Medical Laboratory Science Assessment Program 2

L.1 CLINICAL CHEMISTRY

August 19 , 2022 | Sir Eric E. Carpo, RMT, MS, MT (AMT), (ASVPi)

OUTLINE DISACCHARIDES
● contains 2 monosaccharides covalently bound to each
I. Carbohydrates V. Clinical Enzymology other by glycosidic linkage
II. Lipids VI. Proteins ○ Upon hydrolysis will split into 2 monosaccharides
III. Non-Protein Nitrogen & VII.Liver Function Tests ■ Sucrose(C12H22O11): glucose + fructose
Kidney Function Tests VIII. Blood Gas Analysis ● NON REDUCING SUGAR
IV. Fluid Balance & ■ Lactose: glucose + galactose
Electrolytes ■ Maltose: glucose + glucose
CARBOHYDRATES ○ Inorder to hydrolyse the glycosidic bond, there must
● General formula: CnH2nOn or Cn(H2O)n or (CH2O)n be an enzymes
■ Sucrase - inorder to breakdown sucrose
● “Hydrates of Carbon”
■ Lactase - inorder to breakdown lactose
● Polyhydroxy aldehyde or Polyhydroxy ketone ■ Maltase - inorder to break down maltose
○ Many hydroxyl group ○ This enzyme will breakdown the Disaccharides into
○ To know if the sugar is an aldehyde or ketone, it is monosaccharides to be absorbed in intestine
based on the location of C=O (Carbonyl group) ● Oligosaccharides: composed of 3-10 monosaccharides
functional group covalently bonded to each other
○ usually found in associated with proteins & lipids in
complex molecules that have both structural &
regulatory functions
● Polysaccharides: hundreds to thousands of
monosaccharides
○ Starch - storage form of sugar in plants
○ Glycogen - storage form of sugar in humans

● Those that contain aldehyde group is called an aldose NICE TO KNOW:

Humans consume polysaccharides (disaccharides the
○ Aldose: If there is hydrogen attached to the
least) and not pure glucose. Pure glucose can only be
carbonyl group consumed in powdered form
● Those that contain a ketone group is called a ketose
○ Ketose: If there is two carbon atoms attached to the GLUCOSE METABOLISM
carbonyl group ● Glucose is the primary source of energy for humans
● The simplest/smallest functional sugar contain n=3 ○ converted to ATP
● Glyceraldehyde is the simplest aldose ● The brain is COMPLETELY dependent on glucose for
● Dihydroxyacetone is the simplest ketose energy; nervous tissue cannot concentrate or store CHO
therefore it is critical to maintain a steady supply of
glucose to the tissue; that’s why we feel dizzy in low
CLASSIFICATION OF CARBOHYDRATES blood sugar
A. ACCORDING TO GROUP THEY CONTAIN ● Starch is broken down into smaller sugar units by
● Aldehyde salivary amylase and pancreatic amylase. The
● Ketone disaccharides are hydrolyzed by sucrase, lactase and
B. ACCORDING TO THE NUMBER OF C ATOMS maltase in the intestine.
3 C - triose glyceraldehyde ● Terminologies in glucose metabolism:
4 C - tetrose erythrose ○ Glycolysis - metabolism of glucose to pyruvate or
5 C - pentose ribose lactate for energy
6 C - hexose glucose ○ Glycogenesis - conversion of glucose to glycogen
7 C - haptoses sedoheptulose ○ Glycogenolysis - breakdown of glycogen to
glucose for energy
8 C - nonose neuraminic acid
○ Gluconeogenesis - formation of glucose from
● Pentoses (5 carbon atoms) and Hexoses (6 carbon
non-carbohydrate sources (amino acids, fatty acids
atoms) biologically most important for our existence
& glycerol)
● The blood glucose level is maintained at a fairly constant
C. ACCORDING TO THE NUMBER OF SUGAR UNITS
level by the liver.
(MOLECULAR SIZES)
● RENAL THRESHOLD
MONOSACCHARIDES
○ Glucose is filtered by the glomerulus & reabsorbed
● single/simplest polyhydroxy aldehyde or polyhydroxy
by the tubules to a limit
ketone
○ If the glucose levels exceed 160-180 mg/dL some
○ Can NOT broken down into simpler units by
glucose will escape in the urine in most individuals
hydrolysis
→ Glucosuria
○ Ranges from 3C to 7C; 5C & 6C are most common
○ It varies from individuals, maybe very high FBS with
○ Ex. glucose(C6H12O6), fructose, galactose,ribose &
no glucosuria, maybe normal FBS with glucosuria.
deoxyribose
● the complete oxidation of glucose yields CO2, H2O &
○ Not a product of hydrolysis dahil sila na yun!
ATP

MICO’22 1
● pathways in glucose metabolism: INSULIN
○ Embden-Meyerhof pathway ● Secreted by the beta cells of the Islets of Langerhans in
○ Hexose monophosphate pathway the pancreas
○ Tricarboxylic pathway ● It is a peptide hormone with a mass of approximately
NOTE: 5800 daltons.
● -lysis = Breakdown
● It has a 21-amino acid A chain and 30-amino acid B
● Insulin = most important hormone in BioChem
● 70 - 110 ng/dL = normal blood sugar chain linked by the disulfide bonds (C chain/C peptide: by
● Glycolytic action = decrease blood sugar product of A and B chains).
○ converted to energy (ATP) ● It is initially synthesized as a longer single-polypeptide
○ Insulin is the only hormone capable to convert chain called Pre-proinsulin (11500 daltons), cleavage
glucose to ATP results to Proinsulin (9000 daltons), the immediate
● Glycogen are stored in muscles and liver by precursor of insulin. Proinsulin has only 5% activity of
glycogenesis
insulin.
○ Decrease blood sugar levels
● Glycogenolysis = increase blood sugar
○ By action of glucagon (counters insulin)
● Glucagon maintains the 70-110 mg/dL glucose level
when we are in fasting state
TERMINOLOGIES IN GLUCOSE METABOLISM
(PATHWAYS)
● Glycolysis: Metabolism of glucose to pyruvate or lactate
for energy.
● Glycogenesis: Conversion of glucose to glycogen.
● Glycogenolysis: Breakdown of glycogen to glucose for
energy. NOTE::
● Gluconeogenesis: formation of glucose from ● C-peptide is low/absent in Type I Diabetes and normal
non-carbohydrate sources (amino acids, fatty acids ang to sufficient in Type II Diabetes (normal synthesized
glycerol) insulin)
● Glucose is the only sugar to be directly used for energy ● C-peptide levels in INSULINOMA (cancer of beta cells)
or stored as glycogen. are HIGH
● Glucose does not accumulate in the muscle, it can only ○ cancer → neoplasia → maraming cells secreting
enter the muscle cell with the help of insulin, then it is insulin)
quickly metabolized. ○ INSULINOMA becomes a significant cause of
○ Insulin is the most important hormone in clinical PERSISTENT HYPERGLYCEMIA.
biochemistry because glucose can only enter the ● Too much insulin causes low glucose levels even after
muscle with the help of insulin. It helps the binding eating and correction. To identify the source of excess
process in the receptor. insulin, doctors order a test for C-peptide.
HORMONAL REGULATION ○ Low C-peptide levels → exogenous insulin
● Pancreas: Act as both exocrine and endocrine organ in (medication/injection)
the control of CHO metabolism. ○ High C-peptide levels → hyperfunction of
As an exocrine gland: Produces amylase, protease pancreas (problem with beta cells)
(digestive protein) ● proinsulin is converted to insulin by the enzymatic
As an endocrine gland: Secretes hormones such removal of the 31 amino acid peptide segment that
as insulin, glucagon, somatostatin connects the A & B chains known as C-peptide
● it is the only hypoglycemic agent
● it promotes glycogenesis, lipogenesis & glycolysis; it
decreases glycogenolysis
● it enhances membrane permeability to cells in the liver,
muscle & adipose tissue
● insulin assays may be falsely low in hemolyzed
specimens
GLUCAGON
● secreted by the alpha cells
● it stimulates glucose production - hyperglycemic agent
● it regulates hepatic glycogenolysis, gluconeogenesis &
ketogenesis
● it its released during stress & fasting states
● proglucagon is synthesized by the alpha cells of the
Islets & L cells of the distal small bowel
SOMATOSTATIN
● secreted by the delta cells of the Islets of Langerhans
● it inhibits insulin, glucagon, & growth hormone action
● it is also synthesized in the paraventricular & arcuate
nuclei of the hypothalamus (neuroendocrine hormone)

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 THYROID HORMONES (T3 AND T4) DIABETES MELLITUS
stimulates glycogenolysis & gluconeogenesis ● This is a group of metabolic disease associated with
EPINEPHRINE hyperglycemia due to absolute insulin secretion or
● secreted by the chromaffin cells of the adrenal medulla abnormal insulin action → pertains to insulin resistance
● stimulates glycogenolysis & lipolysis; inhibits insulin (Type 2 DM)
● This is a group of diseases in which blood glucose levels
CORTISOL AND CORTICOSTEROID
are elevated because of deficient insulin secretion and/or
stimulates gluconeogenesis & lipolysis; decreases intestinal
abnormal insulin action
entry of glucose into the cell ● Fasting plasma glucose ≥ 126 mg/dL on more than 1
GROWTH HORMONES testing are diagnostic
● secreted by the anterior pituitary
● stimulates glycogenolysis glycolysis; insulin antagonist SYMPTOMS OF DIABETES MELLITUS
ADRENOCORTICOTROPIC HORMONE (ACTH) Primary symptoms: Acute episodes of DM
● secrete by the anterior pituitary ● Hyperglycemia ● Ketonemia
● stimulates glycogenolysis & gluconeogenesis; insulin ● Glucosuria - exceed the ● Ketonuria
antagonist renal threshold
CLINICAL CONDITIONS OF CHO METABOLISM ● Polyuria
HYPERGLYCEMIA ● Polydipsia
● increase of blood glucose concentration ● Polyphagia
○ FBS ≥ 126 mg/dL ● Sudden weight loss
○ RBS > 200 mg/dL
○ 20hr PPBS > 200 mg/dL ADDITIONAL INFORMATION
○ HBA1c > 6.5%
○ Lab findings:
■ increase glucose in plasma and urine
■ increase in urine specific gravity
■ (+) ketones in serum and urine
■ decrease blood and urine pH
■ electrolyte imbalance (low Na, high K, low
HCO3)
HYPOGLYCEMIA
● decrease glucose levels
○ imbalance between glucose utilization and
production
○ symptoms are related to the CNS (brain is
completely dependent on blood glucose levels)
○ patients with hypoglycemia must meet the
Whipple’s triad:
1. low blood glucose
2. typical symptoms associated with hypoglycemia
3. symptoms are alleviated by glucose
administration ● In diabetes mellitus there is insulin resistance and
○ symptoms: hyperglycemia
■ neurogenic - tremors, palpitations, anxiety & ● 3P’s of Diabetes Mellitus:
diaphoresis ○ Polyuria - excessive urine output
■ neuroglycopenic - dizziness, tingling, blurred ■ In the principle of osmolality, when there is
vision, confusion & behavioural changes an increased level of dissolved substances
○ Lab findings: which is the glucose (considered to be a
■ 65-70 mg/dL levels result in release of hyperosmolar substance). The intracellular
glucagon & other glycemic hormones fluid in the cell will draw out water going to
■ 50-55 mg/dL levels result in observable the vascular component. The water content
symptoms of hypoglycemia in the blood vessel will be increased causing
an increased level of urine output which is
CLASSIFICATION (HYPOGLYCEMIA):
termed as the polyuria. .
● Drug administration: insulin, alcohol (hungry feeling ○ Polydipsia - increase in water intake
when drunk), salicylates, sulfonamides, pentamidine ■ When a person keeps on urinating,
● Critical illness: hepatic failure, renal failure, cardiac obviously he/she will feel dehydrated,
failure, sepsis, malnutrition therefore there is an extreme feeling of
● Hormonal deficiency thirstiness.
● Endogenous hyperinsulinism: pancreatic beta-cell ○ Polyphagia - excessive food intake
disorders ■ The glucose in the blood vessels cannot
● Autoimmune hypoglycemia: insulin autoantibodies enter the cells due to lack of insulin,
● Non-beta cell tumors: leukemia, hepatoma, lymphoma therefore this cannot be converted into ATP
● Hypoglycemia of infancy or childhood: galactosemia, which will serve as the source of energy.
Reye’s syndrome ● Low sodium levels (decreased sodium levels)
● Alimentary (reactive) hypoglycemia - post-gastric ○ This is associated with polyuria. The sodium is
surgery considered to be an extracellular cation (found
● Idiopathic (functional) postprandial hypoglycemia outside the cell). When there is an increase in
urine secretion, the sodium will also be excreted

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 along with the urine resulting in the decrease of ● In severe DM, the ratio of B-hydroxybutyrate to
sodium levels. acetoacetate is 6:1
● Low in pH (decreased in pH levels) = acidosis ● The entire process of ketosis can be reversed by insulin
(Type1) administration
○ High levels of hydrogen ions ● Patients will present with a fruity breath odor and deep
○ Low levels of bicarbonate caused by acidosis respirations
○ This is due to the decrease in insulin levels ○ Deep respirations is a compensatory action of the
because the muscles are depleted of the source body to release carbon dioxide which is an acid
of energy, so maghahanap siya ng ibang source
of energy which is the fatty acids. The fatty acid
will now be the source of ATP and will undergo
beta oxidation process producing 3 ketone
bodies which are:
■ β-hydroxybutyric acid (78%)
■ Acetoacetic acid (20%)
■ Acetone (2%)

CLINICAL CONDITIONS OF CARBOHYDRATE

METABOLISM TYPE 2 DIABETES MELLITUS
Hyperglycemia - increased of blood glucose concentration ● Formerly known as “Non-insulin Dependent DM”
FBS ≥ 126 mg/dL (NIDDM)
RBS > 200 mg/dL ○ Adult type
2hr PPBS >200 mg/dL ○ Maturity onset
HBA1c >6.5% ● Most common type of DM which represent 90-95% of
cases
LABORATORY FINDINGS: ● Characterized by hyperglycemia due to insulin
● Increase glucose in plasma and urine resistance; there is minimal insulin deficiency
● Increase in urine specific gravity ● Ketosis is rare
● Mechanisms involved in the development of type 2 DM
● Positive with ketones in serum and urine
○ As a compensatory mechanism for glucose
● Decrease blood and urine pH tolerance the pancreas has to secrete insulin
● Electrolyte imbalance ○ Hyperglycemia is also toxic to the Cells of the
○ low sodium: together with insulin (tinatangay ni pancreas disrupting function and impairing insulin
insulin) secretion
○ High potassium RISK FACTORS:
○ Low bicarbonate ● Obesity
● Sedentary lifestyle
CLASSIFICATION OF DIABETES
● Family history
TYPE 1 DIABETES MELLITUS
● Formerly known as “Insulin Dependent DM” (IDDM) ● Advanced age
○ Juvenile onset DM ● Ethnicity
○ Ketosis-prone DM ● History of gestation DM
● Represent 5-10% of cases ● Impaired glucose metabolism
● Results from the autoimmune destruction of the beta ● Hypertension
cells causing absolute insulin deficiency (Insulinopenia) ● Dyslipidemia
○ Absolute dependence on insulin
● Has milder symptoms compared to type 1. However,
● Genetic susceptibility is related to the inheritance of
untreated type 2 DM will result to nonketotic
specific immune response genes associated with HLA
hyperosmolar coma (ketosis if type 1)
DR/DQ on chromosome 6
○ Overproduction of glucose (>500 mg/dL)
● Trigger events for immune destruction of beta cells
○ Severe dehydration, electrolyte imbalance, and
include viral infection, toxic exposure, or stress
increased BUN and creatinine
MARKERS FOR BETA CELL DESTRUCTION:
■ Hyponatremia (Outflux of sodium with water)
● Islet Cell Antigen 512 autoantibodies (ICA 512)
■ Renal failure
● Glutamine Acid Decarboxylase antibodies (GAD 65) ○ Type 2 is more manageable than type 1 as long as
○ More common in adult who develop type 1 DM you take oral meds like metformin or insulin
● Insulin Autoantibodies (IAA) injection
○ More common in young children who develop type 1 ● Laboratory findings
DM ○ Increased glucose in plasma and urine
● Tyrosine Phosphatase-like Protein Antibodies (IA-2) ○ Increased urine specific gravity
or Insulinoma Associated Antigen-2 and 2 beta ○ Increased serum and urine osmolality
autoantibodies (IA-2A, IA-2BA) ○ Ketones in serum and urine (Ketonemia and
● Ketosis is more common; plasma insulin concentration is ketonuria)
low or absent; family history of DM is less common; ■ Acetoacetate, B-hydroxybutyrate and acetoin
habitus is lean is produced from FA
● Ketosis develops in DM from excessive synthesis of ○ Decreased blood and urine pH (Acidosis)
acetyl-CoA as the body attempts to obtain required ○ Electrolyte imbalance
energy from stored fat in the absence of an adequate ■ Decreased sodium: Polyuria and shift of
supply of carbohydrate metabolites water from cells
○ The presence of ketone bodies is a frequent finding ■ Increased potassium: Displacement from
in individuals with severe, uncontrolled diabetes cells in acidosis

