayed testing
- Urine left @ room temp -> increased multiplication
of bacteria; bacteria can decompose urea
- Refrigeration has a Bacteriostatic effect
- Will not allow bacterial multiplication
- There are bacteria that can produce urease and can be
present in urine and cause decomposition of urea.
Pathophysiology of UREA
● Azotemia - increased urea in the blood
● Uremia - very high urea in the plasma
- accompanied by renal failure
○ Patients with uremia are also called uremic/uremic
syndrome
○ Both are increased urea in plasma
Increased concentration
- Differentiation of pre, renal, and post renal are tested by BUN:
creatinine ratio in the plasma
- Normal Value: 10:1 TO 20:1 (urea:creatinine)
Different types of Azotemia
- Differentiation of the three azotemia can be done with the use of
BUN-Creatinine Ratio
- Normal: 10 (BUN):1(creatinine) - 20:1
● Prerenal azotemia- before it reaches the nephrons (in the kidneys)
➔ BUN:CREA ratio high; but Normal creatinine
- Only BUN is increased. Therefore, the ratio also
increases.
➔ Caused by reduced blood flow
➔ Congestive heart failure, shock, hemorrhage, dehydration,
increased protein catabolism, high protein diet
➔ The Urea-Nitrogen ratio is high. The overall ratio is high
because the urea nitrogen level is high and the creatinine is
normal.
● Renal azotemia
➔ BUN:CREA ratio high; Elevated creatinine
➔ Damage of filtering structures of the kidney
➔ Renal failure and renal disease (glomerulonephritis, tubular
necrosis)
- In renal and post renal (same characteristics) the urea nitrogen ratio
is high also and the creatinine level is also high
● Postrenal azotemia- after filtered in the kidneys
➔ BUN:CREA ratio high; Elevated creatinine
➔ Due to structures after urea is filtered from the glomerulus
➔ Urinary tract obstruction
➔ Renal calculi, tumors of the bladder or prostate
In what conditions can we find a low urea nitrogen ratio:
● Low protein intake - dietary protein can affect urea
● Acute tubular necrosis - reabsorption is abnormal
● Severe liver disease - urea cycle is affected
Decreased concentration of urea nitrogen
○ Low protein intake
- vegetarian
 ○ Severe vomiting and diarrhea ○ Uric acid is excreted
- Poor absorption of protein ○ Most of the uric acid is reabsorbed in the proximal
○ Liver disease convoluted tubules. Reabsorption happens in the PCT
- Abnormal liver = functions altered including urea tubules and is reused.
cycle; conversion of ammonia to urea will not be ● In lower forms of animals, uric acid is further converted to a more
possible soluble product called “allantoin” (more soluble form of uric acid)
- Low urea level in the plasma produced from purine catabolism
○ Pregnancy - the developing fetus is consuming the protein ○ End product of purine catabolism in lower forms of
from the mother mammals.
Low urea level is less significant than high urea level since it is associated ○ Humans: uric Acid
with renal function. ● In the plasma, uric acid exists in the form of monosodium urates
URIC ACID - At plasma pH of 7, urates are relatively insoluble.
● Concentration >6.8 mg/dL urate crystals may precipitate in tissues
Physiology
and joint cavities
● Major end product of purine (adenine, guanine; derived from
○ Tophi/ tophus - Precipitated urate crystals present in the
exogenous source) catabolism primarily in the liver
tissues and may accumulate in the tissues and in joint
● Catalyzed by enzyme: Xanthine oxidase (XO)
cavities
- enzyme that catalyzes the interconversion of purine
■ Patients with gout (joints are inflamed) are because
derivatives into uric acid
of the precipitated crystals.
- Purine bases: adenine and guanine
■ The pointed part of the urate crystals can cause
- Derived from the breakdown of ingested
tissue damage which leads to inflammation in the
nucleic acids (from diet) and destructed of
tissues.
tissues and cells
Gouty arthritis- due to increased accumulation of tophi in the joint cavities.
○ majority /most of the purine bases that are converted into
uric acid are derived from exogenous sources/diet. (internal ● Most uric acid is reabsorbed in the proximal tubules and reused
organs, seads, beans) ● Relatively insoluble in plasma
○ Can also be derived from tissue destruction. This is called ● XO – xanthene oxidase; Reabsorption – 98 to 100% of the uric acid
endogenous ○ Occurs in the proximal convoluted tubules (PCT)
● Small amounts of uric acid are secreted by the distal convoluted
EXOGENOUS SOURCE: Nucleic acids obtained from diet are converted into
tubules (DCT) into the urine
uric acid
● Renal excretion accounts for about 70% of uric acid elimination
- higher nucleic acids coming from this.
● In acidic urine (pH <5.75) = uric acid is the predominant species and
ENDOGENOUS SOURCE: Nucleic acids obtained from destroyed tissues or uric acid crystals may form
cells
Concentration of uric acid in the plasma is determined by following factors:
➔ When uric acid is produced in the liver, it is carried in the blood to the
(Factors that determines the uric acid concentration in the plasma)
kidneys. The important enzyme responsible for the conversion is XO
● Catabolism of ingested (exogenous) nucleoproteins - consuming
(xanthine oxidase)
food with higher nucleoproteins, more uric acid is produced
➔ In the kidneys, Uric acid is filtered in the glomerulus and is readily
- All foods that have cells. (red meat, beans, internal organs
secreted by the DCT (Distal convoluted tubules) tubules and is
of animals, liver, lungs have high nucleoproteins)
excreted in the urine.
 - Can lead to gouty arthritis ● It is readily automated and can be applied in automated techniques.
● Catabolism of endogenous nucleoproteins - the more cells/ tissues ● First step: uric acid + O2 _ 2H2O → uricase→ allantoin + CO2 +
are destroyed, the more uric acids is produced H2O2 (hydrolyzed)
- Nucleoproteins from tissues or destroyed cells ○ Spectrophotometric (blauch and koch) - decreased in the
● Direct transformation of endogenous purine nucleotide absorbance at 293 NM is measured. UV region.
- Allantoin is measured
Clinical Application of URIC ACID - URIC ACID VS. ALLANTOIN
- The trend must be decreasing. If it does not go
● Assess inherited disorders of purine metabolism
down, uricase has a problem or your uric acid levels
● Confirm diagnosis and monitor treatment of gout
are really low
● Diagnosis of renal calculi - stone formation contribution
- Inversely proportional; decreased absorbance =
- Can contribute because urates are insoluble
more uric acid are converted to allantoin
● Prevent uric acid nephropathy during chemotherapy
○ Coupled enzyme (I) - H2O2 + reagent → catalase → colored
- other cells (i.e normal cells) may be destroyed aside from
compound for hydrogen peroxide measurement
cancer cells, purines from the body are released which
○ Coupled enzyme (III) - H2O2 + indicator dye → peroxidase
causes elevated uric acid
→ colored compound
- Can contribute to endogenous nucleoproteins.