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 DRUG THAT INTERFERE W/ INSULIN RELEASE FROM
B CELLS
● Cyclosporine
● Thiazides and phenytoin inducing insulin resistance
● Glucocorticoids
● Oral contraceptives

LABORATORY METHODS FOR GLUCOSE ANALYSIS

1. SPECIMEN HANDLING AND STORAGE
● Glucose is measured in whole blood, plasma, serum,
CSF, pleural fluid and urine for a variety of diagnostic and
management purposes
GESTATIONAL DIABETES ● The standard clinical specimen is venous plasma
● Impaired inability to metabolize glucose during glucose
pregnancy due to insulin deficiency, metabolic or ● Serum should be separated from the red cells within 30
hormonal changes
minutes, but if serum is in contact with cells for >30
● Increased risk of developing type 2 DM
● Screening is a common practice between 24-28 weeks of minutes, sodium fluoride should be added
gestation ● Glucose is metabolized at room temperature at a rate of
● GDM converts to DM within 10 years in 30-40% of cases 7 mg/dL per hour; at 4oC the loss is approximately at 2
mg/dL per hour
● The rate of metabolism is higher with bacterial
contamination & leukocytosis
● Whole blood specimens tend to give 10-15% lower
glucose readings than plasma glucose
● If whole blood is refrigerated, 2 mg NaF per mL of blood
prevents glycolysis for 48 hours
● When refrigerated serum & plasma glucose is also stable
for 48 hours
● With long term storage even at -20oC, glucose values
decrease significantly & progressively
● In serum specimens without bacterial contamination of
leukocytosis results remain clinically acceptable even
after a delay of 90 minutes before separation of serum &
cells

2. LABORATORY TESTS FOR GLUCOSE

A. FASTING BLOOD SUGAR (FBS)
● no calorie intake for 6-8 hours
● values have diurnal variation with the mean FBS higher
in the morning than in the afternoon
SECONDARY DIABETES MELLITUS ● it is a measure of overall glucose homeostasis
Caused by other conditions and diseases
● reference value: 70-110 mg/dL
PANCREATIC DISEASE B. RANDOM BLOOD SUGAR (RBS)
● Acromegaly (GH excess) ● usually requested during insulin shock & hyperglycemic
● Cushing's disease (Excess cortisol)
ketotic coma
● Pheochromocytoma (Excess catecholamines)
● Glucagonoma (Excess glucagon) ● reference value: <200 mg/dL
● Somatostatinoma (Excessive production of somatostatin) ● used in emergency room
● Primary aldosteronism C. 2HOUR POSTPRANDIAL BLOOD SUGAR (2PPBS)
● Severe liver disease ● measures how well the body metabolizes glucose
● Administration of certain drugs, hormones, and ● reference value: <140 mg/dL
chemicals
MATURITY ONSET-TYPE DIABETES OF THE YOUNG D. GLUCOSE TOLERANCE TEST (GTT)
(MODY) ● either 75g (standard) or 100g glucose load
Occurs in younger individuals who have impaired pancreatic ● multiple blood sugar tests (fasting specimen, 1hr, 2hr,
B cells and still can produce insulin but demonstrate insulin 3hr)
resistance
● it is used to determine how well the body metabolizes
GENETIC DISEASE glucose over a required period of time, same with 2PPBS
● Down syndrome ● it should be performed to diagnose GDM
● Klinefelter’s syndrome ● it is not generally recommended for routine clinical use in
● Rabson-Mendenhall syndrome
● Huntington’s chorea diagnosis of diabetes
● Turner’s syndrome

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 KINDS OF GTT: I. C-PEPTIDE
1. Oral glucose tolerance test (OGTT) ● the amount of circulating C-peptide provides reliable
a. Janney-Isaacson Method (single dose) - most indicators for pancreatic & insulin secretions
common ● it can be used to monitor individual responses to
b. Exton Rose Method (divided dose or double dose) pancreatic surgery
2. Intravenous Glucose Tolerance Test - used for DM ● this mainly evaluates hypoglycemic & continuous
patients with GI disorders; 0.5g of glucose per kg body assessment of beta cell function
weight is given within 3 minutes intravenously ● specimen: fasting serum
● increased in: insulinoma, type 2 DM, ingestion of
REQUIREMENTS FOR GTT: hypoglycemic drugs
1. Patient should be ambulatory ● decreased in: type 1 DM
2. Unrestricted CHO diet of 150g per day for 3 days prior ● Reference value: 0.90-4.3 mg/mL
testing
3. No smoking or drinking alcohol prior testing ADDITIONAL INFORMATION:
4. Fasting for 6-8 hours ● Serum is mostly used specimen in laboratory
● Gold tube is the alternative tube for red tube in serum
5. Glucose load - 75g or 100g: 1.75g of glucose per kg
● In GTT, if 75g then upto 2hr only but if 100g then upto
body weight for children (maximum of 75g) 3hr

PROCEDURES FOR GTT: GLUCOSE METHODOLOGIES

1. Fasting specimen is first collected, followed by drinking I. CHEMICAL METHODS
(within 5 minutes) of 75g or 100g oral glucose load in No hospital or laboratories employ this method anymore, it is
300-400ml water now of historical interest, yet being asked in the board exam.
A. OXIDATION - REDUCTION METHODS
2. Collect specimens after 1hr, 2hr, 3hr
3. Patient should avoid exercise, eating, drinking & smoking 1. Alkaline Copper Reduction Method
during the test ● Principle: Glucose (a reducing agent) reduces cupric
E. GLYCOSYLATED HEMOGLOBIN (HBA1C) ions in alkaline solution forming → cuprous oxide
● is formed from the non-enzymatic, irreversible ● The solution loses its deep blue color and a red
attachment of glucose to HbA1c precipitate forms (when mixed with urine).
● reflects the blood glucose levels for the past 2-3 months ● Benedict’s and Fehling’s reagents, which contain alkaline
● it is useful in monitoring effectiveness of treatment & solution have been stabilized by citrate and tartrate
compliance of diabetic individual to treatment protocol respectively and have been used to detect reducing
● should be performed every 3-6 months in DM patients agents in blood and urine.
monitoring glycemic control Alkaline Copper Tartrate → cuprous ions
● specimen is non-fasting EDTA collected blood A. Folin Wu: cuprous ions + phosphomolybdate →
● affected by diseases associated with shortened red cell phosphomolybdenum blue
survival, hemolytic anemias B. Nelson - Somogyi: cuprous ions +
● reference value: 4.5%-6.5%; effective treatment range: arsenomolybdate → arsenic molybdenum blue
<7% C. Neocuproine method: cuprous ions +
● methods are: affinity chromatography, immunoassay, neocuproine (2, 9 dimethyl 1, 10 phenanthroline
electrophoresis, HPLC hydrochloride) → cuprous-neocuproine complex
F. FRUCTOSAMINE (yellow or yellow orange)
● it is known as glycosylated albumin/plasma protein ADDITIONAL INFORMATION:
ketoamine ● Sucrose is not a reducing sugar
● Benedict’s and Fehlings are colored blue, when you
● reflects blood glucose levels for 3-6 weeks
add a urine or serum with high amounts of glucose, it
● it should not be measured in cases of hypoalbuminemia will change its color from green, yellow, orange or red,
(<30g/L) depending on the amount of glucose in the sample.
● reference value: 205-285 umol/L ● Brick red is the indication of having the highest
● methods: affinity chromatography. HPLC, photoelectric amount of glucose in the sample
G. KETONE BODIES
● recommended when plasma glucose reached 300 mg/dL 2. Alkaline Ferric Reduction Method (Hagedorn
● used to confirm diabetic ketoacidosis (DKA) Jensen)
● specimen is fresh urine or serum Involves the reduction of a yellow ferricyanide to a colorless
H. MICROALBUMIN (MICRAL TEST) ferrocyanide by glucose (inverse colorimetry)
● useful to assist in diagnosis at an early stage & before B. CONDENSATION METHOD
the development of proteinuria (kidney function) Ortho-Toluidine Method
● performed on randomly collected urine specimens with ● Principle: Condensation with aromatic amines
simultaneous assay of creatinine ● Glucose in hot acetic solution condenses with aromatic
amines forming glycosylamine and schiff base (emerald
green)
● Classical method for measuring glucose

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 ○ Glucose + Aromatic Amines → Glycosylamine + C. GLUCOSE DEHYDROGENASE METHOD
Schiff base ● Glucose is reduced to produce a chromophore is
II. ENZYMATIC METHODS measured spectrophotometrically or an electric current
● This method is being employed in modern laboratories ● The amount of NADH generated is proportional to the
● High degree of specificity can be achieved using these glucose concentration
methods ● It provides results in close agreement with Hexokinase
● MORE ACCURATE than colorimetric reactions
procedures
A. GLUCOSE OXIDASE METHOD
● Measures β-D glucose ● Mutarotase facilitates the conversion of α-D-glucose to →
● Most specific enzyme to measure glucose β-D glucose
● Glucose Oxidase converts glucose to → gluconic acid

1. Colorimetric Glucose Oxidase Method (Saifer

Gerstenfeld Method)

Table 1. Diagnostic Criteria for DM

Test NORMAL Values
FBS 70-110mg/dL ≥ 126mg/dL; for 100-125mg/dL (impaired
fasting glucose)
RBS <140mg/dL ≥ 200mg/dL (with symptoms of DM)
2PPB ≥ 200mg/dL; for 140-199mg/dL (impaired
S glucose tolerance)
HBA1 4.5 - 6% ≥ 6.5%
C
Table 2. Diagnostic Criteria for GDM (OGTT)
● FALSELY DECREASED: Increased levels of uric acid,
Test Values
bilirubin, and ascorbic acid; because these substances Fasting ≥ 95mg/dL
can be oxidized by peroxidase
1-hr ≥ 180mg/dL
● FALSELY INCREASED: Strong oxidizing agents like
2-hr ≥ 155mg/dL
bleach
● Commonly used chromogenic agents: (good to know 3-hrC ≥ 140mg/dL
Dapat bumababa as serial measurement with regards to time it goes
only)0
down. dapat bumaba yung blood sugar. Kapag mataas pa rin it is
○ 3-methyl-2-benzothiazoline hydrazine considered as diagnostic for GDM
○ N,N-dimethylaniline

2. Polarographic Glucose Oxidase Method LIPIDS AND LIPOPROTEINS

● Lipids, commonly referred to as fats, are classified as
● Measures rate of oxygen consumption which is
organic substances INSOLUBLE in blood and water but
proportional to glucose concentration SOLUBLE in organic solvents (chloroform and ether).
● Glucose oxidase in the reagent catalyzes the oxidation of ○ Very important biologically because the cell
glucose by oxygen under the first order conditions, membrane is composed of phospholipids bilayer
forming H2O2 ○ Lipids also become a stored form of energy
● The enzymatic conversion of glucose is quantitated by ○ Precursor of hormones (steroid hormones): Lipid
the consumption of oxygen in an oxygen-sensing hormones such as sex hormones which the
buildings is cholesterol; cortisol, vitamin D
electrode ● Provide rich sources of energy and an efficient way for
● The H2O2 is prevented from re-forming oxygen by the body to store excess calories
adding molybdate, iodide, catalase and ethanol ● Provide stability to cell membranes and allow for
B. HEXOKINASE METHOD transmembrane transport.
● Reference method for glucose assays; detects evet al ● important precursors of steroid hormones,
low levels prostaglandins, leukotrienes.
● Less subject to interferences; most sensitive method ● Major Lipids
○ Fatty Acids
● EDTA, Heparin, Citrate and Oxalate samples may also
○ Cholesterol
be used ○ Triglycerides
● FALSELY DECREASED: Gross hemolysis and elevated ○ Phospholipids
bilirubin levels
MAJOR LIPIDS
FATTY ACIDS
● Simplest Lipid Class
● Linear chains of carbons-Hydrogen bonds that terminate
with a carboxyl group.
● Short Chain fatty acid contains 4-6 carbon atoms
● Medium Chain fatty acid contains 8-12 carbon atoms
● Long Chain fatty acid has >12 carbons atoms
● Small amounts of fatty acids exist in the free or
unesterified form bound to albumin

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● Mostly found as constituents of phospholipids and ● Reference Values:
triglycerides. ○ <200mg/dL = Desirable
● Two Types ○ 200-239mg/dL = Borderline High
○ Saturated (without C=C double bonds): single bonds ○ ≥240mg/dL = High
and solidifies at room temperature; such as animal
fats, butter ADDITIONAL NOTE
○ Unsaturated (with C=C double bonds): liquid at We need the lipoprotein because
room temperature. The more double bonds the ● fats and lipids are nonpolar solvents of the blood
harder it will solidify; such as plant oils ● we need carrier molecule such as lipoprotein
● Functions: Building blocks for triglycerides,
phospholipids and sources of metabolic energy
● Examples “LOLA Palmi”
○ Lauric Acid
○ Oleic Acid
○ Linoleic Acid
○ Arachidonic Acid
○ Palmitic acid

CHOLESTEROL
● Unsaturated steroid alcohol contains 4 rings and has
a single carbon-hydrogen side chain tail similar to fatty
acids.
○ Cholesterol is a steroid
● Amphipathic molecule, meaning that the hydroxyl group
in the A-ring is the hydrophilic part.
○ Amphipathic: having both hydrophilic and
hydrophobic parts
● Found on the surface of lipid layers.
● Functions
○ Precursor of Steroids - glucocorticoids, TRIGLYCERIDES/TRIACYLGLYCEROLS (TAG)
mineralocorticoids, androgens, estrogens, ● Contains 3 molecule of fatty acids and 1 molecule of
testosterone. glycerol attached via ester bonds
■ Anything in excess, food get deposited in the ● Comprising 95% of all fats stored in adipose tissue
peripheral tissue causing risk for (main storage of lipids in man).
atherosclerosis etc ● It allows the body to compactly store long carbon chains
○ Important constituent of the cell membrane and bile (fatty acids) for energy that can be used during fasting
acids. states.
■ Maintain the cell membrane integrity that’s why ● When TAG are metabolized, their fatty acids are released
the cell membrane is rigid and stable into the cells and converted into energy then recycling
■ Without cholesterol the cell membrane is less the glycerol into TAG.
rigid ● Broken down by lipoprotein lipase (LPL), epinephrine and
○ Can be transformed to Vitamin D3 in the skin by cortisol.
sunlight. ● Can be classified as exogenous TAG (dietary) and
● Diagnostic Significance endogenous TAG (synthesized by the liver)
○ It evaluates the risk of atherosclerosis, myocardial ● Diagnostic Significance
and coronary arterial occlusion. ○ It evaluates suspected atherosclerosis and
○ There is a direct relationship between elevated measures the body’s ability to metabolize fat.
serum cholesterol and MI. ○ Fasting TAG ≥200 mg/dL is at risk for coronary artery
○ Essential in the diagnosis and management of disease.
lipoprotein disorders. ○ TAG and cholesterol are the MOST IMPORTANT LIPIDS
○ Used to monitor effectiveness of lifestyle changes in the management of coronary heart disease (CHD).
and stress management. ● Reference Values
● Forms of Cholesterol ○ <150mg/dL: Normal
○ Cholesterol Ester (70%) - Hydrophobic form wherein ○ 150-199mg/dL: Borderline High
cholesterol is bound to fatty acids; Located in the ○ 200-499mg/dL: High
core of particles. ○ >500mg/dL: Very High TAG
○ Free Cholesterol (30%) - Polar, Non Esterified ● Transported by Chylomicrons (CM) and Very Low
alcohol; free cholesterol and phospholipids are Density Lipoprotein (VLDL)
found on the surface of lipoprotein. ○ CM: dietary (Exogenous)
■ Low Density Lipoprotein (LDL) is the primary ○ VLDL: produced by the liver (endogenous)
carrier of cholesterol.