■ For hydrogen peroxide measurement
- Chemotherapy = increased uric acid
■ More hydrogen peroxide is formed; more uric acid
● Detect kidney dysfunction
is present.
- Is also considered a kidney function test
● Readily automated and highly specific test
● Reference method for uric acid is the same as UREA: IDMS
Methods of Analysis for URIC ACID
Chemical method: DIRECT METHOD
Reference Intervals:
● Phosphotungstic acid (Caraway method) - based on the oxidation of
● Adult male (P/S) -3.5 -7.2 mg/dL, (0.21-0.43 mmol/L)
the uric acid in a protein-free filtrate
● Adult female (P/S) - 2.6-6.0 mg/dL, 0.16-0.36 mmol/L
- Disadvantage: Non-specific; requires protein removal
● Child (P/S) - 2.0-5.5 mg/dL, 0.12-0.33 mmol/L
(deproteinization method)
● Adult (urine/24 hour) - 250-750 mg/day, 1.5-4.4 mmol/day
- Uses PFF sample
● Principle:
○ Uric acid + H3PW12O40 (Dodecatungstophophoric acid) +
O2 → Na2CO3/OH → allantoin + tungsten blue + CO2
○ Tungsten blue (colored product): Measured colorimetrically.
○ Allantoin - this is a more soluble form of uric acid. The form
after uric acid. Humans do not have the capacity to turn uric
acid into allantoin.

Enzymatic method: INDIRECT METHOD

- Readily automated, very specific method
● This is a very specific method which utilizes uricase as the primary
enzyme. Uricase is part of the reagent

Specimen Consideration
● Uric acid testing: May be measured using heparinized plasma,
serum or urine (green top tube)
● Avoid gross lipemia, high bilirubin concentration and hemolysis
- Can be interfering agents
- Patients with problems in lipid metabolism, inform the
physician of the characteristics of the sample.
● Avoid EDTA or fluoride additives (affects uricase method)
- Inhibits uricase activity
- Causes false decreased
● Salicylates and thiazide can falsely elevate values for uric acid
Pathophysiology
Increased concentration (hyperuricemia)
ELEVATED URIC ACID CONCENTRATION
● Enzyme deficiencies
○ Lesch-nyhan syndrome
- decreased hypoxanthine guanine
phosphoribosyltransferase - HGPRT
- Increased de novo synthesis of purine -> uric acid
formation
● Phosphoribosylpyrophosphate synthetase deficiency
○ Glycogen storage disease type 1 (G6P deficiency)
↑ triglycerides ↓ urate excretion
- Most common glycogen storage disease
○ Fructose intolerance (fructose-1-phosphate aldolase
deficiency
↑ lactate ↓ urate excretion
○ Hemolytic and proliferative process (ex. Leukemia,
lymphoma, etc.)
↑ endogenous uric acid formation
↑ Increased metabolism of cell nuclei
○ Treatment of myeloproliferative disease w/ cytotoxic drugs
○ Chronic renal disease
↓ uric acid filtration and secretion
○ Toxemia of pregnancy and lactic acidosis
↑ binding to renal tubules
● Drugs and poisons
● Purine-rich diet ( ex. Liver, kidney, shellfish)
● Increase tissue catabolism or starvation
Decreased uric acid concentration (hypouricemia)
● Liver disease
- Uric acid synthesis is in the liver; conversion of purine base
to uric acid is affected; low production of uric acid
● Defective tubular reabsorption (fanconi syndrome)
- Low reabsorption of uric acid
● Chemotherapy with azathioprine or 6-mercaptopurine
● Overtreatment with allopurinol
○ Drug used to treat tophi precipitate
○ Causes decreased de novo purine = decreased uric acid
level
URIC ACID MEASUREMENT: DO NOT REQUIRE FASTING
CREATININE
Physiology of creatinine
● 5% in the plasma and 4.5% in the urine
● Chief product of muscle metabolism
● Unlike urea, it is not affected by protein content in the diet
- FASTING NOT REQUIRED
- Recent consumption does not affect
● In the kidneys, creatinine is excreted into the plasma at a constant
rate.
The creatinine level in the plasma is related to muscle mass and
muscle/physical activity
- More physically active, higher creatinine in the plasma
- Muscle mass and muscle activity contribute to the
creatinine concentration in the plasma
- Males have higher normal values
➔ Creatinine comes from Creatine.
➔ Creatine is formed primarily in the liver.
◆ Formed from different amino acids such as arginine, glycine
and methionine.
 ◆ It is then transported to the tissues. (such as muscles)
◆ As it goes to the muscles, creatine is converted into
creatine phosphate (serves as a high-energy source) by the
catalytic enzyme called creatine-kinase (“kinase” promotes
transfer of phosphate group to another).
● In the muscle, creatine phosphate serves as the
high energy source/reservoir of muscles.
● When an individual requires energy because of
muscle activity, creatine phosphate will now lose
the phosphoric acid as energy is required.
➔ When creatine phosphate is utilized, it loses phosphoric acid
➔ When phosphoric acid is lost, a cyclic compound is formed which is
CREATININE.
● 2nd pathway:
○ Creatine in the liver, when dehydrated, can directly be
converted into creatinine.
○ The creatine in the liver can undergo dehydration (loses
water) and converts into a cyclic compound creatinine.
● When creatinine is formed in either of the 2 of the pathways:
○ Creatinine diffuses into the plasma/blood and is transported
to the kidneys where it is excreted at a constant rate.
● MOST OF CREATININE IS EXCRETED IN THE URINE
Is it normal for us to have increased creatine in the plasma? NO.
it is normally found in the muscle and liver. If it is in the blood, there is
something wrong in the muscles
- Muscle trauma, muscle disease/damage
Clinical Application of CREATININE
● Determine sufficiency of kidney function
● Determine severity of kidney damage
● Monitor the progression of kidney disease
● Measure completeness of 24-hour urine
○ Creatinine is measured to determine the glomerular
filtration function of the kidneys since it is excreted at a
constant rate
■ 24 hour urine is used as the specimen.
- 24 hour urine is collected on ambulatory patients only.
- You need to instruct the patient to collect the sample.
- Take note of the time.
- The first urine specimen must not be included or must be
voided or discarded.
- The succeeding urine specimen must be collected in a
wide-mouthed sterile container.
- Storage is important, they must place the urine bottle with
the collected specimen in a cold temperature (refrigerator)
to prevent bacterial growth and decomposition of the
chemical constituents of the urine specimen.
- Transport the specimen immediately to the laboratory as
soon as the 24 hour urine is finished.
- If the laboratory is far, transport the specimen with
ice. (cooler with ice)
- In the laboratory, Inspect the urine for discoloration or
contamination, you can accept the specimen.
- After accepting, You need to measure the total volume of
the urine collected by the patient.
- The total volume is part of the calculation.
- Pour the urine specimen in the cylinder, and
measure the total volume.
 - Return to the urine container and mix.
- Then get an aliquot. Aliquot is a small volume of specimen
taken from the well mixed specimen
- You get a 10mL (minimum allowable) worth of
aliquot which represents the 24 hour urine.
- You discard the rest of the urine specimen.
Renal clearance and glomerular filtration rate
● In GFR, creatinine is constantly secreted, plasma creatinine is
inversely proportional to the GFR.
● Plasma creatinine commonly assess the renal filtration function of
kidneys
● The higher the plasma creatinine, the slower the GFR. if the plasma
creatinine is low, GFR is fast
A= surface area = 1.73= constant for standard surface area
Ucr = Urine Creatinine
Pcr = plasma creatinine
Methods of Analysis for CREATININE
Chemical method: DIRECT METHOD
● Direct Jaffe Reaction - creatinine + picrate (reagent) → red-orange
complex (measured colorimetrically)
● Jaffe-Kinetic (rate of change of absorbance) - rate of change of
absorbance (color formation) is detected to avoid interferences of
non creatinine chromogens
● Jaffe with adsorbent - creatinine in protein-free filtrate is adsorbed
onto fuller’s earth (aluminum magnesium silicate) or lloyd’s reagent
(sodium aluminum silicate) then eluted and reacted with alkaline
picrate
○ Uses adsorbent substance
○ Adsorbent - is used to adsorb on creatine present in the
specimen.
■ With the use of adsorbent, what specific test
characteristic is enhanced?
■ SPECIFICITY is enhanced with adsorbent.
● Jaffe without adsorbent - creatine in protein-free filtrate reacts with
alkaline picrate to form colored complex
Enzymatic method: INDIRECT METHOD
● Creatininase-CK
○ Creatinine + H2O → creatininase → creatine
○ Creatine + ATP ←CK→ creatine phosphate + ADP
○ Phosphoenolpyruvate + ADP → PK → pyruvate + ATP
○ Pyruvate + NADH + H <--LD→ lactate + NAD+ (oxidized)
decreased absorbance of NAD at 340 NM.
● Creatininase-H2O2
○ Creatinine + H2O → creatininase → creatine
○ Creatine H2O --creatininase → sarcosine + ADP
○ Sarcosine + O2 + H2O ADP → sarcosine oxidase → glycine
+ CH2O + H2O2
○ H2O2 + colorless substrate → peroxidase → colored
product + H2O
- Increased absorbance is measured
- Increase absorbance is directly proportional to the
absorbance of creatinine
Reference Values AMMONIA
Population Plasma, Serum, Jeff Method Enzymatic ● Smallest fraction of NPN in plasma
or Urine Method ○ Because it is further converted into urea in the urea cycle
● Byproduct of amino acid deamination
Adult Male Plasma or 0.9-1.3 mg/dL 0.6-1.1 mg/dL ● Basically a toxic gas.
Serum (80-115 (55- 96 umol/L) NEUROTOXIC AGENT/GAS
umol/L) - Can cause irreversible
damage in the brain
Adult Female Plasma or 0.6-1.1 mg/dL 0.5-0.8 mg/dL ● Product of free amino acid
Serum (53-97 umol/L) (40- 66 umol/L) deamination
● Remove from the circulation and
Child Plasma or 0.3-0.7 mg/dL 0.0-0.6 mg/dL converted to urea in the liver
Serum (27-62 umol/L) (0-52 umol/L) In the plasma, since ammonia is gas, it is
combined with water:
Adult Male Urine 24H 800-2,000
mg/day (7.1- ● Ammonia mixed with water
17.7 mmol/day) becomes ammonium hydroxide
to be less toxic. It is removed from
Adult Female Urine 24H 600-1,800 the body.
mg/day (5.3- In the liver, most of the ammonia is
15.9 mmol/day) converted to urea. Some of the ammonia
- In general, male have greater muscle mass becomes ammonium hydroxide. Urea is
now excreted in the kidneys
Specimen Consideration of CREATININE
● Falsely increased (positive interference) Clinical application of AMMONIA:
○ Due to the presence of Glucose, alpha keto acids, ascorbate, ➔ Diagnosis of hepatic failure and hepatic coma
uric acid, cephalosporins, dopamine. - Ammonia is from deamination, when the liver is
● Falsely decreased (negative interference) abnormal/diseased, the ammonia that is produced by
○ ↑ Bilirubin, hemoglobin (hemolyzed), lipemic specimens deamination, is no longer converted to urea. From the
● BUN creatinine ratio: plasma, ammonia can be transported to different tissues
○ Comparison of the BUN and creatinine levels is a better including the CNS. neurotoxic
indicator of the source of elevation in either substance - Can lead to comatose (hepatic coma)
○ The normal BUN-creatinine ratio is 10:1 to 20:1 - Comatose due to damage to the brain tissues
● Pathophysiology of Creatinine: because of the increased level of ammonia in the
○ Increased concentration circulation due to hepatic failure.
■ Renal failure (glomerular function) ➔ Reye’s syndrome - acute metabolic disorder of the liver
■ Increased plasma concentration = decreased GFR ➔ Inherited deficiencies of urea cycle.
(inversely proportional)
- Excretion rate of creatinine in glomerulus is slow, plasma creatinine
will not be excreted fully, will be trapped in the plasma.
Methods for AMMONIA:
- Blood with tubes must be surrounded by ice to maintain
temperature. An increase in the temperature can decrease the
ammonia concentration

Chemical method: DIRECT METHOD

● Ion selective electrode (ISE)
○ Diffusion of NH3 through ion selective membrane into
NH4Cl causes the pH change which is measured
potentiometrically.
○ Membrane would react with ammonium chloride and
changes in pH and measured potentiometrically
● Spectrophotometric
○ NH3 + bromphenol blue → blue dye
○ Presence of ammonia makes the pH increase (more
alkaline)

Enzymatic method: INDIRECT METHOD

● GLDH (Glutamate dehydrogenase) - decrease in absorbance is
measured at 340 nm
○ NH4 + 2-oxoglutarate + NADPH + H ←GLDH→ glutamate +
NADP + H2O
○ Reduced to oxidized NADP
Reference Values:
Adult Plasma 19-60 µg/dL (11-30 µmol/L)

Child (10days-2 Plasma 63-136 (40-80 µmol/L)

years) µg/dlg/dL

Specimen consideration:
➔ May be measured using heparinized and EDTA tubes
- Green top/ Lavender top may be used.