8
 APOLIPOPROTEINS
● specialized proteins located on the surface of
lipoprotein particles
● help maintain the structural integrity/stability of the
lipoprotein complex
● help to keep the lipids in solution during circulation
through the bloodstream
● serve as activators or inhibitors of the specific lipase
enzymes involved in the synthesis or breakdown of fatty
acid esters
● interact with specific cell-surface receptors & direct the
PHOSPHOLIPIDS
● Similar in the structure to TAG except that they only lipid to the correct target organs & tissues in the body
have 2 esterified fatty acids.
○ it has phosphate group which is polar that interact in MAJOR LIPOPROTEINS:
water 1. CHYLOMICRONS
● The third position on the glycerol backbone contains a ● largest & the least dense produced by the intestine
phospholipid head group. that transports lipids of dietary TAG to the tissues of the
● Several types of phospholipid head groups (which are all body including liver
hydrophilic in nature) “ICES” ○ 2 sources of TAG:
○ Inositol
■ Dietary (exogenous): ingested fats will be
○ Choline
○ Ethanolamine packed into TAG in chylomicrons, then
○ Serine transported to the circulation
● Amphipathic Lipids: contains polar hydrophilic head ■ Endogenous
groups and nonpolar hydrophobic fatty acids. ● TAG rich but relatively poor in free cholesterol,
● Function: phospholipids, & protein
○ Surfactant: decreases surface tension within the
● Apolipoproteins: Apo B-48, Apo A-1, Apo C, Apo E
alveolar space, thus allowing effective gas exchange
and prevents alveolar collapse during expiration. ○ helps maintain structural integrity and serves as
■ Type I: Pneumocytes which secretes surfactant receptors
para di magcollapse yung lungs 2. VERY LOW DENSITY LIPOPROTEIN (VLDL)
○ Participates in cellular metabolism and blood ● produced by the liver & supply the tissues of the body
coagulation with TAG of endogenous origin
○ Important component of cell membrane ○ Accumulation of TAG in liver → fatty liver
● Forms ● Delivers TAG to the tissues
○ Phosphatidylcholine: 70%
● rich in TAG but to a lesser extent than CM
○ Sphingomyelin: 20%
○ Cephalin: 10% ○ when TAG is removed, only cholesterol content is
left → LDL
● Have a higher buoyant density because of their lower
lipid/protein ratio
● prolonged consumption of high fat diet leads to elevated
TAG & VLDL particles
● Apolipoproteins: Apo B-100, Apo C, Apo E
3. LOW DENSITY LIPOPROTEIN (LPL)
● synthesized by the liver
● major end product from the catabolism of VLDL
● constitutes 50% of the total lipoprotein mass in human
plasma
● major source of cholesterol for tissues
● most cholesterol-rich of the lipoproteins & most
atherogenic
LIPOPROTEINS ● in fasting plasma samples, DLD contains the cholesterol
● Lipids carriers; transports lipids within the tissue that is not present in HDL or VLDL
● are large macromolecular complexes of lipids which are ● it transports cholesterol to the peripheral tissues
synthesized by the liver or intestine specifically ● it carries most of the circulating cholesterol & transports it
responsible for moving lipid classes throughout the to hepatic & extrahepatic tissues, where it is taken up by
aqueous environment of the blood LDL-mediated endocytosis
● MAIN PURPOSE: To transport TAG & cholesterol to sites ○ LDL transports cholesterol to the circulation making
of energy storage & utilization it atherogenic component
● the protein provides a coat for the lipid mixture, ● Apolipoproteins: Apo B-100, Apo E
cholesterol & other lipids aggregate forming a large ○ Apo B-100: same w/ VLDL, because when VLDL
complex is metabolised → LDL

9
 4. HIGH DENSITY LIPOPROTEIN (HDL) SEPARATION OF LIPOPROTEINS
● smallest but most dense 1. ULTRACENTRIFUGATION
● “good cholesterol” ● based on buoyant density
● produced by the liver & intestine ● lipids have the density of 1.0 g/mL while proteins have
1.4 g/mL
● involved in reverse cholesterol transport, transport
○ the density of a lipoprotein particle is determined
excess cholesterol from the tissue & return it to the liver mostly by its protein and TAG content
→ to be deposited ○ the higher the lipid content, the less dense the
● HDL2 transports effectively the lipids to the liver & is lipoprotein particle
cardioprotective
● it is involved in anti-inflammatory, anti-oxidant,
anti-thrombotic, & nitric-oxide inducing mechanisms →
cardioprotective
● Apolipoproteins: Apo A-1, Apo A-II, Apo C
○ Apo A-1: only apolipoproteins that returns to its
lipoprotein

LIPOPR CHOLE. FREE PROTEI PHOSPH

TAG
OTEINS ESTERS CHOLE N OLIPID
CM 80-95% 2-4% 1-3% 1-2% 3-6%
VLDL 45-65% 16-22% 4-8% 6-10% 15-20%
LDL 4-8% 45-50% 6-8% 18-22% 18-24%
HDL 2-7% 15-20% 3-5% 45-55% 26-32%
● HDL has more proteins than fat → “good cholesterol”
● LDL has more cholesterol → “bad cholesterol”
● more proteins = more dense
○ HIGH LDL → also seen in the circulation
● CM has more TAG than VLDL 2. ELECTROPHORESIS
● separated using agarose gel electrophoresis
● pinaka mabilis makapunta sa opposite pole
MINOR LIPOPROTEINS: ○ CM remains at the origin; VLDL at the pre-beta
1. INTERMEDIATE DENSITY LIPOPROTEINS (IDL) region; LDL at the beta region; HDL at the alpha
● product of VLDL catabolism/ VLDL remnants region (migrates fastest)
● IMMEDIATELY converted to LDL → “subclass of LDL”
○ however in dyslipidemias, IDL stays longer and
doesn’t immediately convert into LDL
● defective clearance of IDL in type 3 Hyperlipoproteinemia
is probable due to deficiency of Apo E-II
● Apolipoproteins: Apo B-100
● Atherogenic: because it contains high amount of
cholesterol
● VLDL → IDL → LDL ● more protein = faster migration
2. LIPOPROTEIN (A) LIPOLYTIC ENZYMES
● similar to LDL in density & composition LIPOPROTEIN LIPASE
● contains molecule of Apo (a) linked to Apo B-100 by ● hydrolyzes TAG & CH esters from lipoproteins
disulfide bonds ● hydrolysis of TAG releases fatty acids & glycerol
● known as “sinking pre-beta lipoprotein” ● It is present on the surface of capillary endothelial cells of
● increased levels may indicate premature coronary heart adipose tissues, cardiac & skeletal muscles.
HEPATIC LIPASE
disease & stroke
● Hydrolyzes TAG & phospholipids from HDL
● independent risk factor for atherosclerosis ● Hydrolyzes lipids on VLDL and LDL
● Atherogenic ENDOTHELIAL LIPASE
● Apolipoproteins: Apo B-100, Apo (a) Hydrolyzes TAG & phospholipids from HDL
ABNORMAL LIPOPROTEINS: Lecithin Cholesterol Acyl Transferase (LCAT)
1. LIPOPROTEIN X ● catalyzes the esterification from HDL
● found in obstructive jaundice & lecithin cholesterol ● enables HDL to accumulate Ch and Ch ester
acyltransferase (LCAT) deficiency ● cholesterol in tissues need to be esterified, LCAT is
responsible for that
● lipids account for more than 90% of its weight
ATP BINDING CASSETTE PROTEIN A1 (ABCA1)
● proteins such as Apo C and albumin constitute the 10% for efflux of Ch from peripheral cells
2. BETA-VLDL (FLOATING BETA LIPOPROTEIN)
● accumulates in Type 3 Hyperlipoproteinemia ADDITIONAL INFORMATION:
● richer in cholesterol than VLDL & apparently results from ● ABCA1 transfers Ch to HDL
defective catabolism of VLDL ● in the HDL, the LCAT enzyme will esterify the Ch

10
 apolipoprotein receptors, or enzymes of lipoprotein
metabolism.
Table 2. Lifestyle Factors and Secondary Causes of Dyslipidemia
Lipoprotein Profile Secondary Causes Lifestyle Factors
Obesity
Chole-HIGH Hypothyroidism
Excess dietary
LDL-HIGH Nephrotic syndrome
cholesterol/saturated
HDL-LOW Chronic obstructive liver disease
fat
Obesity
Chronic renal failure and Physical inactivity
TAG-HIGH
nephrotic syndrome Cigarette smoking
LDL-NORMAL
Insulin resistance/Diabetes Excess alcohol
HDL-LOW
Mellitus intake
High CHO diet
Chole-HIGH Severe hypothyroidism
TAG-HIGH Diabetes mellitus Obesity
ADDITIONAL INFORMATION: HDL-LOW Nephrotic syndrome
● ano ang role ni APO-A1 in HDL? Physical inactivity
○ it activates the LCAT Increased body
HDL-LOW (isolated) Certain medications weight
○ ligand/receptor for ABCA1 High CHO diet. low
● ano ang role ng APO-E fat diets
○ the recognition factor that targets the CM ● Thyroid hormones increase metabolism. Low thyroid
remnants to VLDL remnants back to the liver hormones = slow metabolism = “taba-in”
● ano ang role ng APO-C2
○ binds to lipoprotein lipase para maactivate eto ● As you can see, kidney and thyroid diseases (secondary
and ma hydrolize yung TAG to adipose tissue causes) are involved in having dyslipidemia profiles
para magamit as a source of energy ● In lifestyle factors, obesity and sedentary lifestyle are
always involved, along with the vices (smoker/alcoholic)
● It was known before that only food intake can affect our
●DISORDERS ASSOCIATED W/ LIPIDS & LIPOPROTEINS
FREDRICKSON CLASSIFICATION: lipid profile. But later found out that it was a complex
Type Lipoprotein TAG Ch system. For example, mahina kumain pero may
I CM very high normal hypothyroidism naman. So tataas pa rin ang lipid profile
IIa LDL L-N H niya
IIb LDL & VLDL H H
III IDL H H LABORATORY METHODS FOR LIPID ANALYSIS
IV VLDL very high normal SPECIMEN COLLECTION AND STORAGE
V CM very high normal ● Fasting for 12 hours (12-16 hours)
● But if FBS + lipid profile, 10 hours fasting (if more than 10
ADDITIONAL INFORMATION: hours, glucose for FBS will be affected)
● TYPE I ● Capillary samples are slightly lower than venous samples
○ mababa ang risk factor sa CVD ● Plasma or serum (most common; no laboratory in the PH
○ greatly improves with proper diet and exercise
use plasma for lipid analysis except probably for
● TYPE IIa
○ also known as familial hypercholesterolemia research purposes only) samples can be used for
○ high risk of CVD analysis however for electrophoresis and
● TYPE IIb ultracentrifugation methods, plasma is used because it
○ AKA familial combined hyperlipidemia can be cooled at 4C
○ high cardiac risk ● EDTA is the preferred anticoagulant for plasma samples
● TYPE III (for theoretical purposes only) because it prevents
○ high cardiac risk
oxidative and enzymatic alterations that occur in the
○ dietary control
○ familial Dysbetalipoproteinemia lipoproteins during storage
● TYPE IV ● Citrate exerts large osmotic effects that result in falsely
○ lower cardiac risk than type II & III low plasma lipid and lipoprotein concentrations
○ familial hypertriglyceridemia ● Heparin because of its high molecular weight can alter
○ may or may not be associated w/ premature electrophoretic mobilities
coronary disease
● TYPE V
CHOLESTEROL
○ low cardiac risk
○ may be associated with premature coronary ● Total cholesterol (TC) is measured rather than its forms
disease (cholesterol ester and free cholesterol)
○ something to do w/ metabolism chylomicrons. ● TC may be assayed from non-fasting blood samples
(fasting has little effect on TC)
● Complex systems of lipid classification have evolved ● TC levels increase with age but women have lower
because of our improved understanding of lipids, values than men except after the age of 50 years old due
lipoprotein metabolism, and genetics. to menopause (hormonal changes like decrease)
● They can arise from lifestyle or secondary causes as well ● Hemolysis can falsely increase levels of TC
as mutations in genes encoding apolipoproteins, ● Icteric specimens
● Avoid water contamination

11
 NOTE: ● Unesterified Ch are extracted using petroleum ether
● According to Henry’s, cholesterol is more stable in ● Measured with Liebermann-Burchard reagent
blood. Regardless if the patient did fasting or not, ● Modified Abell-Kendall method is the reference
method for Ch (it uses hexane extraction after
kung mataas talaga ang cholesterol mo, mataas na
hydrolysis with alcoholic KOH followed by reaction
yun. However, in practice, we still require our patient with Liebermann-Burchard reagent)
to fast. Walang nagpapa-random cholesterol 4. Colorimetric, Extraction, Saponification, and
● In contrast with triglycerides, it is greatly affected by Precipitation (Four-Step Method) -
the recent food intake Schoenheimer-Sperry, Parekh, and Jung
● Technically, the real reason for fasting for lipid profile NOTE:
is for triglycerides (unlike cholesterol) ● General methods still need to be discussed because it
● Bihira lang sa bata ang inaatake sa puso due to usually appears in the board exam, although rarely
hypercholesterolemia or hypertension, although it is done in practice since enzymatic machines are
possible already being used
● As a child, physiologic consequences are not yet felt. ● But in the ASCPi exam, it is no longer asked
But when you reach your 30s - 40s, symptoms start to
appear II. ENZYMATIC METHODS
● It can be noted that heart attacks are often seen ● Measures TC directly in plasma or serum through a
series of reactions in which cholesteryl esters are
earlier in males than females but when females reach
hydrolyzed
the age of 50 years old, they also have greater risk ● The 3-OH group of cholesterol is oxidized, and hydrogen
(TC levels increase with age) peroxide, one of the reaction products, is quantified
enzymatically
I. CHEMICAL METHODS ● Focus more on enzymatic methods since these are the
● The presence of double bonds and hydroxyl groups in ones present in the reagents/bottles/kit forms
the sterol’s structure makes it possible for Ch to carry out
colorimetric reaction
● Principle: Dehydration and oxidation of Ch to form a
colored compound
● 2 General Reactions in The Colorimetric Ch ● Cholesterol esters will be acted upon by the enzyme
Determination: cholesteryl esterase liberating cholesterol and free fatty
acids
Liebermann-Burchard ● Cholesterol will now be acted upon by cholesterol
● For historical purposes only oxidase
● End product: Cholestadienyl monosulfonic acid (green) ● If hydrogen peroxide is present as a substrate,
● Reagents of color developer mixture: peroxidase is the enzyme used therefore it is subject for
○ Glacial acetic acid - oxidizer interfering substances
○ High levels of ascorbic acid = falsely decreased TC
○ Acetic anhydride - oxidizer
○ Bilirubin can interfere because of its own spectral
○ Concentrated sulfuric acid - dehydrating agent and properties. Bilirubin is absorbed at 500nm thus
color developer (turns to green) possibly lowering TC levels; however bilirubin is also
Salkowski oxidized by hydrogen peroxide (H2O2) which results
● End product: Cholestadienyl disulfonic acid (red) to losing its absorbance at 500nm, bilirubin
GENERAL METHODS exceeding 5 mg/dL decreases Ch levels by
5-15%
○ Hemoglobin has an inherent color = falsely
increased TC
○ High levels of uric acid
● Reference range:
○ <200 mg/dL (desirable 140-200 mg/dL)
○ 200-239 mg/dL (borderline high)
○ ≥ 240 mg/dL (high)

TRIGLYCERIDE
1. Colorimetry (One-Step Method) - Pearson, Stern, and I. CHEMICAL METHODS
Mac Gavack A. Colorimetric- Van Handel & Zilversmith
● Denaturation of CHON upon the addition of color
developer mixture (a strong acid) ● TAG are saponified with alcoholic KOH into glycerol and
2. Colorimetric and Extraction (Two-Step Method) - fatty acids. Glycerol is then oxidized by periodate
Bloor’s solution into formaldehyde. Formaldehyde reacts with
● Extraction of lipid using ethanol ether (Bloor’s chromotropic acid to form a blue color.
reagent) ● For historical purposes only
3. Colorimetric, Extraction, and Saponification
(Three-Step Method) - Abell-Kendall
● Reference method for cholesterol
● Liebermann-Burchard reagent is still used
● Saponification is done by adding alcoholic KOH
(saponifies Ch ester)

12
B. Fluorometric method- Hantzsch Condensation
● For historical purposes only LOW DENSITY LIPOPROTEIN
● Maybe calculated or measured directly
● Normally computed in lipid profile

A. Usually calculated using Friedewald formula (indirect

II. ENZYMATIC METHODS method, not valid for TAG over 400 mg/dL)
A. In this reaction the disappearance of NADH (conversion
to NAD) is measured at 340 nm.
● NADH to NAD conversion