➔ Samples should be centrifuged at 0 to 4 deg C within 20 minutes of
collection and the plasma or serum removed.
- Increased temp (warm) can falsely decrease ammonia level
in the sample if violated.
➔ Avoid cigarette smoking for several hours
- Can falsely increase the ammonia level
January 13, 2022
Clinical Chemistry Lecture
LIVER FUNCTION
INTRODUCTION (VIDEO 1)
Anatomy
LIVER is the Largest internal organ
- One of the most abused organs in the body
- Plays a critical biochemical role in the metabolism, digestion,
detoxification, and elimination of substances from the body.
● Large and complex organ and has many roles
○ weighs approximately 1.2-1.5 kg in a healthy adult
● Anatomically speaking, the liver is attached and located in the
diaphragm.
○ Diaphragm- muscle that separates the heart and the lungs
with the abdomen.
○ The liver is protected by the lower ribcage
○ The liver is held in place by the ligamentous attachment.
● The liver is divided unequally/asymmetrically in the right and the
left lobe.
○ The falciform ligament divides the lobes.
○ The right lobe is 6 times bigger than the left lobe.
● In terms of blood supply, the liver is a highly vascular organ. It has
many networks of vessels.
● The blood supply to the liver comes from the hepatic artery and
portal vein.
○ Hepatic artery - supplies 25% of the blood to the liver
○ Portal vein - remaining 75% is provided by the portal vein.
- When these 2 blood supplies merge, they now flow into the
“sinusoids”, which then course between individual
hepatocytes.
- Sinusoids- courses between cells in the live;
courses between individual hepatocytes
In terms of excretory system, the excretory system of the liver begins at the
bile canaliculi
● Bile canaliculi - start of the excretory system of the liver; these are
small spaces between the hepatocytes that form the “intrahepatic
ducts”.
○ intrahepatic ducts- where the excretory products of the cell
can drain. -> excretion of substance
The liver is composed of different basic microscopic units called “lobules”.
- Lobules actually divide the liver
2 major group of cells present in the liver:
○ Hepatocytes - this is the major cell in the liver.
■ Responsible for the biochemical functions of the
liver
○ Kupffer cells - these are phagocytic cells present in the liver.
They are considered as macrophages. Immunologic
protection
■ These cells protect the liver from harmful agents.
■ Provides immunity/ protection against infectious
agents.
■ Phagocytose bacteria, particulate agents present in
the liver.
■ Responsible for immunologic functions of the liver.
LIVER BIOCHEMICAL FUNCTIONS (Video 2)
- Liver has many contributions/roles to perform in the body
Functions: Overview
● Excretory and Secretory function
● Synthetic Function
● Detoxification/ drug metabolism function
● Storage function
● Digestion function
Excretory and secretory function
● Liver is basically the site for processing and excretion of
endogenous and exogenous substances.
○ In the liver, specifically in the hepatocytes, these substances
are processed and later on removed/excreted from the liver.
○ Example of a substance that is processed and
excreted is BILIRUBIN.
○ The liver is the only organ capable of processing
and excreting, degrading bilirubin.
Synthetic/Storage function
- In the liver, there are many biologic compounds that are synthesized
- Major biomolecules are formed in the liver: carbohydrates,
lipids and proteins
Carbohydrate Metabolism:
○ Glycogenesis - glycogen formation from glucose
○ Glycogenolysis - breakdown of glycogen to produce glucose
○ Gluconeogenesis - formation of carbohydrates/glucose
from non-carbohydrates sources
- These three occur in the liver, thus, the liver is
important in regulating glucose level.
Protein Metabolism - almost all proteins are synthesized in the liver
EXCEPT antibodies
○ Major plasma proteins: Albumin
○ Majority of the Alpha and Beta globulins
○ Acute phase reactants ex. C-reactive protein (CRP)
○ Coagulation proteins ex. Fibrinogen
- If there is a problem in the liver, formation of the
protein may develop hemorrhage.
- Patient may have profuse bleeding.
- Clot formation is affected if liver has damage
- Without coagulation proteins, patients with liver
disease may also develop bleeding disorders.
Lipid metabolism:
○ Major site for removal of chylomicron remnants
○ Conversion of acetyl-COA to fatty acids, TGL, and
cholesterol
■ Endogenous lipids - cholesterol formed in the liver
○ Metabolism of cholesterol into bile acids (cholesterol
metabolism)
■ Cholesterol is the precursor for the formation of
bile acids
○ Lipoproteins (VLDL, HDL) and phospholipids are
synthesized in the liver
Urea metabolism
● Urea is produced from the deamination of free amino acid in the
urea cycle (which also happens/occur in the liver)
Ketone bodies
● Ketone bodies are formed from the degradation of lipids.
Enzymes (AST, ALT, ALP, 5NT, GGT, LDH)
● AST- aspartate aminotransferase
● ALT- Alanine aminotransferase
● ALP- alkaline phosphatase
● 5NT = 5 nucleotidase
● GGT = gamma glutamyl transferase
● LDH - Lactate dehydrogenase
● Enzymes as biocatalysts are intracellular proteins (proteins inside
the cell).
○ ↑ enzyme level in plasma = cell injury/damage in liver
○ Because enzyme is no longer inside the cell, which is not
normal
○ Enzyme measurement is also useful in determining injuries
to cell/tissues
● Enzymes are also produced in the hepatocytes
Fat soluble vitamins (A,D,E,K) and several water soluble vitamins (B12)
Storage depot for glycogen.
● Glycogen can also be stored in the muscles.
Detoxification and Drug Metabolism ● Is a pigment. The principal pigment in bile
First Pass ● Also formed from destruction of heme-containing proteins such as:
First pass is a mechanism wherein all substance absorbed from the small minor heme-containing proteins.
intestine must first pass through the liver. ○ Cytochromes
○ Peroxidase
- The liver will screen this substance whether this substance is toxic,
○ Myoglobin
harmful or requires detoxification.
- These may also serve as a source of bilirubin.
- Liver serves as “gatekeeper” before distribution/circulate
- Majority of heme is from hemoglobin.
substances
● Things needed to be detoxified: (foreign materials)
- Drugs Liver is the only organ that can process and excrete bilirubin.
- Poison/intentional poison
- Bilirubin and ammonia R-E SYSTEM: BILIRUBIN
Mechanism: how they are detoxified
● Bind the material reversibly
○ Reversible binding of the material is to inactivate the toxic
compound
● Chemically modify the compound
○ So that it can be excreted.
○ The modification of the compound makes it possible for the
kidneys to excrete it.
○ Form of the compound is changed into an excretable
compound.
Processes on how liver can detoxify the substances:
○ Oxidation
○ Reduction
○ Hydrolysis
○ Hydroxylation Carboxylation
○ Methylation
- In the liver there is an important drug metabolizing system that is
specifically responsible for drug metabolism: Cytochrome P-450.
- Patients with chronic illnesses are checked for liver damage as well
- Alcohol intake are detoxified in the liver
Bilirubin metabolism:
BILIRUBIN ○ At approximately 126 days, old RBCs (deformed) are phagocytosed
- A pigment product of hemoglobin metabolism: in the reticuloendothelial system specifically in the spleen.
● End product of hemoglobin metabolism. - In the spleen, the old red cells are destroyed/lysed
- Specifically derived from the heme portion of the ○ When RBCs are phagocytosed, RBCs will be hemolyzed. Hemoglobin
hemoglobin. is released and will undergo degradation and will be catabolized.
- A large portion of bilirubin is from hemoglobin ■ From hemoglobin, globin is released and is recycled in the
 bone marrow and the liver.
■ When globin is released, heme is also released.
- From heme, iron is liberated/released.
■ Iron in the heme is released and will bind to transferrin and
will now transport iron in the liver or bone marrow during
erythropoiesis (to be recycled)
When heme and iron are released, heme is converted to biliverdin through
the catalytic activity of the enzyme heme oxygenase. (catalyst)
● When biliverdin is formed, biliverdin is further
transformed/converted into bilirubin through the catalytic activity of
the enzyme biliverdin reductase.
- Bilirubin that is formed from biliverdin is called B1 aka
indirect bilirubin/unconjugated/water insoluble (non-polar)
- It is also called as the pre-hepatic bilirubin because it is
formed outside the liver
■ B1 is now released from the reticuloendothelial (phagocytes) into
the plasma. It is insoluble. B1 needs a transporter protein which will
be ALBUMIN.
- ALBUMIN will bind to bilirubin to transport B1 in the liver
specifically in the hepatocytic surface.
On the surface of hepatocytes, albumin will be detached from bilirubin 1
- B1 (water insoluble) can enter the hepatocytes.
- Cytosol is a plasma/water like structure which will mean B1 will
need a new transporter protein which is called ligandin carrier
protein inside the hepatocyte.
■ Ligandin brings the bilirubin 1 to the smooth
endoplasmic reticulum.
- Once it reaches the Smooth ER, ligandin
detaches from B1 so that it can enter the
smooth endoplasmic reticulum.
■ B1 inside the Smooth ER, will undergo the process
of conjugation/esterification.
The conjugation process requires the specific action of UDPGT
- uridine diphosphoglucuronyltransferase (UDPGT)
- UDPG-T: enzyme responsible for the conjugation of B1
The catalytic mechanism of UDPGT: It will catalyze the transfer of
glucuronyl/glucuronide to each of the 2 propionic side chains of
bilirubin. 2 glucuronyl will be transferred.
From B1, bilirubin after conjugation, becomes bilirubin 2/B2.
- B2 is also known as conjugated bilirubin/ bilirubin diglucuronide/
water soluble bilirubin/ polar bilirubin/ direct bilirubin.
- Since this bilirubin is produced in the liver, it is also called the
HEPATIC BILIRUBIN.
B2 is now excreted from the hepatocyte, the excretion of B2 happens in the
bile canaliculi and then B2 will now be mixed with bile.
- When bile is secreted from the liver, bile is temporarily stored in the
gallbladder. (pigment mixed with the bile)
- When the individual consumes fatty foods, the bile is an emulsifying
agent.
The bile is emulsified and expelled to the small intestine (Duodenum) for
absorption
- Bile will proceed to the large intestine. In the large intestine, bilirubin
2 is converted/processed by a microbial species.
- Intestinal bacteria specifically in the COLON will now process B2.
Bilirubin 2 in the large intestine will be processed by intestinal bacteria to
become meso bilirubin.
- Mesobilirubin in the intestine is further reduced to form
mesobilirubinogen.
- Mesobilirubinogen is further converted to urobilinogen. This
happens in the colon.
- Urobilinogen is usually a colorless product.
Majority/most of the urobilinogen roughly around 80% is oxidized to
become an orange colored product known as urobilin or stercobilin.
- Urobilin and stercobilin is excreted along with the feces. It is the
pigment present in the stool sample in the feces.
The remaining 20% urobilinogen, majority of the 20% remaining
urobilinogen is reabsorbed in the extrahepatic circulation and it is recycled
and is re-excreted.
- After it is recycled in the liver, it is re-excreted
- The small quantity of the remaining urobilinogen will now enter the
systemic circulation and from the blood it will be filtered by the
 nephrons of the kidneys and will be excreted in the urine ➔ UNCONJUGATED BILIRUBIN
➔ Water insoluble
End form of bilirubin metabolism are the pigments as part of the feces or - requires albumin and ligandin as transporter protein
urine. ➔ Non-polar bilirubin
➔ Indirect reacting
- This pigment can also influence the color of the urine and stool
➔ Hemobilirubin
sample.
- direct product of hemoglobin degradation
- Increase in Hgb degradation, B1 first increase
Is it possible for bilirubin to be excreted in the form of bilirubin as is? ➔ Free bilirubin/slow
Yes. If bilirubin levels are high, it can be directly filtered as bilirubin in the ➔ Prehepatic bilirubin
kidneys. ➔ Unconjugated bilirubin
If you shake the urine sample, it will form a yellow foam which signifies high Bilirubin 2
bilirubin. Water soluble or B2 can be directly excreted in the urine as it is. ➔ CONJUGATED BILIRUBIN
B1 cannot be excreted as is since it is not water-soluble, unlike B2 ➔ Water soluble
- Do not require protein carriers
2 FRACTIONS OF BILIRUBIN: ➔ Polar bilirubin
- In the laboratory, we measure the total bilirubin. ➔ Direct reacting
- Total bilirubin is simply B1 + B2. ➔ Cholebilirubin
- If either of these 2 fractions is increased, automatically, total - B2 is basically part of bile (B2 is a pigment of bile)
bilirubin is affected, increased or decreased. - cholesterol is the precursor of bile
➔ One minute/prompt/fast bilirubin
➔ Post hepatic/obstructive/regurgitative bilirubin
Bilirubin 1 Bilirubin 2 ➔ Conjugated bilirubin
Unconjugated Bilirubin Conjugated Bilirubin
Delta Bilirubin - bilirubin tightly bound to albumin
Water insoluble Water soluble - A B2 that is tightly bound to albumin.
● It has a longer half-life than other forms of bilirubin
Non-polar bilirubin Polar bilirubin - Due to the albumin transporter.
- Having a protein transporter in the plasma makes it more
Indirect reacting Direct reacting
stable and makes longer circulation life.