B. In this reaction the chromogenic substances are added

● Reference value: 50-130 mg/dL
and measured at 500 to 600nm.
● Hydrogen peroxide peroxidase method
B. Homogenous assay uses detergent to block HDL and
VLDL from reacting with the dye to form a colored
chromogen product. An enzymatic cholesterol analysis
is performed with only LDL able to react.
● They use a combination of two reagents: the first
reagent usually selectively removes non-LDL
Reference values: lipoproteins ( and or stabilizes or inhibits LDL from
● < 150 mg/dL (normal: 60-150 mg/dL) reacting with enzymes) and the second reagent
● 150-199 mg/dL (borderline high) releases cholesterol from LDL so that it can be
measured enzymatically.
● 200-499 mg/dL (high)
● >500 md/dL (very high- acute and recurrent pancreatitis) NONPROTEIN NITROGEN COMPOUND

HIGH DENSITY LIPOPROTEIN

A. Chemical Precipitation
● Precipitate LDL and VLDL with dextran
sulfate-magnesium chloride or heparan
sulfate-sulfate manganese chloride, then assay the
supernatant for cholesterol. The cholesterol is present in
the HDL
NOTE:
● Serum contains different lipoproteins are added by
precipitating reagents (dextran sulfate or heparan ● They are called NPNs because they have nitrogens but
sulfate) they are not proteins.
● If the precipitating agents precipitate the lipoprotein, ● Waste products of the body (end product of metabolism).
magiging mabigat ang lipoprotein and kapag mabigat ● Kidneys must be functioning to remove the wastes in the
na, kapag sinentri maiiwan sa supernatant yung HDL body.
● Included on kidney function test
and siya yung kukunin for analysis
● For manual: very tedious COMPOUND Approximate Approximate Urine
B. Homogeneous assays plasma Concentration
● the most popular method and are used in automated concentration (% of Excreted N)
(% of Total NPN)
analyzers
UREA 45-45 86.0
● These assays do not require pre-treatment and
AMINO ACIDS 25 —
separation techniques; make use antibody to Apo B-100
URIC ACID 10 1.7
to bind LDL and VLDL; an enzymatic cholesterol analysis CREATININE 5 4.5
can then be performed with only HDL able to react. CREATINE 1-2 —
NOTE: AMMONIA 0.2 2.8
● For automated analyzers ● Urea- highest concentration both in urine and blood
● It requires antibody techniques ○ Kapag tumatagal ang ihi nagiging ammonia kaya
● Apo B-100 is present in LDL and VLDL, so if may pumapanghi ang ihi
antibody against it siya yung matatanggal sa solution
and maiiwan ang HDL BLOOD UREA NITROGEN (BUN)
● Reference values: ● It is the major end product of protein (dietary) and amino
catabolism.
○ 40-75 mg/dL
○ From meats
○ <35 mg/dL is considered high risk for CHD ○ If vegetarian: beans and tofu (tokwa)
○ >60 mg/dL is cardio-protective

13
● Highest NPN compound in the blood- 45-50%. waste product catabolism
● It is synthesized in the liver from CO2 and ammonia from
the deamination of amino acids via the Krebs-Henseleit HIGH BUN:CREA Ratio with
cycle or Ornithine cycle. NORMAL PLASMA
● It is freely filtered at the glomerulus but reabsorbed CREATININE
substantially at the PCT and the inner collecting duct RENAL ● Disease of the renal
○ If we have renal failure, blood urea is the first ● indicates that the vessels, glomerulus,
compound that will rise. kidneys itself are renal tubules and renal
● It is the first metabolite to elevate in kidney diseases and dysfunctional interstitium
easily removed by dialysis. ● Acute and Chronic renal
● The concentration of urea in the plasma is determined by failure
renal function, protein content of the diet and the rate of ● acute glomerulonephritis
protein catabolism. POST-RENAL ● Kidney stones
○ Tumataas ang urea sa dugo kapag mahina na ang ● results from anatomic (nephrolithiasis)
kidneys since hindi na niya nailalabas, affected by obstruction to urine flow ● Congenital anomaly
renal function, protein content of the diet. out of the kidney ● Inflammatory lesion
○ Kapag mahina na ang kidneys iwas-iwasan na ang ● Neoplasm
pagkain ng maraming karne. HIGH BUN:CREA Ratio with
● The concentration of urea is expressed only by the ELEVATED PLASMA
nitrogen content of urea so to get the concentration of CREATININE
urea from BUN:
○ UREA = BUN x 2.14 BLOOD UREA NITROGEN (BUN)
● BUN:Creatinine Ratio is 10:1 - 20:1 Specimen requirements:
○ Clinical Significance: ● Non-fasting sample (serum, plasma & urine)
■ To evaluate renal function ● Citrate and fluoride inhibit urease
● Avoid hemolyzed sample
■ to assess hydration status
LABORATORY METHODS:
■ to determine nitrogen balance
CHEMICAL METHOD (DIRECT METHOD)
■ to aid in the diagnosis of renal disease
● Diacetyl Monoxime ● uses
■ to determine adequacy of dialysis Method FERROTHIOCYANATE
● Azotemia - means elevated concentration of NPNs in the as color developer
blood
● Uremia - clinical term that describes the patient’s signs UREA + DAM → Yellow
and symptoms when symptomatic end stage renal failure diazine derivative (540nm)
is present ENZYMATIC METHOD (KINETIC/INDIRECT METHOD)
Do not measure Urea directly, but measures the byproduct of
● Decreased BUN is due to:
urea → ammonia
○ Low protein intake
● Hydrolysis of urea by
○ Severe vomiting urease
○ Diarrhea ○ the ammonia
○ Liver Disease produced is
○ Pregnancy measured by various
methods to calculate
the concentration of
UREA CYCLE
urea in the original
sample
METHODS USED TO MEASURE AMMONIA:
● By use of coupled
enzyme method to
measure the rate of
disappearance of NADH
to NAD read at 340nm
● Using the Berthelot
Reaction

● Using indicator dye

UREA CYCLE is found on the LIVER ● Measuring the
LIVER will convert AMMONIA to become UREA conductivity of
ammonium ions
CLASSIFICATION AND CAUSES OF AZOTEMIA Reference range of 6-20 mg/dL / 2.1-7.1 mmol/L
CLASSIFICATION CAUSES Ammonia: (serum/plasma)
PRE-RENAL ● Congestive heart failure
● refers to conditions with ● Hemorrhage 12-20 mg/dL / 0.43-0.71
reduced blood flow to ● Shock mmol/L (24hr urine)
the kidneys thereby ● Dehydration Conversion factor of 0.357
reducing urine output ● High protein diet Ammonia:
and causing retention of ● Increased protein

14
 BLOOD URIC ACID ADDITIONAL INFORMATION:
● It is associated with gout
● End product of Purine (adenine and guanine)
metabolism. Derived from:
○ Dietary source
○ Breakdown of nucleus of the cell. It means cell
destruction ex. chemotherapy drugs (they are
anti-nucleus or anti-DNA. sinira yung DNA para di
na mag undergo ng mitosis as a consequence nag
rerelease ng maraming purine bases. kaya
pinapamonitor yung mga Uric acid level ng mga
cancer patients) ● sa internship watch out baka raw merong case na
○ Direct transformation of endogenous purine may hyperuricemia. Minsan pag sobrang taas na ng
nucleotides uric acid ng patient iniihi na nila and makikita mo ito
● it is formed from xanthine by the action of xanthine parang karayom and no wonder it is very painful.
oxidase in the liver and the intestine. Imagine natutusok niya yung glomerulus kaya
pwedeng mag cause ng gouty nephropathy
● It is freely filtered, partially reabsorbed and secreted in
the renal tubules.
● It is a weak acid; at pH 7.4 and is relatively insoluble
>95% exists as monosodium urate (MSU) but at
concentration >6.8mg/dL in the plasma saturated thus,
precipitates in the tissues, MSU precipitates in acidic
urine as uric acid crystals.

CLINICAL SIGNIFICANCE:
GOUT
● This is the tophi may where in may deformities na
● disease primarily found in men 30-50 y/o, post
menopausal women
● pain and inflammation of the joints caused by I. CHEMICAL METHODS
precipitation of MSU A. OXIDATION- REDUCTION
● Persons with gout are highly susceptible to
nephrolithiasis
● In severe cases deposition of uric acid and urates in
tissues occur known as tophi causing deformities
1. Archibald, Caraway, Henry-sodium Carbonate
2. Folin, Brown, Newton Benedict- sodium cyanide
INCREASED NUCLEAR METABOLISM
II. ENZYMATIC METHODS
● occurs in patients on chemotherapy for proliferative A. Uricase Method- specific method
disease such as leukemia, lymphoma, multiple myeloma, ● uric acid has high absorbance at 293nm
polycythemia, hemolytic and megaloblastic anemia
● monitoring of uric acid levels is important to avoid
nephrotoxicity
B. COUPLED ENZYME METHODS- measures the
hydrogen peroxide produced in uricase reaction by using
CHRONIC RENAL FAILURE
catalase and peroxidase. The color produced is
causes hyperuricemia because filtration and secretion is proportional to the uric acid in the specimen.
impaired ● Bilirubin and ascorbic acid destroys the peroxidase if
present in sufficient quantity
ENZYME DEFICIENCIES ● Commercial reagents use KCN and ascorbate
● inherited purine disorders oxidase to minimize these interferences. these are
● Lesch Nyhan Syndrome- an inborn error of purine stabilizers
metabolism Reference range:
○ X-linked genetic disorder caused by deficiency of ● Male: 3.5-7.2 mg/dL / 0.21-0.43mmol/L
hypoxanthine-guanine phosphoribosyltransferase, ● Female: 2.6-6.0 mg/dL / 0.16-0.36mmol/L
an important enzyme in the biosynthesis of purines
Conversion factor: 0.0595
DECREASED URIC ACID SECRETION
● most common cause of toxemia of pregnancy CREATININE
(preeclampsia), lactic acidosis. ● End product of muscle metabolism. The amount
generated in an individual is directly proportional to the
HYPERURICEMIA mass of skeletal muscle present.
less common, usually secondary to liver disease or defective ● Formed from creatine and creatinine phosphate in
tubular reabsorption, fanconi syndrome. Chemotherapy using muscle and excreted into the plasma at a constant rate
Azathioprine and 6-mercaptopurine and over treatment with related to muscle mass.
allopurinol.
○ tumataas ang creatinine pag nag-eexercise kasi
tumataas yung muscle mass

15
● Creatine- is produced by skeletal muscle, kidneys and ● Ammonia is neurotoxic and is associated with
pancreas and is then transported to the tissues encephalopathy
especially in the skeletal muscles and brain via the ● Clinical significance: Prognostic indicator of hepatic
bloodstream. Within cells, creatine phosphocreatine failure, severe liver disease, Reye’s syndrome (acute
provides a ready, rapid-source of energy. metabolic disorder of the liver)
● It is synthesized primarily in the liver from arginine,
glycine and methionine KIDNEY FUNCTION TEST
● It is not reused in the body’s metabolism, solely as a ANATOMY & PHYSIOLOGY OF THE KIDNEYS
waste product and not easily removed by dialysis. ● Pair of kidney-bean shaped organs located just above
● It is commonly used to monitor renal function, an index of the waist between the peritoneum and the posterior wall
overall renal function. of the abdomen (retroperitoneal)
● It measures the completeness of 24-hour urine collection. ● Located between the levels of the last thoracic vertebrae,
● It is used to evaluate fetal kidney maturity- as gestation a position where they are partially protected by the 11th
and 12th pair of ribs
progresses, more creatinine is excreted by the fetus into
● The right kidney is slightly lower than the left because the
the amniotic fluid (2mg/dL) liver occupies considerable space on the right side
CLINICAL SIGNIFICANCE: superior to the kidney
1. Increase in impaired renal function, chronic nephritis and ● Each kidney is 10-12 cm long (4-5 in.), 5-7 cm wide (2-3
congestive heart failure in.), and 3 cm (1 in.) thick
2. Decreased in decrease muscle mass, advanced and ● Each kidney weighs 135-150 grams
severe liver diseases, pregnancy and inadequate dietary ● The internal anatomy has two distinct regions: Renal
protein cortex and Renal medulla
I. CHEMICAL METHODS ○ The Renal medulla consists of several cone-shaped
A. Jaffe reaction renal pyramids, the base (wider end) of each
● Classic assay for serum and urine creatinine were pyramid facing the cortex, and its apex (narrower
creatinine reacts with picric acid in alkaline solution end) called the Renal papilla points toward the
to form a red-orange complex with absorbance at Hilum (Indentation).
proximity 490-505 nm. ○ The Renal cortex is divided into an outer cortical
● Non-specific method; the presence of ascorbic acid, zone and an inner juxtamedullary zone.
glucose, protein and acetoacetate give falsely ■ Those portions of the renal cortex that extend
elevated values; bilirubin and hgb give falsely between the renal pyramids are called Renal
decrease values. columns
B. Kinetic Jaffe ● Together, the renal cortex and renal pyramids of the renal
● 2-step measurement medulla constitute the parenchyma (Functional units).
● hindi lang once mo siya immeasure sa ○ Functional units of the kidney: about 1 million
spectrophotometer, kundi dalawang beses. yung nephrons
first reading im-minus sa second reading. ● Urine formed by the nephrons drains into large papillary
ducts, which extend through the renal papillae called
II. ENZYMATIC METHODS Major and Minor Calyces.
Creatinase H2O2 method ● From the major calyces, urine drains into a single large
● adapted for dry-chemistry; potential to replace Jaffe cavity called Renal pelvis and then out through the
method because it is more specific; no interference ureter to the urinary bladder.
from acetoacetate or cephalosporins.
FUNCTIONS OF THE RENAL SYSTEM
● Elimination of waste products
● Maintenance of water balance
○ Maintained by ingestion of water (controlled by the
Brain Thirst Center) and excretion/reabsorption of
Reference values: water in the renal tubules under hormonal control by
● male: 0.6-1.1mg/dL(enzymatic) / 0.9-1.3mg/dl (jaffe) ADH
● female: 0.5-0.8mg/dL (enzymatic) / 0.6-1.1mg/dL( jaffe) ● Maintenance of Electrolyte balance
○ Sodium, Potassium, Phosphate, Calcium and
Conversion factor: 88.4 magnesium is maintained by tubular reabsorption
under hormonal control.
AMMONIA ○ Chloride is passively reabsorbed with sodium
● Formed by the deamination of amino acids during protein ● Maintenance of Acid-Base balance - controlled by the
metabolism kidney conservation of bicarbonate ions and removal of
metabolic acids (H+) to conserve blood pH level
● It is removed from the circulation and converted to urea
○ Kung mataas ang Hydrogen levels, the kidney will
in the liver secrete the hydrogen in the urine to compensate for
○ Kapag hindi nangyayari yung conversion, it means the acidosis. Then kapag need ng hydrogen,
may problem yung liver. Kaya kapag mataas yung i-rereabsorb.
ammonia, nagiging toxic and nagkakaroon ng ● Endocrine function
hepatic associated coma ○ Kidney synthesizes EPO, prostaglandins, active
● Its production is not dependent on renal function but on form of vitamin D, and one enzyme, Renin.
liver function

16
 STRUCTURES OF THE NEPHRON TUBULAR REABSORPTION
GLOMERULUS ● As filtered fluid flows along the renal tubule and through
● Made of arterioles surrounded by distended end of the the collecting duct, tubule cells reabsorb about 99% of
renal tubule in the renal cortex the filtered water and many useful solutes.
● Glomerular function is to strain proteins from plasma to ● The water and the solutes return to the blood as it flows
produce a “protein-free” filtrate that becomes urine through the peritubular capillaries and vasa recta.
○ Kapag may proteins na sa urine, sira na si ● Reabsorption refers to the return of substances into the
Glomerulus bloodstream
TUBULAR SECRETION
PROXIMAL CONVOLUTED TUBULE (PCT) ● As fluid flows along the renal tubule and through the
● Reabsorbs sodium, potassium, chloride, water, collecting duct, the tubule and the duct cells secrete
bicarbonate, amino acids, proteins, and other ions. other materials such as wastes, drugs, and excess ions
● It secretes sulfate, glucuronides, hydrogen ions and into the fluid
drugs. ● Notice that tubular secretion removes a substance from
● Some substances have a maximum concentration in the the blood
plasma, so the tubule cannot resorb it all. All excess
substances spills over into urine (e.g. Glucose) TESTS FOR GLOMERULAR FILTRATION RATE (GFR)
● The PCT allows for the elimination of Urea and ● A measure of clearance of normal molecules that are not
Creatinine bound to protein and are freely filtered by the glomeruli,
neither secreted, nor reabsorbed by the tubules
● Considered the best overall indicator of the level of
DESCENDING LOOP OF HENLE kidney function
● It promotes the reabsorption of WATER
CLEARANCE TESTS
ASCENDING LOOP OF HENLE ● Measures the rate at which the kidneys are able to
● Reabsorbs solutes such as sodium, chloride, calcium remove (clear) a filterable substance from the blood
and magnesium ● It represents the volume of the plasma that would
contribute all the solute excreted
DCT AND COLLECTING TUBULES ● It is expressed in mL/min, representing the volume of the
● Reabsorbs sodium, secretes potassium, ammonia, and plasma that would be totally cleared of the solute in 1
hydrogen ions. minute
● The collecting tubule is the final site for either ● Plasma concentration and clearance is inversely
concentrating or diluting urine. proportional
● It is under hormonal control for resorption of water and ○ As clearance of the substance declines, its
sodium concentration in plasma increases
● To ensure that glomerular filtration is measured
accurately, the substance analyzed must be the one that
THREE MAIN MECHANISMS INVOLVED: is neither secreted nor reabsorbed by the tubules
1. Glomerular filtration ● Measurable waste products excreted by the kidney
2. Tubular reabsorption include creatinine, urea, and uric acid
3. Tubular secretion ● With a decline in GFR, these waste products are retained
and their circulating concentrations rise