Hemobilirubin Cholebilirubin ● It is formed due to prolonged elevation of conjugated bilirubin in
biliary obstruction.
Free bilirubin/Slow One-minute/Prompt bilirubin - Malignancy, tumor,
● It helps in monitoring the decline of serum bilirubin following
Prehepatic Bilirubin Post surgical removal of gallstone
hepatic/Obstructive/Regurgitative
● It reacts with diazo reagent in the direct bilirubin assay
bilirubin
- For bilirubin measurement
● It is computed by using this formula:
Bilirubin 1 ○ Total B - Direct B + Indirect B = Delta B.
 ● It is not calculated on neonatal patients 1. Pre-hepatic hyperbilirubinemia
- less than or equal to 14 days) 2. Post-hepatic hyperbilirubinemia
● Reference values: 3. Hepatocellular Combine Hyperbilirubinemia
○ Less than/ < 0.2 mg/dL
○ (< 3 micromol/L) Prehepatic hyperbilirubinemia
- “Before” the liver
Jaundice - Before bilirubin reaches the liver, the abnormality exists
- Is a sign of a problem in the body. It is not normal. It manifest in an Mechanism: too much destruction of RBCs
abnormality - There is premature hemolysis of red blood cells
● Also called icterus or hyperbilirubinemia - 126 days normal lifespan of rbcs.
We use “Jaundice” to describe a patient and we use “icterus” to describe a - <126 days, increased degradation of hemoglobin.
sample Bilirubin assay: elevated indirect bilirubin/B1.
- Jaundice- patient that is abnormally yellow - Total bilirubin is also high; but B2 is unaffected/normal
- Icterus- sample that is abnormally yellow discoloration. Conditions
Jaundice is a yellow discoloration of the skin, sclera and mucous ● Intravascular hemolysis - lysis of rbcs
membranes. ● Hemoglobinopathies -hemoglobin in red cell is abnormal;
- Sign: objective, seen by other people susceptible to lysis
● It is clinically evident when bilirubin levels exceeds 2 or 3 mg/dL ● Spherocytosis
- When you are tasked to do a phlebotomy on a patient with jaundice, ● G6PD deficiency- important in protecting cell membrane from lysis
you must be extra careful because the patient may have an from certain drugs
infectious disease. - Deficient G6PD: prone to lysis
● Autoimmunity- antibodies attack his own cells (beta cells)
Classification of Jaundice: - Autoantibodies are abnormal
According to the cause: ● Hemolytic transfusion reaction (HTR)- incompatible red cell of
- To differentiate between physiologic and pathologic jaundice, look donor to patient.
at the patient’s sclera in the eye. - Transfusion reaction from wrong blood transfusion
○ Physiologic - the cause is due to increased consumption of ● HDFN - hemolytic disease of fetus and newborn
pigmented food or beverage/ due to increased level of vitamin A in - Red cells of the fetus/newborn are attacked by antibodies of
the blood. Hypervitaminosis. the mother.
- Differentiate: If sclera is white normal but the skin is yellow, - Immune system of the mother vs. the red cells of the baby
it is physiological - Happens when the blood type of mother is different from
○ Pathologic - cause of abnormality in the processing of bilirubin. the baby because of the father’s blood type (that is more
- Accumulation of bilirubin dominant and is passed to the infant)
- Differentiate: if the sclera, skin and mucous membrane are ● Red cell degradation
all yellow it is pathologic. ● Inefficient erythropoiesis
● Pernicious anemia (Vitamin B12 def.)
- due to absence of intrinsic factor
● Defective hepatocellular uptake or conjugation
Pathologic subdivision of Jaundice : - Problem in the conjugation process (B1 is not conjugated
 and stays as is)
● Viral hepatitis
● Hereditary enzyme deficiency
● Hepatic immaturity in newborns
- Liver not fully developed in newborns.
Common denominator in most of the conditions is that there is a premature
increase of red blood cell destruction
Post-Hepatic Hyperbilirubinemia
- After the liver
- In the pathway, abnormality exists in the gallbladder, bile duct
Mechanism: failure of bile to flow to the intestine/ impaired bilirubin
excretion
Bilirubin assay: Elevated direct bilirubin/B2
Total bilirubin is also high; but B1 is unaffected/normal
- Since the problem exists in the excretion of B1
Conditions:
● Intrahepatic disruption
- Bile canaliculi anatomy problem
● Viral hepatitis- destruction of hepatocytes, globules
● Alcoholic hepatitis- alcohol can damage the liver and can affect
excretion of bilirubin
● Chlorpromazine
● Cirrhosis- stages of liver damage: fatty liver -> hepatitis -> cirrhosis
(fibrous)
● Bile duct disease- abnormality in bile duct
● Biliary disease- gall bladder disorder
● Biliary cirrhosis
● Cholangitis - inflammation of the bile duct
● Biliary atresia
● Extrahepatic bile duct destruction
● Gallstones - can block the daluyan of bile
● Carcinoma of gallbladder, bile ducts, or head of pancreas
● Bile structure inflammation or surgical misadventure
- iatrogenic - doctor-induced problem
- Bile flow obstructed by mistake
Hepatocellular combine hyperbilirubinemia
- This involved the hepatocytes (cell responsible for the biochemical
function of the liver)
Mechanisms: hepatocyte injury caused by viruses, alcohol, and parasites
Bilirubin assay: elevated direct and indirect and total bilirubin
↑B1; ↑B2; ↑TOTAL BILIRUBIN
Conditions:
● Impaired hepatocellular uptake
● Defective conjugation
● Abnormal secretion of bilirubin by the liver cells
DERANGEMENTS OF BILIRUBIN METABOLISM
1. Gilbert's Syndrome
2. Crigler-Najjar Syndrome
3. Dubin Johnson syndrome/ Rotor syndrome
4. Lucey Driscoll Syndrome
Gilbert’s syndrome- mild type of abnormality
- There is bilirubin transport deficit
- It is characterized by intermittent unconjugated hyperbilirubinemia
- There is absence of hemolysis and absence of underlying
liver disease.
- This is non-fatal/non-severe type of abnormality of bilirubin
metabolism
● Impaired cellular uptake of bilirubin
- Diagnosed in young adults (20-30 yrs. old)
- Commonly observed in caucasians; very rare in Filipinos
● Affected individuals may have no symptoms but may have mild
icterus. (nonsevere; mild type of abnormality)
- The molecular basis is related to the enzyme UDPGT.
- uridine diphosphoglucuronyltransferase (UDPGT)
- There is an abnormality in this enzyme. A
superfamily of enzyme responsible for encoding the
enzyme responsible for conjugation of bilirubin
- UDPGT comes from the superfamily of UGT
● BILIRUBIN ASSAY:
○ Elevated Bilirubin1/ B1
○ Elevated total bilirubin
- The liver’s conjugation system is partially working at approximately
30% level.