1. CREATININE CLEARANCE
● It provides an estimate of the amount of plasma that
must flow through the glomeruli per minute
● It is an excellent measure of renal function because
creatinine is freely filtered by the glomerulus and not
reabsorbed
● Production and excretion of creatinine is related directly
to muscle mass
○ When renal function is normal or stable, creatinine
excretion is almost equal to its production, which
depends primarily on muscle mass

BASIC RENAL PROCESSES

GLOMERULAR FILTRATION ● Reference value: 85-125 mL/min (males); 75-112
● In the first step of urine production, water and most mL/min (females)
solutes in the blood plasma move across the wall of ○ Kapag mababa yung clearance rate, hindi
glomerular capillaries into the glomerular capsule and nagagampanan ng kidney yung kanyang normal
then into the renal tubule function kasi may sira na sa kidney.
● The fluid that flows through the glomerulus is called the
Glomerular filtrate (120-130 ml/min) 2. INULIN CLEARANCE
● The volume of blood filtered per minute is the ● It is considered to be the reference method
Glomerular Filtration Rate (GFR) and its determination ● Polymer of fructose
is essential in evaluating renal function ● Not routinely used because of the necessity for
continuous IV infusion

17
3. UREA CLEARANCE COMMON GLOMERULAR DISEASES
● Same lang sa creatinine, 24-hour collection 1. ACUTE GLOMERULONEPHRITIS
● It can demonstrate progression of renal disease or ● A pathologic lesions in the Glomerulus
response to therapy ● Rapid onset of hematuria, proteinuria, decreased GFR,
● It does not give reliable estimates of the GFR since urea elevated BUN, creatinine, Oliguria, sodium and water
is freely filtered by the glomerulus but variably retention
reabsorbed by the tubules ● Numerous hyaline and granular casts are seen
○ Not a very indicator for clearance kasi binabalik sa ● Often related to Group A - Beta Hemolytic Strep.
katawan because of immune complexes deposited on the
basement membrane
4. CYSTATIN C
● A low molecular weight protease inhibitor and produced 2. CHRONIC GLOMERULONEPHRITIS
at a constant rate by all nucleated cells Lengthy glomerular inflammation
● It is freely filtered by the glomerulus, not secreted by the
renal tubules but reabsorbed 3. NEPHROTIC SYNDROME
● It is completely reabsorbed and catabolized by the PCT, ● Massive proteinuria (>3.5 g/day), hypoalbuminemia,
hence its presence in the urine denotes damage of that hyperlipidemia, lipiduria
tubule ● Caused by Glomerular disease states
● Rises more rapidly than creatinine in Acute Kidney Injury
(AKI)
○ Useful in detection early changes in kidney function 4. IgA NEPHROPATHY
● Recommended for pediatric patients, persons with
diabetes, the elderly and critically ill patients TUBULAR DISEASES
● Occur as the progression of all renal diseases as GFR
5. BETA-TRACE PROTEIN/BETA-2 MICROGLOBULIN falls
● It dissociates from human leukocyte antigens at a RENAL TUBULAR ACIDOSIS
constant rate and is rapidly removed from the plasma by ● Primary tubular disorder affecting acid-base balance
glomerular filtration ○ Impaired tubular secretion of hydrogen ion and
● A rise in plasma level of beta-2 microglobulin has been impaired reabsorption of bicarbonate ions
shown to be a more sensitive indicator of a decrease in
GFR than creatinine clearance INTERSTITIAL NEPHRITIS
● Not reliable in patients who have history of immunologic ● Acute or chronic inflammation of the tubules that can be
disorders or malignancy caused by radiation toxicity, infections, medications.
○ Increased levels of Beta-2 are seen in
myeloproliferative and lymphoproliferative disorders
URINARY TRACT INFECTIONS
● Measurement of serum Beta-2 macroglobulin is used
clinically to assess renal tubular function in renal ● Kidneys: Pyelonephritis
transplant patients ● Urinary Bladder: Cystitis
○ Increased levels indicates organ rejection ● >10^5 CFU/mL in urine cultures with positive urine
leukocytes, nitrite, leukocyte esterase, WBC Casts are
OTHER MARKERS seen in pyelonephritis
1. MYOGLOBIN
OBSTRUCTION
● A low molecular weight protein that transports oxygen
from Hgb to contractile muscles ● Caused disease in two ways:
● Associated with acute skeletal muscle and cardiac ○ Increased intratubular pressure until nephrons renal
muscle injury failure ensues
○ Repeated Urinary Tract Infection
● Can occur in the Upper Urinary Tract
2. MICROALBUMIN ○ Kidneys and Ureters
● Important in patients with DM to detect nephropathy ● Can occur in the Lower Urinary Tract
● 30-300 mg albumin in 24 hours is diagnostic of ○ Bladder and Urethra
albuminuria ● Causes of obstruction include neoplasms (Kidney,
● In random urine, albumin to creatinine ratio of >30 Bladder, Prostate, Lymph node constricting tumors)
mg/g is diagnostic of albuminuria ○ Congenital deformities
● Myoglobin is toxic to the kidneys ○ Kidney stones or Renal calculi
■ formed from crystallization of various
3. NEUTROPHIL GELATINASE-ASSOCIATED substances
LIPOCALIN (NGAL) ■ CaOx, Magnesium ammonium phosphate,
● Expressed by neutrophils and epithelial cells Calcium phosphate, Uric acid, Cystine
● Can be measured within 2-6 hours of Acute Kidney Injury ○ Crystallization is due to reduced renal blood flow
● Has been shown to be useful early predictor of AKI ■ Decreased fluids and large amounts of these
● Limited specificity because it may also rise in systemic insoluble substances
stress without AKI

18
 LIVER FUNCTION TESTS ● Other Ferroxidase located at the Basal
ANATOMY: lateral membrane of the intestinal cell =
● Largest (1.2-1.5kg) & most complex organ of the GI tract. HEPHAESTIN
● 2 major lobes: right & left c. The low level is known as Wilson’s disease.
● 2 smaller lobes: Caudate & Quadrate d. Copper deposits at the edge of the iris are
● 2 blood sources: called Kayser-Fleischer rings
○ Hepatic artery: which brings oxygen 4. Clotting Factors
○ Hepatic portal vein: which supplies nutrient-rich a. Coagulation proteins are synthesized in the
blood from the digestive tract liver.
○ The 2 blood supplies eventually merge and flow into b. Most common coagulopathy is disseminated
the sinusoids. intravascular coagulation.
● Blood flow is approx. 1,500 mL/min
DETOXIFICATION
3 SYSTEMS: 1. AMMONIA
● Biochemical Hepatic System: ALL metabolic activities. ● It arises from deamination of amino acids, which occurs
○ ex. Glucose metabolism, lipid metabolism both mainly through the action of digestive and bacterial
involve the liver. enzymes (bacterial proteases, ureases, and amine
● Hepatobiliary system: metabolism of bilirubin oxidases) on proteins in the intestinal tract.
○ excretion of bilirubin ● The primary site for ammonia production is the small
● Reticuloendothelial System (RES): Kupffer cells act for intestine but ammonia can only be metabolized by the
immunity and breakdown of Hgb liver because it contains the critical enzymes for the
→which serve as sentinels in the liver; the permanent Krebs-Henseleit Urea cycle.
macrophages in the liver ● It is also released from metabolic reactions that occur in
○ The liver itself is the largest and most complex skeletal muscles during exercise.
organ in the GI tract. ● The liver normally removes most of this NPN via the
portal vein circulation and converts it to urea, which is
FUNCTIONS:
eliminated by the kidneys.
1. Synthesis: CHO, CHONs, lipids
2. Metabolism: bilirubin (waste product) CLINICAL SIGNIFICANCE
3. Detoxification: ammonia (it becomes a nontoxic ● hepatic coma and Reye syndrome
product), drugs ● in severe liver disorder, it accumulates and reaches the
4. Excretion: bile (bile acids/salts, pigments & cholesterol), systemic circulation, which is then converted to glutamine
bile acids (cholic acid and chenodeoxycholic acid in the brain, thus compromising the Krebs cycle leading
conjugated with the amino acids glycine & taurine to form to coma due to lack of ATP for the brain (ammonia
bile salts) increases CNS pH)
● Bile→to emulsify the fats that we eat ● elevated plasma levels of ammonia are neurotoxic and
5. Storage: fat-soluble vitamins, water-soluble vitamins, are often associated with encephalopathy.
glycogen, and also lipids. Also includes hormones and ● increased levels seen in cirrhosis, hepatitis, Reye
thrombopoietin. syndrome, chronic renal disease and acetaminophen
poisoning
● REFERENCE VALUE: 19-60 ug/dL (11-35 mmol/L)
SYNTHETIC FUNCTIONS:
A. Protein Synthesis 2. DRUGS
● the liver is the site for most ● many xenobiotics are metabolized in the liver via the
● plasma CHON synthesis except for Ig & vWF cytochrome P450 oxidase system
○ Ig are produced by plasma cells, ● often involves 2 phases:
○ vWF are produced by endothelial ○ phase 1 reactions involve oxidations, hydroxylations
cells & Weibel-Palade Bodies ○ phase II reactions conjugate the metabolite to polar
(WPB) of the platelets. compounds such as glucuronic acid, taurine,
● greater than 90% of all CHONs & 100% of albumin glycine, and sulfate
synthesis occurs in the liver
1. Albumin BILIRUBIN METABOLISM:
a. major CHON produced by the liver at about 120 ● heme waste product; converted to bilirubin in 2-3 hours
mg/kg/day ● approx. 200-300 mg bilirubin/day
b. a transport CHON for many substances both ● bilirubin is a waste product of breakage of the rbc and
endogenous and exogenous (ex. drugs, hemoglobin metabolism
hormones, bilirubin, etc.)
c. one of the major prognostic features in patients
with cirrhosis BILIRUBIN SYNTHESIS/FORMATION:
2. Alpha-1-antitrypsin (AAT) ● senescent/old(120 days)/damage rbc is taken up by
a. the most abundant alpha-1 globulin and the macrophage
most important protease inhibitor in the plasma. ● broken or destroyed rbc releases heme and globin
3. Ceruloplasmin ○ heme: made up of iron + porphyrin ring
a. the major copper containing transport CHON in ○ iron is recycled, porphyrin ring now is metabolized
serum by the enzymes in macrophage then goes to the
b. is a ferroxidase (from ferrous to ferric), liver to be converted to bilirubin
essential for converting iron to the ferric state to ○ albumin: transport proteins
allow binding to transferrin.

19
 NOTE:
● albumin: transport protein
● urobilin: pigment for urobilinogen
● stercobilin: found in the stool

CAUSES OF ELEVATED UNCONJUGATED IN SERUM

1. Hemolysis
2. Gilbert’s Syndrome
● mild B1 hyperbilirubinemia
● caused by mutation at UGT1A1 gene resulting in
lower transcription rates and overall enzymatic
activity
3. Crigler-Najjar Syndrome
STEPS: ● high levels of B1 hyperbilirubinemia
1. Inside the macrophage, heme oxygenase breaks down ● with multiple mutations
this porphyrin ring to biliverdin ● types: type 1 and 2
2. Biliverdin is reduced by biliverdin reductase to form ● type 1: more serious; homozygously non-functioning
bilirubin/B1/unconjugated/non-polar bilirubin proteins (>5-20 mg/dL)
(nonpolar/hydrophobic) ● type 2: less severe; low enzyme levels
3. B1 cannot enter the liver because its hydrophobic;
therefore it will bound to albumin, since albumin is a
transport protein CAUSES OF ELEVATED CONJUGATED IN SERUM
4. B1 will now enter the liver, to be conjugated by the Elevated levels of B2 (meaning na-conjugate na), this
enzyme called “Uridyl Diphosphate happens when there is Excretion deficit (may problem sa
Glucuronyltransferase” forming B2 daanan)
5. B2/polar/conjugated bilirubin is now water-loving thus it 1. EXCRETION DEFICITS
can now be excreted
● Dubin-Johnson Syndrome:
6. B2 passing thru the intestine will be converted to
urobilinogen with the aid of bacteria, leading to formation ○ inborn error of metabolism; blockage of excretion of
of stercobilin which can be found in the stool bilirubin due to defects in ATP-binding cascadel;
associated with intense dark pigmentation of the
liver due to accumulation of lipofuscin
2. ROTOR SYNDROME
same with Dubin-Johnson where there is blockage in the
excretion of B2 w/o liver pigmentation; due to viral origin
NOTE:
According to Henry’s, there’s pigmentation if it is
Dubin-Johnson Syndrome, while in Rotor Syndrome
there’s NO pigmentation.
3. BILIARY OBSTRUCTION
● Gallstones - very common, usually due to cholesterol.
○ Cholelithiasis
note: mas informative itong diagram
■ most common cause in adults of
OTHER NAMES FOR BILIRUBIN hyperbilirubinemia.
B1 B2 ■ This condition results from the presence of bile
unconjugated conjugated stones (bilirubin or cholesterol) most commonly
water insoluble water soluble in the common bile duct (choledocholithiasis).
non-polar polar ● Inflammatory conditions
hemobilirubin cholebilirubin ● Hepatitis
Delayed/slowed reacting prompt reacting ○ toxic destruction of hepatocytes resulting in blocking
pre-hepatic/hepatic post-hepatic of conjugation & in excretion of B2
indirect reading direct reading ● Tumors

Clinical Significance of Altered Bilirubin levels: LIVER FUNCTION ALTERATIONS DURING DISEASE
1. Increased formation of bilirubin from red cell turnover JAUNDICE
● mabilis nasisira ang rbc ● “yellow”, icterus is used to describe yellow discoloration
● example condition/s: HDN,hemolytic anemias of the skin, eyes, & mucous membranes most often
2. Decreased conversion of unconjugated to conjugated
forms of bilirubin resulting from retention of bilirubin
● lack of Uridyl Diphosphate ● Most commonly classified based on the site of disorder:
Glucuronyltransferase(UDPGT) Pre-hepatic, Hepatic, & Post-hepatic
● liver problems a. Pre-hepatic
3. Impairment of bilirubin excretion ● when the problem causing jaundice occurs prior
● B2 cannot be released due to blockage to liver metabolism
● Hemolytic anemia