Crigler-Najjar syndrome
- There is conjugation deficit
 - A syndrome of chronic nonhemolytic unconjugated CANALICULAR MULTISPECIFIC ORGANIC ANIONIC
hyperbilirubinemia TRANSPORTER.
- An inherited disorder of bilirubin metabolism resulting from a - This protein transporter is deficient in Dubin
molecular defect with the gene involved in the bilirubin conjugation Johnson syndrome
- The ability of the liver to uptake and conjugate bilirubin is
● Infants are treated by means of phototherapy: functional.
Functions of phototherapy: Rotor syndrome:
- Promotes the photooxidation of bilirubin 1 to a less toxic ● The problem/defect is associated with the transporter
form: biliverdin. B1 oxidized -> biliverdin protein called ligandin.
- Promotes the destruction of excess B1 in the peripheral - The mechanism of DJS and RS are similar or of the same type.
circulation.
Purpose of phototherapy is to correct hyperbilirubinemia (spec. Unconjugated How do we differentiate these 2 conditions:
hyperbilirubinemia B1) ★ By examining the liver biopsy sample of the patient. When the liver
2 types of crigler-najjar: is stained under the microscope.
- Type 1 Crigler-Najjar Syndrome- There is complete ○ In patients with DJS, their liver biopsy sample/slide, has
absence of enzymatic bilirubin conjugation. darkly stained granules due to the pigmented lysosomes.
- No B2 is formed at all. ○ In patients with RS, there is no pigmentation or dark stained
- The color of the patient's bile is colorless. granules in the liver biopsy.
- A rare condition.
- Type 2 Crigler-Najjar Syndrome- There is mutation in the Lucey driscoll syndrome
genes causing a severe deficiency of the enzyme ➔ Familial form of unconjugated hyperbilirubinemia
responsible for conjugation. ◆ This is an inherited type
- Enzymes (B2) are formed but severely deficient. ➔ B1 is elevated/increased along with total bilirubin.
- Markedly low B2 are formed. - also known as the transient familial hyperbilirubinemia
➔ This is a rare condition wherein B1 and total bilirubin level of the
Dubin Johnson syndrome and Rotor syndrome newborn is abnormally increased.
- Have the same pathogenesis, origin, and mechanism ↑ Total Bilirubin ; ↑ B1
Mechanism: there is bilirubin excretion deficit.
- There is problem in B2 excretion Type of Jaundice Total bilirubin Conjugated (B2) Unconjugated (B1)
- B2 is already conjugated but has abnormal excretion
Prehepatic increased/high unaffected /normal increased/high
➔ Blockade of the excretion of the bilirubin into the canaliculi
➔ Characterized by elevated total bilirubin and B2 Hepatic
↑ Total Bilirubin ; ↑ B2
Gilbert’s dse increased unaffected /normal increased/high

Dubin Johnson syndrome Crigler-Najjar increased/high decreased/low increased/high

syndrome
● This is a rare inherited disorder
● There is a deficiency of protein transporter which is called C-NS Type 1 increased/high ABSENT Severely high
“MDR2-cMOAT”: CANALICULAR MULTIDRUG RESISTANCE/
 Dubin Johnson increased/high increased/high unaffected /normal
- Conjugated bilirubin (B2) that is covalently bound to
albumin
Rotor syndrome increased/high increased/high unaffected /normal - B2 that is attached to protein
- Seen only when there is significant hepatic obstruction
Jaundice of increased/high unaffected /normal increased/high
Newborn - Not filtered in the kidneys
- When attached to albumin, it is too large for the
Posthepatic increased/high increased/high unaffected /normal glomerulus to filter
- Not excreted in the urine
- Measurement of three fractions = total bilirubin
Liver Function Tests (Laboratory)
- Directly affected by the changes of the
LIVER is the Largest internal organ concentration of the fractions.
- One of the most abused organs in the body - If any of the fractions is either increased/
- Plays a critical biochemical role in the metabolism, digestion, decreased, TP will be affected.
detoxification, and elimination of substances from the body.
BILIRUBIN Specimen collection and Storage
> A pigment product of hemoglobin metabolism: ● Serum or plasma can be used for method using diazotized
Two fractions: Conjugated (Direct) and Unconjugated (Indirect) sulfanilic acid
- Conjugated (direct): B2 ○ If the method is evelyn -malloy: serum is preferred over
- aka POLAR/WATER SOLUBLE TYPE plasma
- Commonly known as B2 ○ In EM, the accelerator is alcohol which can precipitate
- Found in the plasma in the “free state” (no protein carrier proteins in the plasma and cause interference
nor attachment) ● Serum is preferred in EM Method
- In the Lab: DIRECTLY reacts with the diazo reagent ● Fasting sample is preferred
- Diazo reagent: DIAZOTIZED SULFANILIC ACID ○ Lipemia will falsely increase the bilirubin concentration
SOLUTION ○ Lipemia will falsely increase bilirubin concentration;
- The direct reaction of B2 to diazo is due to absence because it will indicate that the patient did not undergo
of ACCELERATOR fasting
- Accelerator: serves as solubilizer ○ Bilirubin measurement is performed with FBS, lipid profile
- Insoluble substance needs accelerator to be ○ Bilirubin is performed in the morning
soluble ● Hemolyzed sample should be avoided
- Unconjugated (Indirect): B1 ○ Hemolyzed specimen may decrease the reaction of bilirubin
- Aka NON-POLAR/ WATER INSOLUBLE TYPE with the diazo reagent.
- Commonly known as B1 ○ Falsely decreased
- In the plasma, indirect bilirubin is bound to protein carrier: ● Specimen should be protected from light
Albumin (carrier protein) ○ Bilirubin is photosensitive substance
- In the Lab: the reaction with diazo reagent is INDIRECT ○ When exposed to light, the bilirubin is converted
- Due to the requirement of accelerator reagent, so B1 (photoconversion) and the level can be decreased by
is solubilized and could react with the diazo reagent. 30-50% per hour of exposure.
■ Cover with carbon paper
- DELTA Bilirubin ■ When stored in freezer, cover
 ■ At room temperature, serum/plasma can be stored
in the dark for 2 days; bilirubin will still be stable.
(room temp: 20-24degC)
■ At 4degC, bilirubin is stable for up to 1 week in a
dark storage.
■ At -20degC or colder, bilirubin can be stored and
preserved in the dark indefinitely.