20
 b. Hepatic 5. GGT
● when the problem resides in the liver (intrinsic ● membrane localized enzyme found in high
liver disease) concentrations in the kidneys, liver, pancreas,
intestine & prostate but not in the bone
c. Post-hepatic
● a microsomal hepatic enzyme, therefore ingestion
● results from biliary obstructive disease (stones, of ethanol, certain drugs (barbiturates, tricyclic
tumors) antidepressants, anticonvulsants) elevates GGT
● Problem in excretion due to obstruction 6. LD
● serving as a general, non-specific marker for
Type of Unconjugated Conjugated Total cellular injury
Jaundice Bilirubin Bilirubin Bilirubin ● Not frequently requested not until Covid (LD is
Pre-Hepatic High Normal High used as a marker for covid)
Hepatic High High High
Post-Hepatic Normal High High NOTE:
Ferritin
Clinical Manifestations of Liver Disease ● is a storage of iron.
1. Jaundice ● Not frequently requested, not until Covid.
2. Portal hypertension ● Used as an inflammatory marker for Covid.
● occurs when there is obstruction of portal flow ● Increased in Covid cases, sa sobrang inflammation
3. Disorders in hemostasis tinago nang tinago yung iron to deprive
● Clotting factors are produced in liver microorganism esp. bacteria
4. Enzymes released from diseased liver tissue
● Cytoplasmic enzymes: enzymes seen in the cytoplasm
Enzyme tests for liver injury ○ Cytoplasm of the liver:
● Liver enzymes pay an important in the assessment of ■ ALT, AST, LDH
liver function because injury to the liver resulting in ● Mitochondrial enzyme:
cytolysis & necrosis will cause release of enzymes in the ○ AST has mitochondrial isoenzymes
circulation ● Enzymes in the canalicular
● for differentiating hepatocellular (functional) from ○ GGT, ALP → obstruction is the indication if any of
obstructive (mechanism) them is increased
● ALT, AST, ALP, LD, 5’-nucleotidase (5’NT), GGT ● Enzymes in blood increases when there is leakage due
to obstruction or cell destruction
1. ALT formerly called Serum Glutamate Pyruvate Enzyme Half-life
Transaminase SGPT
Cytoplasmic ALT 47 hrs
● found mainly in the liver
Cytoplasmic AST 17 hrs
● Kapag ALT and talking about liver
disease,obstruction ang tinitingnan Mitochondrial AST 87 hrs
2. AST formerly called Serum Glutamate Oxaloacetate ● Liver injury:
Transaminase SGOT ○ ALT > AST
● widely distributed in the heart, kidneys, muscles ○ ALT is HIGHER in due to longer half-life
3. ALP ● Alcoholic Hepatitis:
● zinc metalloenzymes that are widely distributed in ○ ALT < AST
all tissues, highest seen in the liver. bone, ○ leakage of ALT and AST
intestines, kidneys & placenta ○ mitochondrial organelle is destroyed and leakage of
● seen as a marker for extrahepatic biliary mitochondrial AST
obstruction ○ Deritis (AST:ALT ratio)
4. 5’NT ■ 3-4:1 → higher AST
● found in wide variety of cells; increased in ■ mitochondrial AST is HIGH due to longer
hepatobiliary disease half-life
● has no bone source so it is good in differentiating ■ presence of cytoplasmic AST also
ALP elevations
● Bihira i-request MOST COMMON DISEASE PROCESSES AFFECTING
● Only found in liver for obstruction LIVER
● Si ALP nakikita rin kapag may Obstruction. So, in order
5’NT is used para malaman kung liver talaga yung 1. Hepatitis: acute or chronic inflammation in which there is
problem. damage and destruction of hepatocytes
2. Cirrhosis: fibrous formation which leads to decrease in
CASE/S: hepatocytes
1. ALP - High & 5’NT - Normal 3. Tumor: hepatocellular carcinoma
a. Liver disease
b. Other sources
answer: B
Explanation:
5’NT is normal and it is only found in the liver, while
ALP is high and it can be seen in other organs.

21
 5. Infections
6. Toxins/drugs
7. Blocked bile ducts

HEPATIC TUMOR/HEPATOCELLULAR CARCINOMA

● 2nd most common site of metastases
● primary liver tumor is HCC which is the 5th most
common cancer worldwide

LABORATORY METHODS FOR BILIRUBIN ANALYSIS

SPECIMEN COLLECTION AND STORAGE:
● Serum or plasma may be used, however serum is
preferred in order to minimize possible turbidity or
interferences by plasma proteins
○ Plasma: presence of fibrinogen (200-400 mg/dL)
● Lipemia causes FALSE INCREASE in bilirubin
concentrations
● Hemolyzed specimen must be avoided because of
complex interferences with the reagent; decrease
bilirubin reaction with diazo reagent
● It should be protected from light → 30-50% reduction
hour
○ Bilirubin is photosensitive
● Stable for 2 days at RT, 1 week at 4C, indefinite at -20C
○ We processed immediately the sample because even
the fluorescent light can affect the bilirubin levels
CASE/S:
● Newborn jaundice is caused by immature liver →
physiologic jaundice. So if it undergoes phototherapy the
bilirubin becomes water soluble. Bilirubin can get
HEPATITIS
● Can be viral or non-viral in etiology photoisomerize by light
● Specimen transport of bilirubin: the bottle should be
● acute or chronic in duration
covered by foil or carbon paper. Do not use bond paper→
A. ACUTE HEPATITIS 30-50% reduction per hour
1. Viral: Hepatitis A, B, C
2. Acute Alcoholic hepatitis 1. EHRLICH (1883)
3. Toxic hepatitis: due to toxins or drugs; (ex: Bilirubin + Diazotized Sulfanilic Acid → AZOBILIRUBIN (Pink-Purple)
acetaminophen) ● Diazotized Sulfanilic Acid: 0.1% sulfanilic acid + 0.5%
4. Reye syndrome NaNO3
5. other causes: Wilson’s disease, autoimmune hepatitis ● Sample: Urine
2. VAN DEN BERGH (1913)
B. CHRONIC HEPATITIS (> 6 mos) Diazotized sulfanic acid may be used to serum in the
1. Viral: Hepatitis B and C presence of an ACCELERATOR(Alcohol)
2. Non-alcoholic Steatohepatitis 3. MALLOY-EVELYN (1937)
3. Autoimmune Hepatitis Bilirubin + Diazotized Sulfanilic Acid + Accelerator → 2 AZOBILIRUBIN
4. Alcoholic Liver disease ● 1st clinically useful method for Bilirubin
● Uses 50% methanol as an accelerator/dissociating
NTK: agent; pH is 1.2
● Hepatitis B: etiologically the number one cause of ● Sample: Serum
Liver disease in the Philippines ● Absorbance: at 560nm
● unvaccinated individual will pass the virus to his/her ● Product: Pink-red to purple color compound
offspring 4. JENDRASSIK-GROFF(1938)
● Most common; more sensitive and precise
CIRRHOSIS ● Product: Pink-blue colored compound
● diffuse fibrosis with nodular regeneration ● Caffeine benzoate: accelerator/dissociating agent
● represents end stage of scar formation and regeneration ● Sodium acetate: buffer
in chronic liver injury ● Ascorbic acid: destroys excess diazo reagent
● Tartrate: alkalinizes the solution; it converts purple to the
● Caused by virtually ALL chronic hepatitis
blue azobilirubin
1. Chronic alcoholism ● Absorbance at 600 nm
2. Hepatitis B and C ● Whenever we do bilirubin analysis we need 2 reaction
3. inherited disorders: Wilson’s AAT deficiency, A. Bilirubin + Diazotized Sulfanilic Acid + Accelerator→ 2
hemochromatosis, galactosemia AZOBILIRUBIN (Total Bilirubin)
B. Bilirubin + Diazotized Sulfanilic Acid → 2 AZOBILIRUBIN
4. Non-alcoholic steatohepatitis (Conjugated Bilirubin)

22
 CASE/S:
1. First Reaction: Test Tube contains serum (Bilirubin) + ULN (UPPER LIMIT than NORMAL)
Diazotized Sulfanilic Acid + Accelerator (Malloy 50% ● 5x higher the upper limit
methanol or Jendrassik Caffeine sodium benzoate) ● Example: 5-10 IU/L (Reference value)
then incubate. Spectrometric reading→ Total ○ Patient result: 12 IU/L - not considered
Bilirubin. Accelerator is used to extract the B1 from significantly elevated because there are
the albumin physiologic variations to be considered (age,
○ How to compute B1 gender, etc.)
● 𝐵1 = 𝑇𝑂𝑇𝐴𝐿 𝐵𝐼𝐿𝐼𝑅𝑈𝐵𝐼𝑁 − 𝐵2
2. Second Reaction: Test Tube contains serum ENZYMES
(Bilirubin) + Diazotized then incubate. NO ● are specific biologic proteins that catalyze biochemical
ACCELERATOR. Spectrometrical reading→ B2 or reactions in different organs in the body and which may
Conjugated Bilirubin also known as Direct also be located in different organelles and structures
Reading/PROMPT REACTING within a cell.
● The catalyzed reactions are frequently specific and
B1 B2 essential to physiologic functions, such as the hydration
Unconjugated Conjugated of CO2, nerve conduction, nutrient degradation and
Water insoluble Water soluble energy use.
Non-polar Polar ● Found in all body tissue, enzymes frequently appear in
serum, following cellular injury or sometimes, in smaller
Indirect reading Direct reading
amounts, from degraded cells.
Hemobilirubin Cholebilirubin
Delayed/slow reacting Prompt Reacting E + S ↔ ES ↔ E + P
Pre-hepatic Hepatic or post-hepatic ● Enzymes are not consumed in the reaction. Rather,
they are released once again.
UNCONJUGATED BILIRUBIN ● E is the enzyme, S is the substrate, ES is the
Unconjugated Bilirubin = Total Bilirubin – Conjugated Bilirubin enzyme-substrate complex, and P is the product.
or IB = TB – DB
3 BILIRUBIN FRACTIONS GENERAL PROPERTIES
polar, water reacting that is found in 1. A catalyst accelerates the rate of chemical reaction.
CONJUGATED the plasma in a free state thus 2. Enzyme specificity defines the capacity of a protein
(DIRECT) / B2 reacting to diazo reagent without an catalyst to recognize and bind only 1 or few molecules,
accelerator the substrate, excluding all others, a process referred to
non-polar, water insoluble that is as binding specificity. Reaction specificity is a unique
UNCONJUGATE
found in plasma bound to albumin. chemical process in which a solitary type of covalent
D (INDIRECT) /
Because of this, it will only react in the bond is broken or formed. Most enzymes exhibit absolute
B1
presence of an accelerator. reaction specificity that is no by-products formed.
conjugated bilirubin covalently bound 3. Enzymes DO NOT affect the value of equilibrium
to albumin constant. In a reversible reaction, the accelerate the
*DELTA forward and reverse by the same relative amount
only seen in hepatic obstruction
BILIRUBIN 4. Enzymes contain a surface region known as active site
too large to be filtered by the
glomerulus and excreted in the urine where binding and catalysis
*TB = DB + IB + Delta Bilirubin ENZYME COFACTORS
● enzymes requires cofactors
REFERENCE VALUES: 17.1 (conversion factor) ● ⅔ all enzymes contain cofactors that are a group of the
heat-stable substances required for catalysis.
TOTAL BILIRUBIN 0.3-1.2 mg/dL 3-17µmol/L
● They are low molecular weight organic molecules and
DIRECT BILIRUBIN <0.2 mg/dL 0-3 µmol/L inorganic ions.
INDIRECT BILIRUBIN 0.2-0.8 mg/dL 3-14 µmol/L ● Cofactors such as: (commonly used)
>2 mg/dL= JAUNDICE ○ nicotinamide adenine dinucleotide (NAD)
>20 mg/dL = KERNICTERUS (Bilirubin in the CNS); ○ pyridoxal phosphate
irreversible ● Inorganic ions such as:(called Activators)
● NICU - always request for bilirubin (STAT) ○ chloride
○ magnesium ions
CLINICAL ENZYMOLOGY ● When cofactors bound (covalent bonds) tightly to the
● Often useful in the diagnosis of particular diseases or enzyme it is called a prosthetic group.
physiologic abnormalities ● The enzyme portion that is purely proteins called
● Some enzymes are formed specifically for release into apoenzyme, in combination with its cofactor forms a
the circulation since this is where they carry out their complete and active system called a holoenzyme.
functions ● Catalytic site or active site of holoenzyme
● MOST enzymes detected are from cells, NOT from the ENZYME NOMENCLATURE & CLASSIFICATION
body fluids ● The nomenclature of commonly measured enzymes was
● Enzyme levels vary by pre-analytical variables standardized by the Enzyme Commision (EC) of the
● Their release into the body fluids may be due to: International Union of Biochemistry (IUB).
increased cell turnover/damage — the old cells are dying ● Each enzyme was designed according to the reaction it
& breaking down catalyzed.
● Other causes of increased enzymes: ● some characteristic of the reactions was identified
○ Neoplasia, increased enzyme synthesis, obstruction (usually the substrate) & the suffix-ase was added (ex.
to secretion, decreased clearance lactate dehydrogenase).
○ Cancer cells: many cells → many enzymes

23
● Another way is by using the systematic name to each ENZYMES KINETICS
enzyme, defining the substrate acted on, the reaction ● the basic objective of clinical enzymology is the
catalyzed & possibly the name of any coenzyme involved demonstration of the total concentration of specific
in the reaction (ex. L-Lactate: NAD+ oxidoreductase). enzymes in serum & other body fluids
● In addition to naming enzymes, the IUB identifies each ● By adding the substrate, say to serum & observing either
enzyme by numerical code, the first digit by its class, the its disappearance or the appearance of the product, the
second and third its subclass and sub subclass, presence of the enzyme can be ascertained.
respectively, the final number is the serial specific to ● Thus the rate at which the substrate disappears or
each enzyme in a subclass (ex. 1. 1. 1. 27) product appears can be directly used to determine the
concentration of enzyme present.
TYPE OF RXN ● E + S ↔ ES ↔ E + P
CLASS CATEGORY
CATALYZED ● Each molecule of the substrate must combine with a
oxidation/reduction molecule of enzyme to form a molecule of product.
1 Oxidoreductase
reactions ● In measuring enzyme activity we set up assay conditions
transfer of functional groups so that there are many more substrate molecules than
2 Transferases
from 1 substrate to another enzymes.
hydrolysis of various bonds ● In this situation, each enzyme molecule binds a substrate
3 Hydrolases (cleavage of bonds with and converts it, then accepts another substrate molecule
water) for further reaction.
removal of groups w/o ● the substrate cannot be converted any faster than the
4 Lyases hydrolysis to form double number of enzyme molecules present allows.
bonds ● Thus the enzyme level is rate-limiting, meaning the
convert 1 isomer to another enzyme concentration alone determines how fast the
5 Isomerases
(isomerizations) reaction proceeds when substrate is present in excess.
bond formation coupled
6 Ligases NOTE:
w/ATP hydrolysis

ENZYME STRUCTURE
● proteins that work as a catalyst
● speed up chemical reactions without being altered
themselves

as we add substrate the faster product formation forms.

TWO THEORIES However as we increase the substrate it forms a plateau -
LOCK AND KEYS meaning it slows the rate because you don’t add
very specific and rigid enzymes. So the enzymes are bound to a previous
substrate, aantayin pa matapos sa previous substrate
before siya mag bound sa new substrate.

FACTORS AFFECTING REACTION RATE

1. SUBSTRATE CONCENTRATION
● as the substrate level increases, the enzyme reaction
rate also increases.
● There is a point where further increase in substrate
concentration produces no more enhancement of the
reaction rate.
INDUCED FIT MODEL ● At this point the enzyme is saturated with the substrate. It
The enzyme here can alter shape to fit in the similar substrate follows the Michaelis-Menten equation stating that the
substrate readily binds to free enzymes at a low
substrate concentration.
2. FIRST-ORDER KINETICS
the reaction rate is directly proportional to substrate
concentration
3. ZERO-ORDER KINETICS
when the resultant free enzyme immediately combines with
the excess free substrate and depends on enzyme
concentration
4. ENZYME CONCENTRATION
the higher the enzyme level, the faster the reaction will
proceed because more enzyme if present to bind with the
substrate

24
 5. pH REACTION RATE MEASUREMENT OF ENZYME ACTIVITY
prefers optimum pH (7.0 - 8.0). pH extreme changes may ● E + S ↔ ES ↔ Product + Enzyme
denature an enzyme or influence its ionic state, resulting in ● Paano imemeasure yung Enzyme if di siya affected ng
structural changes or a change in the charge on an amino reaction?
acid residue in the active site. ○ Enzyme is a catalytic protein
6. TEMPERATURE ○ We are measuring the RATE (gaano ka-bilis)
● Increasing temperature usually increases the rate of a ○ THE RATE OF:
chemical reaction by increasing the movement of ■ PRODUCT FORMATION
molecules, the rate at which intermolecular collisions ● The faster the product formation there is,
occur, & the energy available for the reaction. it means the higher the enzyme levels.
● This is the case of the enzymatic reactions until the ● Kasi fixed yung concentration ng
temperature is high enough to denature protein substrate, kasi laboratory conditions na
composition of the enzyme. then per minute.
7. COFACTORS ■ SUBSTRATE CONSUMPTION
● non-protein entities that must bind to a particular enzyme ● Gaano kabilis naubos yung substrate
before a reaction occurs. ● In 1 minute, gaano karaming substrate
● Common activators are metallic (Ca++, Fe++, Mg++, yung naubos because fixed yung
Zn++, K+) & non-metallic (Br, Cl). conditions
● Activators function by altering the spatial configuration of ■ DISAPPEARANCE OF
the enzyme for proper substrate binding. COFACTORS/COENZYMES
● Increasing the cofactors will increase the velocity of an ● Pwede gamitin basehan to measure the
enzymatic reaction in a manner similar to substrate enzyme activity
concentration. ● Hindi natin kaya imeasure yung enzyme
● Without cofactors there are no enzymatic reactions. directly
● Enzyme quantitation is based on catalytic activity
8. INHIBITORS ● Common methods might measure an increase in product
are molecules that decrease the rate of enzyme reactions. It concentration, a decrease in substrate concentration, a
may bind to the active site blocking the access of substrate to decrease in coenzyme concentration or an increase in
the enzyme. the concentration of altered coenzymes.
● Enzyme concentrations are always performed in zero
Noncompetitive inhibitor order kinetics
Competitive inhibitor
● binds the enzyme at a ● Optimum pH & temperature must be observed.
● occurs when the inhibitor
place other than the active
binds at the same active site
site.
as the substrate. TYPES OF ENZYME ASSAYS
1. FIXED TIME/ ENDPOINT
● After 1 minute, i-mmeasure.
● The reactants are combined, the reaction proceeds for a
designated time, then the reaction is stopped & a
measurement of the amount of reaction is made. The
reaction is assumed to be linear.

2. CONTINUOUS MONITORING/ KINETIC

● MAY INTERVAL
○ If the entire procedure asks for 2 minutes, in
between magmemeasure ka. After 1 minute,
magmemeasure and then tanggalin, continue
Uncompetitive inhibitor: incubation and then another 60 seconds ibalik ulit
● binds only to the ES complex and not to free. ● Multiple measurements, usually absorbance change, are
● nagkita na yung enzyme and substrate. Meron na ES made during the reaction, either at specific time intervals
complex, but because of the presence of the inhibitor, (30s or 60s) or continuously by a continuous-recording
nagpupumilit siya. So mayroon nang ESI complex and spectrophotometer. More advantageous because
hindi na maglelead ng product formation because there's linearity may be verified so any deviations may be
no reactions involved anymore. observable.

9. BUFFER UNITS OF ENZYMATIC ACTIVITY MEASUREMENT

● It plays an important role in regulating the pH. It also ● The IUB & IUPAC proposed that enzymes be reported in
affects the rate of reaction. International Units (IU). It is the amount of enzyme which
● An example of a buffer contributing to the enzyme catalyzes the conversion of 1 umol of substrate in 1
reaction rate is the ALP system. Here the buffer serves minute under specified conditions.
as an acceptor of the phosphate group removed from the ● Enzyme concentration is usually expressed in units per
phosphate group. liter (IU/L)
● Without this acceptor present, the reaction is markedly ● The SI recognizes the unit for enzyme activity as katal
lower. (mol/s). Enzyme concentration is then expressed as
katals per liter (kat/L) (1 IU = 17 nkat).

25
 STANDARDIZATION OF ASSAY CONDITIONS
Many enzymes have a Selected Reference Method or an
assay approach defined by an international group. These
methods have been approved after a careful study &
evaluation of all parameters of the reaction. LIGASE
● An enzyme that catalyzes the bonding together of two
OXIDOREDUCTASE molecules into one with the participation of ATP.
● Catalyzes oxidation-reduction reaction ● ATP involvement is required because such reactions are
○ Reduction is the addition of H to = generally energetically unfavorable and they require the
○ Oxidation is the removal of H to = simultaneous input of energy obtained by hydrolysis
● Oxidation-reduction are not independent precesses but reaction in which ATP is converted to ADP.
linked processes that must occur together, an
oxidoreductase requires a coenzyme that is oxidized or
reduced as the substrate is reduced or oxidized.
● LDH is an oxidoreductase that removes H atoms from a
molecule.

ENZYMES OF CLINICAL SIGNIFICANCE

There are 6 groups of enzymes based on the type of reaction
they catalyze which are mainly focused on biochemistry, so
we are only focusing on the clinically significant enzymes,
including their mode of action, where they can be found, etc.
TRANSFERASES CLASSIFICATION OF FREQUENTLY QUANTITATED ENZYMES
● Catalyzes the transfer of a functional group from one Lactate dehydrogenase (LDH)
Glucose 6-phosphate dehydrogenase
molecule to another. Oxidoreductases
(G6PD)
● Two major subtypes of transferases: Glutamate dehydrogenase (GLD)
○ TRANSAMINASES Aspartate aminotransferase (AST)
■ Catalyzes the transfer of an amino a group Alanine aminotransferase (ALT)
from one molecule to another Transferases Creatine Kinase (CK)
○ KINASES Gamma glutamyl transferase (GGT)
■ Plays a major role in metabolic energy Pyruvate-kinase (PK)
production reactions Alkaline phosphatase (ALP)
Acid phosphatase (ACP)
■ Catalyzes the transfer of a phosphate group
Amylase (AMS)
from adenosine triphosphate (ATP) to give Hydrolases Cholinesterase (PCHE)
adenosine diphosphate (ADP) & Chymotrypsin (CHY)
phosphorylated product (a product containing 5’-Nucleotidase (5-NT)
an additional phosphate group) Lipase (LPS)
● Amino acid I + keto acid II -> keto acid I + amino acid II Lyases Aldolase (ALD)
● Alanine + alpha-ketoglutarate -> pyruvate + glutamate Isomerases Triosephosphate isomerase (TPI)
Ligase Glutathione synthetase (GSH-S)
CREATINE KINASE (CK)
● ATP-Creatine-N-Phosphotransferase
● Phosphorylates ATP
○ Adds a phosphate group in the ATP

LYASE
● Catalyzes the addition of a group to a double bond or the
removal of a group to form a double bond in a manner ● Generally associated with ATP regeneration in contractile
that does not involve hydrolysis or oxidation. of transport systems (muscle contraction)
● A dehydratase affects the removal of the components of ● Physiologic function occurs in muscle cells where it is
water from a double bond & a hydratase affects the involved in the storage of high energy creatine phosphate
addition of the components of water to a double bond. ○ It is very much involved in energy production, etc.
● When muscle contraction occurs, ATP is hydrolyzed to
ADP to produce chemical energy for the contraction
process.
● Muscle action is the significance
● Occurs as a dimer consisting of 2 subunits: M and B
ISOMERASE ● TISSUE SOURCES:
● An enzyme that catalyzes the isomerization ○ Skeletal (MM)
(rearrangement of atoms) of a substrate in a reaction, ○ Heart (MB)
converting it into a molecule isomeric with itself. ○ Brain (BB)
● There is only one reactant & one product in reactions
where isomerases are operative.

26
CK ISOENZYMES (based on the tissue sources) ● Light-sensitive, serum storage should be in dark place
1. CK-1 / CK-BB ○ The effect of light in CK is not as drastic as
● Migrates the FASTEST compared to Bilirubin
2. CK-2 / CK-MB ● Inactivation in the serum can be reversed by adding
● Sensitive indicator for AMI
sulfhydryl compounds to the assay reagent, such as
3. CK-3 / CK-MM
● Major isoenzyme in healthy people N-acetylcysteine, mercaptoethanol, thioglycerol &
● Migrates the SLOWEST dithiothreitol
NOTE: NOTE
● CK isoenzymes are numerically arranged based on the Serum – mostly used sample in clinical chemistry
electrophoretic mobility, with CK-1 being the fastest.
Pinakamabagal naman (CK-3) yung malapit sa origin.
● When it comes to subunits, CK-1 is the CK-BB since ASSAYS:
the brain yung nasa pinakamataas na part of the body, 1. Tanzer-Gilvarg
followed by the heart (CK-2/CK-MB) and skeletal ● Forward assay
muscle (CK-3/CK-MM). ● Coupled enzyme system in which creatine is converted
● CK-1 is not commonly tested nor is the CK-3 routinely to → creatine phosphate
requested. Instead, puro CK-2 (CK-MB) ang ● Optimum pH 9.0
naeencounter sa laboratory kasi nandito yung clinical ● Change in absorbance at 340 nm is determined
significance ng CK. It is what the physicians want to ● If NADH to → NAD is present, absorbance is always 340
know. However, it still depends on the protocol of the nm
laboratory.
● The value of CK detection is for Myocardial
damage.

CLINICAL SIGNIFICANCE
1. ACUTE MYOCARDIAL INFARCTION
● CK-MB rises within 4-8 hours
● CK-MB peaks at 12-24 hours 2. Oliver-Rosalki
● CK-MB returns to normal within 48-72 hours (3 ● Reverse reaction
days) ● Coupled enzyme system in which creatine is produced
2. Duchenne Muscular Dystrophy, Rhabdomyolysis from creatine phosphate
3. Strenuous exercise ● Optimum pH 6.8
4. Cerebrovascular accident, seizures, nerve ● Faster reaction & less interferences from side
degeneration, CNS shock reactions
NICE TO KNOW:
● Tumataas din ang CK because of their different
isoforms. Kung may stroke and muscle diseases,
tumataas ang CK isoforms.
● (When it comes to stroke, na-dc for a few secs)
● TRIVIA: ● REFERENCE VALUE:
○ Serum CK and CK/Progesterone ratio is used to ○ Total CK:
diagnose ectopic pregnancies ■ Males: 46-171 IU/L
○ Total CK have been used to diagnose V. vulnificus ■ Females: 34-145 IU/L
infections (Henry’s) ○ CK-MB: <5% of Total CK
NOTE:
ATYPICAL FORMS OF ISOENZYMES ● Hexokinase method is the most sensitive method for
1. MACRO-CK glucose determination
● Migrate midway between CK-MM and CK-MB ● Regarding CK-MB’s reference value : if it is more than
● Largely CK-BB with IgG or IgA attached to it 5%, this indicates Acute Myocardial Infarction (AMI)
○ CK-BB is supposed to be the fastest, however
there is IgG or IgA attached to it, so it flowers Isoenzyme Electrophoresis
down the electrophoretic mobility.
2. MITOCHONDRIAL-CK (CK-Mi)
● Bound to the exterior surface of mitochondrial
membranes of muscle, brain and liver
● Migrates to point CATHODAL to CK-MM
● NOT present in normal serum nor in AMI
● Normal CK’s:
SPECIMEN PRECAUTION
○ If the first dotted line is the origin, the fastest is
● Serum sample is used since anticoagulants inhibit
CK1(CK-BB), followed by CK2(CK-MB) then
enzyme activity CK3(CK-MM)
● Hemolysis (first caution in CK) ● Abnormal CKs:
○ Because CK is inside the muscle, so if the blood is ○ Macro-CK: (2nd dotted line) May mga nakakabit na
hemolyzed it may result to cell destruction or cardiac immunoglobulin kay CK-MB kaya medyo mabagal
muscle destruction yung takbo
○ If the sample is hemolyzed – The CK result will ○ Mitochondrial-CK:(1st dotted line) di talaga
tumatakbo sa electrophoresis
increased

27
 LACTATE DEHYDROGENASE (LDH) ● REFRESH:
● We’ve learned LDH as non-specific, very general liver ○ ALL enzymes are proteins but NOT ALL proteins
enzyme are enzymes.
● Catalyzes the oxidation of lactate to pyruvate with the ○ CK, AST, ALT are the cardiac enzymes
mediation of NAD as a H+ acceptor ○ Myoglobin and Troponin are the cardiac proteins
● Zinc-containing enzyme (serves as cofactor) that is part ○ In practice, when there is suspected AMI:
of the glycolytic pathway and is found in virtually all cells ■ Troponin-I is the MOST specific since it is
in the body mainly found in the cardiac muscles and our
○ Non-specific due to its presence in almost all parts only perspective in the laboratory (since
of the body Troponin-T has other distributions and
■ Kahit saang disease, pwedeng tumaas ang Troponin-C is a Ca2+-binding subunit)
levels ng LDH kaya hindi masyadong 2. Pulmonary embolism
pinagtutuunan ng pansin ng mga doktor 3. Liver disease: LDH–4 and LDH–5
i-request kasi dagdag gastos pa sa patient. 4. Muscular dystrophy
5. Hemolytic anemia
6. Acute lymphocytic leukemia

SPECIMEN PRECAUTION
● TISSUE SOURCES: (widely distributed in the body) Specimen Precaution:
○ Heart ● High interference from hemolysis (RBCs contain 150x
○ Liver the serum level of LDH)
○ Skeletal muscle ● Unstable even in storage at different temperature
○ Kidney ● If not assayed immediately, store at 25°C & analyzed
○ RBC within 48 hours
○ Lung ● Loss of activity is faster at 4°C than at 25°C
● Pre-analytical variable to be considered in LDH: ASSAYS:
○ High interference from hemolysis (RBC’s contain LDH activity can be measured using either the Forward
100-150x the serum level of LDH) (Lactate to Pyruvate) or Reverse (Pyruvate to Lactate)
○ Should be free from hemolysis since LDH is inside
1. Wacker method
the cells, along with the following that can be seen in
intracellular namely: Potassium, ACP, Magnesium, ● Utilizes the forward reaction with formation of NADH
Phosphorus from NAD
○ All these five components will INCREASE if the ● Absorbance at 340 nm
serum specimen is hemolyzed. ● Optimum pH 8.3-8.9

LACTATE DEHYDROGENASE ISOENZYMES

LDH–1 HHHH Heart, RBC, 2. Wroblewski-LaDue
Migrates the FASTEST (H4) Renal Cortex
● Utilizes the reverse reaction where NADH serves as a
17 to 27%
LDH–2 HHHM Heart, RBC,
cosubstrate & is consumed during the reaction
Normally HIGHEST concentration (H3M) Renal Cortex ● Faster reaction BUT susceptible to substrate
27 to 27% exhaustion & linearity problems
LDH–3 HHMM Pancreas, lung, ● Optimum pH 7.1-7.4
18 to 25% (H2M2) Lymphocytes, ● REFERENCE VALUE: 125-220 IU/L
Platelet
LDH–4 HMMM Hepatocyte
3 to 8% (HM3)
LDH–5 MMMM Hepatocyte,
Migrates the SLOWEST (M4) Skeletal muscle,
0 to 5% prostate ASPARTATE AMINOTRANSFERASE (AST)
LDH–6 Present in patients ● Formerly Serum Glutamate Oxaloacetate
Cathodic to LDH–5 with Arteriosclerotic Transaminase (SGOT)
6th band of electrophoresis failure
Represents alcohol dehydrogenase enzyme ● Catalyzes the transfer of an amino group from aspartate
Elevated in hepatotoxicity and ethylene glycol to alpha-ketoglutarate to yield oxaloacetate & glutamate
to toxic compounds
Aspartate + alpha-ketoglutarate < —---- >Oxaloacetate +
Concentration in the normal serum: LDH–2 > 1 > 3 > 4 > 5
glutamate
CLINICAL SIGNIFICANCE AST
1. Acute myocardial infarction (AMI)
● LDH–1 > LDH–2 flipped pattern ● Has 2 isoenzyme fractions: cytoplasmic &
● LDH begins to rise within 12-24 hours mitochondrial AST - the cytoplasmic isoenzyme is the
● LDH peaks within 48-72 hours predominant form in serum
● LDH returns to normal within 10 days NOTE:
NOTE: ● Mas mahaba yung sa mitochondria (87 hrs) kesa sa
● LDH is slower than CK cytoplasm (17 hrs)
○ CK-MB: 4-8 hours ● Kapag may alcohol damage, saka magrerelease ng AST
○ LDH: 12-24 hours sa mitochondria
● According to Sir Eric’s experience, walang
nagpapa-LDH kapag may patients with AMI kahit
● Pyridoxal phosphate (Vit B6) acts as coenzyme
mayroong LDH sa lab. Some laboratories do not have
LDH in their cardiac panel but it still depends on the ● Tissue sources: heart, liver and skeletal muscle
protocol of the laboratory.