SOURCES OF ERROR:
● Instrument should be frequently standardized
○ To maintain reliable bilirubin results
○ Reliable results when there is both accuracy and precision
○ Machines should be frequently checked and standardized
using controls
● Careful preparation of bilirubin standards is critical
○ Make sure that it is not directly exposed to light
○ Bilirubin in the sample may undergo deterioration when
exposed to light
● Hemolysis and lipemia should be avoided
○ Can alter bilirubin concentration
○ Hemolysis (decrease) and Lipemia (increase) causes error
● Serious loss of bilirubin occurs after exposure of samples
○ Exposure of sample to sunlight
● Exposure of light be kept to a minimum
○ 30-50% decrease per hour
○ Storage: minimum exposure to light
○ It must be stored in the dark until testing is performed
METHODS:
Jendrassik-Grof
Characteristics:
- Uses the caffeine benzoate as stabilizer
- Used to soluble all insoluble type of bilirubin
- Not affected by pH
- No need for pH adjustment in the test
- Insensitive to a 50 fold variation in the
- Not affected by hyper/hypoproteinemia
- Maintains optical sensitivity even at low bilirubin
concentration
- Has minimal turbidity and relatively constant serum
blank/lock
- Not affected by hemoglobin by up to 750mg/dL
- >750mg/dL Hgb conc. = interference will occur
- Use solubilizer in B1 only, total of B1 and B2 = TB
Evelyn-Malloy Procedure
Characteristics:
- Serum is the preferred specimen
- The diazotized sulfanilic acid reacts at the central methylene
carbon of bilirubin to form azo-bilirubin molecule
- pH dependent, 1.2 pH
- At this pH, the azobilirubin will produce a red-purple color
- Maximum absorbance of 560nm. (spectrophotometrically)
- The accelerator used is: methanol
Additional Characteristics
● Ascorbic acid is added to the aliquot tested
○ Used to destroy the excessed diazo reagent
○ Excess diazo reagent that did not react can cause additional
reading.
● The solution is alkalinized using an alkaline tartrate solution
○ Alkalinization of the soln promotes shift in the absorbance
spectrum of the azobilirubin to a more intense color
■ More intense color= less effect of the interference
of the substance in the sample
■ Commonly performed in Jendrassik-Grof method
● The final blue product is measured at 600 nm (JG)
○ In EM, red-purple @560nm
○ In JG, blue @600nm
- Can we directly obtain the value for indirect (B1) concentration in
the sample? No. because, it needs to be solubilized and converted
into more soluble form
TB = B1 + B2
B1= TB - B2
B1 is not directly measured in the laboratory; TB and B2 is measured
but not B1 because it needs prior conversion
Urobilinogen in Urine and Feces - porphobilinogen
Urobilinogen - a colorless end product of bilirubin metabolism that is - Sulfonamides`
oxidized by the intestinal bacteria to become brown pigment urobilin. - Procaine
- A small portion that is not taken up by the hepatocytes is excreted - 5-hydroxyindoleacetic acid
by the kidney as urobilinogen (can be measured in urine and stool) - Bilirubin in the urine
- In the normal individual, a small portion urobilinogen is excreted in 3. Fresh urine is necessary, and the test must be performed without
the urine and feces while some portion can be reabsorbed and taken delay
up in the blood to be excreted in the urine - If the urine specimen is allowed to stand at room temp,
- Increased urobilinogen = hemolytic diseases, defective liver function there is possible oxidation of urobilinogen to urobilin.
(hepatitis) - To prevent, do not delay the measurement.
- Decreased urobilinogen= biliary obstruction (bara sa bile duct) End color reaction must be measured within 5 minutes in the
spectrophotometer because the end color intensity slowly decreases when
there is delay in the measurement.
Determination of Urine Urobilinogen (semiquantitive)
Serum Bile Acids
Principle:
- Serum Bile Acids analysis is rarely performed because the methods
● Urobilinogen reacts with p-dimethyl aminobenzaldehyde to form a
required are very complex
red color which is then measured spectrophotometrically
- Bile acid analysis involves:
● Ascorbic acid and saturated sodium acetate are added to the
- Extraction with organic solvent
sample aliquots
- Partition chromatography
○ Ascorbic acid = reducing agent to maintain urobilinogen in
- Gas chromatography
the reduced state
- UV light absorption
○ Saturated Sodium acetate = used to stop the reaction and
- Mass spectroscopy
minimized the combination of other reagent with the Erlich’s
- Fluorescence method and radioimmunoassay
■ Erlich’s reagent= paradimethylaminobenzaldehyde
- Not routinely performed method
Specimen
BROMSULFONTHALEIN (BSP) DYE EXCRETION TEST
● A fresh 2-hour urine specimen is collected
- Bile acid method is used for potency of bile ducts and
○ Usually, the time of collection is usually done in the
hepatocellular function
○ afternoon
1. Rosenthal White
○ afternoon= increased urobilinogen concentration
- Known as double collection method
● Strictly follow specimen storage protocol
a. Dose: 2mg/kg body weight of the patient
○ Urine specimens must be placed in a room, cool temp, and
b. Collection: : done after 5 minutes and after 30 minutes after
protected from light.
dye administration in the patient.
Comments and Sources of Error
Normal value: After 5 minutes: 50% dye retention
1. The results of this test are reported in Erlich’s unit rather than in
After 30 minutes: 0% dye retention
milligram of urobilinogen
- Erlich unit = approx. 1 mg of urobilinogen 2. Mac Donald Method
2. Compounds, other than urobilinogen, that may be present in the - Known as single collection method
urine and react with ehrlich's reagent may cause interference and a. Dose: 5mg/kg body weight of the patient
must be removed. b. Collection: done after 45 minutes
Compounds that may interfere: Normal Value: after 45 minutes: +/- 5% dye retention;
 ● Aside from these tests, NPNs are also used to determine liver
function (urea, uric acid, ammonia) as well as protein, specifically
albumin
○ Albumin is commonly used because albumin is the
predominant protein in the plasma which is made in the live
Liver Function Test:
- Urea
- Albumin- most predominant protein in the plasma
- NPNs
- Enzymes:
- Aminotransferases
- ALT (alanine amino transferase), AST (aspartate
amino transferase
- Phosphatases
- alkaline phosphatase
- 5' nucleotidase
- GGT (Gamma glutamyltransferase)
- LDH (Lactate dehydrogenase)
Common denominator: these enzymes are used to assess liver function and
to determine presence of damage/ abnormality in the liver
- Because these are produced and seen in the
hepatocytes
- Increased values: indicates injury and damage to
liver
- Because they are normally seen inside the cell
(intracellular)
Test measuring Haptic Synthetic Ability
● Serum Albumin
○ More commonly measured; Predominant protein
● Serum Globulin
- Used to assess the ability of the liver to synthetically
produce these substances
- Decreased values = severe damage/ severe impairment,
chronic liver diseases
- Tested because all proteins are produced in the liver except
immunoglobulin
-