28
 CLINICAL SIGNIFICANCE
1. Acute myocardial infarction
a. AST begins to rise within 6-8 hours
b. AST peaks at 24 hours
c. AST returns to normal within 5 days
Alkaline Phosphatase (ALP)
NOTE: Basically pinakamabilis si K followed by AST and ● Optimal pH 9.0-10.0
then followed by Lactate dehydrogenase ● Requires Mg++ as activator
● Tissue sources: Intestine, liver, bone, spleen, placenta
& kidney
2. Hepatocellular disorders - highest levels in viral
hepatitis ● In the liver the enzyme is located on BOTH Sinusoidal &
3. Skeletal muscle disorders such as muscular canalicular membranes; bone activity is confined to the
dystrophy & inflammatory conditions osteoblasts
● Elevated in hepatobiliary disorders such as biliary tract
SPECIMEN PRECAUTION obstruction, bone disorders like Paget’s disease,
● Hemolysis osteomalacia, rickets, hyperparathyroidism &
● Stable for 3-4 days at refrigerated temperature
osteogenic sarcoma
ASSAY:
● Normally elevated between 16-20 weeks of pregnancy
1. Karmen method
and persists until the onset of labor
● Coupled enzyme reaction
● Optimum pH 7.5 (7.3-7.8) ● Exists in different isoenzymes which can be separated by
● Read at 340 nm electrophoresis:
● REFERENCE VALUE: 5-35 IU/L ○ The liver isoenzymes migrates the fastest towards
the anode while the intestinal isoenzyme is the
slowest
● Heat stability: (PATTERN: BLIP-LSLS)
NOTE: 1. Bone - labile at 56oC for 10 mins
To interpret enzymes, receiving a value with one unit 2. Liver - stable at 56oC for 10 mins
higher than the normal range, don’t conclude that it has 3. Intestinal - labile at 65oC for 30 mins
complications yet. However, if it is 2x or 3x higher than the 4. Placental - stable at 65oC for 30 mins
normal range, then you may conclude that it has NOTE: Placental isoenzyme is the most heat stable (65oC
complications. for 30 mins)
Alanine Aminotransferase (ALT)
● Chemical inhibition
● Formerly Serum Glutamate Pyruvate Transaminase
1. Phenylalanine inhibits intestinal & placental
(SGPT)
● Catalyzes the transfer of an amino group from alanine to 2. 3M urea inhibits bone ALP
alpha-ketoglutarate to yield pyruvate & glutamate 3. L-leucine inhibits Nagao isoenzyme
(carcinoplacental isoenzyme)
Alanine + alpha-ketoglutarate < —- > Pyruvate + glutamate 4. Phenylalanine inhibits Regan isoenzyme
ALT 5. Levamisole reagent inhibits liver & bone ALP
● Pyridoxal phosphate acts as coenzyme
● Tissue source: highest concentrations in the liver REGAN AND NAGAO ISOENZYMES
● Elevated in hepatic disorders ● Regan & Nagao isoenzymes are referred to as carcino
placental alkaline phosphatases & occurs 3-15% in
SPECIMEN PRECAUTION
● Stable for 3-4 days at Ref. temp cancer patients
● Relatively unaffected by hemolysis ● Regan isoenzyme is an example of ectopic enzyme
ASSAYS: production by a malignant tissue.. It is found in lung,
There are 3 major approaches but all measure the formation breast, ovarian & gynecological cancer; bone ALP-co
of ketoacid produced in the reaction. migrator;
1. Reitman-Frankel ● Most heat stable ALP
● Colorimetric ● Nagao isoenzyme is found in adenocarcinoma of the
● Utilizes dinitrophenylhydrazone which gives a strong pancreas & bile duct, pleural cancer
blue color
SPECIMEN PRECAUTION
● Absorbance at 505 nm
● Hemolysis may cause elevations because ALP is
● Lack of specificity
approximately 6x more concentrated in RBCs than in
2. Reaction with diazonium salts serum
3. Coupled enzyme reaction ● Should be run immediately or within 4 hours of
● Uses LDH as an indicator which catalyzes the reduction collection
of pyruvate to lactate with the simulation oxidation of ● Diet may induce elevations in ALP activity in group B or
NADH O secretores after a high fat meal (25% higher ALP)
● Change in absorbance is measured at 340 nm at pH 7.5 ● REFERENCE VALUE:
● REFERENCE VALUE: 7-45 IU/L ○ Male: 53-128 IU/L
○ Female: 42-98 IU/L

29
 Summary of ALP METHODS ● rapid & non-specific
Methods Substrate Product method
1. Bessey, Lowry & Brock 2. Babson & Reed alpha-
p-nitrophenol + alpha-naphth
● Endpoint or kinetic ● For continuous napthylphospha
p-nitrophenyl PO4+ or yellow ol
method monitoring methods te
phosphate nitrophenoxide
● 405 nm 3. Roy
ion
absorbance ● More specific for thymolphthalein free
2. Browers- McComb prostatic form & is monophosphat thymolphthal
● Uses phosphate used for quantitative e ein
accepting buffer endpoint reactions
● Reference method inorganic
beta-glyceroph
& most specific 4. Bodansky phosphate +
osphate
● Continuous glycerol
monitoring 5. Gutman, King & phenylphosphat
technique that phenol
Armstrong e
requires pH 10.15
● 405 nm GAMMA GLUTAMYL TRANSFERASE (GGT)
absorbance
phenyl ● Involved in the transfer of the gamma glutamyl residue
3. King- Armstrong phenol
phosphate from gamma glutamyl peptides to amino acids, H2O, and
inorganic other small peptides
4. Sinowara, Jones & beta-glyceroph
phosphate +
Reinhart osphate ● Glutathione serves as the gamma glutamyl donor
glycerol
phenolphthalein phenolphthalein Glutathione + Amino acid ←(GGT)→ Glutamyl-peptide +
5. Huggins & Talalay
diphosphate red L-cysteinylglycine
alpha-naphthol
6. Moss alpha-naphthol
phosphate ● Tissue sources: liver, kidney, brain, prostate &
pancreas; but assays are confined primarily to the liver &
ACID PHOSPHATASE (ACP)
hepatobiliary system
● Optimal activity at pH 5.0
● Elevated in alcoholism & all forms of liver disease,
● Tissue sources: Prostate, bone, liver, spleen,
enzyme inducing drugs (warfarin, phenobarbital,
erythrocytes, kidney & platelets
phenytoin)
● The major forms are coded by different genes possess
● Not elevated in bone disease as is ALP
different molecular weights, structures as well as
● Not elevated in muscle or hemolytic disorders as is
sensitivity to tartrate inhibition
AST
● RBC ACP is inhibited by: 2% formaldehyde & 1mM
● Not elevated in Myocardial Infarction (MI) until
cupric sulphate but not inhibited by 20mM tartrate
possibly the 4th day, probably associated w/liver damage
solution unlike the other isoenzymes like prostatic ACP
secondary to cardiac insufficiency
● Tartrate-resistant acid phosphatase (TRAP) is present
●
in certain chronic leukemia & lymphoma most notably
METHODS: SZASS, ROSALKI & TARROW, ORLOWSKI
HAIRY CELL LEUKEMIA ● For ethanol intoxication
● Elevated in prostate disease but is being replaced by ● Most widely used substrate is gamma glutamyl
prostate specific antigen (PSA) because it is more p-nitroanilide
sensitive and specific for prostate cancer ● The gamma glutamyl residue is transferred to
● Used for Forensic Investigation – rape glycylglycine, releasing p-nitroaniline (a chromogenic
product with a strong absorbance at 405-420nm)
NOTE
● Serum activity is stable for 1 week at 4C
Prostatic secretion is acidic, while vaginal fluid is alkaline
● Not affected by hemolysis
● REFERENCE VALUE:
○ M: 6-55 IU/L
SPECIMEN PRECAUTION
○ F: 5-38 IU/L
● Affected by hemolysis; serum must be separated
immediately to prevent leakage of RBC & platelet ACP
● Serum ACP decreases within 1-2hours if left at room 5’ NUCLEOTIDASE (5–NT)
temperature ● It is a cytoplasmic – membrane bound phosphoric
● If not assayed immediately, the serum should be frozen monoester hydrolase
or acidified to a lower pH 6.5. With acidification, ACP is ● Predominantly found in liver
stable for 2 days at room temperature ● Marker for hepatobiliary disease & infiltrative lesions
● REFERENCE VALUE: of the liver
○ Total ACP = 0.3 - 11.7 IU/L
● Used to determine the source of elevated ALP is from the
○ Prostatic ACP = 0 - 3.5 ng/mL
liver or bone
Summary of ACP METHODS
Methods Substrate Product AMYLASE (AMS)
1. Hudson ● One of the most important digestive enzyme
● This reaction
produces colorless
● Catalyzes the breakdown of starch into monosaccharides
p-nitrophenyl ● Smallest enzyme in size and is normally filtered by the
product but with the p-nitrophenol
phosphate
addition of alkali, the renal glomerulus & also appears in the urine
product turns into ● Tissue sources: acinar cells of the pancreas & salivary
chromogens
glands

30
● Elevated in acute pancreatitis, mumps, and
macroamylasemia (results when AMS combines with Ig ASSAYS:
to form a complex that is too large to be filtered by the ● Procedures include estimation of liberated fatty acids
(titrimetric) and turbidimetric methods
glomerulus)
● It uses olive oil as the substrate because other esterases
● AMS isoenzymes in acute pancreatitis: can hydrolyze TAG and synthetic diglycerides
1. Begins to rise w/in 2-12 hours ● Addition of colipase (protein secreted by pancreas) and
2. Peaks at 24 hours bile salts will make assay more sensitive & specific for
3. Returns normal w/in 3-5 days detection
● AMS in urine remains elevated for 7 days ● Triolein (more pure form of TAG= used as substrate for
LPS
● Hemoglobin inhibits activity of LPS= falsely low values
SPECIMEN PRECAUTION
● Stable in serum for 1-3 weeks at 4C
● Test immediately: common request on ER or for
pancreatitis
● Stable in urine and serum 1. Cherry and Crandall
● Little loss of activity= 1 week at room temp. or 4C for 1 Hydrolysis of olive oil after incubation for 24 hours at 37C and
month titration of liberated fatty acids using NaOH
● AMY may be normal in hyperlipidemia because plasma
TAG inhibits/suppress serum AMY activity
● Administration of morphine or other opiates may falsely
elevate serum AMY 2. Tietz and Fiereck
ASSAYS:
1. Amyloclastic 3. Peroxidase Coupling Method
Measures the disappearance of starch substrate (amount of ● Most commonly used method
starch broken down) ● Does not use olive oil
2. Saccharogenic ● Uses peroxidase or glycerol kinase
● REFERENCE VALUE: <38 IU/L
● Measures the appearance of the product (amount of
reducing sugars produced by the hydrolysis of starch)
GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE (G6PD)
● classic reference method expressed in Somogyi units
● An oxidoreductase that catalyzes the oxidation of
3. Chromogenic glucose-6-phosphate to 6-phosphogluconate or the
Measures color produced of the product coupled with a dye corresponding lactone
● Tissue source: RBC, adrenal cortex, spleen, thymus,
lymph nodes, and lactating mammary glands
4. Continuous
● Coupling of several enzymes systems by continuous ● Most of the interest lies on its role in the RBC (hemolytic
monitoring technique anemia)
● Measured at 340 nm with optimum pH of 6.9
NOTE
● In clinical chemistry, G6PD assay is used to diagnose
the G6PD deficiency
● G6PD deficiency – common in the Philippines
(Southeast asia)

● REFERENCE VALUE: ASSAYS:

○ Serum: 28-100 IU/L ● A red cell hemolysate is used to assay for deficiency of
○ Urine: 1-15 U/h the enzyme while serum is used for evaluation of enzyme
elevations
LIPASE (LPS) / TRIACYLGLYCEROL ACYLHYDROLASE ● REFERENCE VALUE: 7.9-16.3 U/g Hgb
● Hydrolyzes glycerol esters of fats to produce alcohol &
fatty acids
Triglycerides ← (LPS) → Glycerol + 3 fatty acids
● It catalyzes partial hydrolysis of dietary TAG in the
ALDOLASE/FRUCTOSE 1,6-DIPHOSPHATE ALDOLASE
intestine to the 2-monoglyceride intermediate with the
● Catalyst in one of the reactions in the glycolytic
production of long chain fatty acids
breakdown of glucose to lactic acid
● Found primarily in the pancreas, not affected by renal
● All blood cells contain this enzyme, the RBC level is 150x
disorders
as high as the serum level
● Clinically elevated in acute pancreatitis, rises 4-8 hours,
● Don’t use hemolyzed sample
peaks at 24 hours, and decrease within 8-14 days
● Clinical significance: Muscle disease & MI (rises in 6-8
● Persist for approximately 5-8 days in acute pancreatitis
hours like AST, stays up for 3-4 days)
NOTE ● Isoenzymes:
LPS assay is more sensitive in diagnosis of Acute
○ Aldolase A – skeletal muscles
pancreatitis
○ Aldolase B – WBC, liver, kidney
○ Aldolase C – brain tissue

31
 LABORATORY METHODS FOR ENZYMES ● So sequence is very important, yung tamang order and
Measurement of enzyme activity: pagkakasunod-sunod. Hindi porket same yung numbers,
● Enzyme quantitation is based on catalytic activity for instance 146 amino acids yung beta chain and you
have 2 beta chains magiging normal. Imagine, it is really
● Common methods might measure an increase in product
fascinating (si sickle cells disease) kasi yung pang-anim
concentration, a decrease in substrate concentration, a lang na amino acid out of 146 amino acids yung beta
decrease in coenzyme concentration or an increase in chain. Yung pang-anim lang naging valine, which is
the concentration of altered coenzyme dapat glutamic and yet yung consequence nito ay
● Enzyme concentrations are always performed in zero terrible. Dahil lang sa isang nagpalit na amino acid,
order kinetics panghabambuhay na yung consequence (na may sakit).
● Optimum pH & temperature must be observed ● Sickle cell disease is predominant in individuals that have
an ethnicity of Middle Eastern and Africans.
NOTE ● In Saudi, there’s a ward called “sicklers” (as in tabi-tabi
pinagsama ko na sila lahat sa Enzymes of Clinical Significance :))
sa ward yung may mga SCD and kapag umaatake raw
yung SCD sumisigaw raw sa sakit. Kasi, look at the
PROTEINS shape, yung moon-shaped dadaloy sa ugat mo edi
● Mainly manufactured in the LIVER. nagkandatusok-tusok/nagkanda-clog clog.
● Composed of CARBON, HYDROGEN, OXYGEN and
NITROGEN. 2. SECONDARY
● Very important role of Proteins: ● winding of the polypeptide chain; alpha-helix sheats,
○ To maintain the structure beta-pleated sheets & random coils, all dependent on
○ As enzymes numerous H-bonds & occasional disulfide bonds.
○ As antibodies ● Nagtatapat-tapat yung amino acids so they form
○ Catalytic metabolism hydrogen bonds. So medyo inikot mo na, kapag inikot
○ Hormones (a lot of hormones are proteins) mo na yung linear mo kanina may amino acids na
● Polymers of amino acids linked together by peptide magtatapat, so dahil nagkatapat sila magfoform sila ng
bonds hydrogen bonds.
○ very short chains are designated as di-, tri-, tetra- or ● JUST LIKE A TELEPHONE CORD, yung magkakatapat
pentapeptides every fourth amino acid ay magfoform ng hydrogen
○ >5 residues in length are oligopeptides, between bond.
6-30 are polypeptides
● Synthesized mainly in the liver except for Ig 3. TERTIARY
● Amphoteric- can bear positive and negative charges ● involves the intramolecular folding of the polypeptide
because of their acid and basic amino acid compositions chain into a compact 3-dimensional structure with
● Structure: specific shape.
1. fibrous (mainly structural) such as fibrinogen, ● More complicated folding, mas maraming
troponin, collagen and myosin nagkakatapat-tapat giving you a different bond
2. globular such as hemoglobin, enzymes, peptide formations.
hormones and plasma proteins ● Such as disulfides (kasi halimbawa dalawang sulfur
● Functions: catalysts, buffers, regulates metabolism, containing amino acids they will form disulfide bonds),
immunity, repair body tissues, plasma proteins and hindi lang hydrogen bonds and so on.
maintain osmotic pressure
4. QUATERNARY
● refers to the association of several polypeptide chains
or subunits into a larger “oligomeric” aggregate subunit.
● Just like the hemoglobin, it is already quaternary kasi
apat na yung polypeptides and nagkabuhol-buhol na sila.
● The proteins are linked by the peptide bonds, because
that’s one way of measuring the protein concentration.
● THE HIGHER THE PEPTIDE BONDS, THE HIGHER
THE PROTEINS.
● But structurally, it can be fibrous (pahaba na structure)
and also globular (maraming folds kaya pabilog).

GLOBULAR PROTEINS
● Most globular proteins retain biologic activities (in their
structure) at optimal conditions.
● Globular proteins have the following four structures:

1. PRIMARY
● linear sequence of the amino acid; refers to the identity
& specific order of amino acid residues in the polypeptide
chain. (LINEAR- like a rosary; beads)
● Again, in hematology kasi doon unang naka discover ng
mga ganito. Yung nagkapalit-palit na position ng amino
acids sa beta chain. What is that very common
hemoglobinopathy? Na kung saan nagkaroon ng palit
palit na sequence? ANSWER: SICKLE CELL DISEASE
(sixth glutamic acid to valine)

32
● The function of proteins depends on its structure. So Alpha 1 Alpha 2 Beta Gamma
kapag ang structure ng protein ay nasira, they become *Alpha 1- *Haptoglobin *Transferrin *Immunoglobuli
useless as well. Its biologic activity is rendered useless. Antitrypsin *Ceruloplasmi *Complement ns
● So it is very important to maintain its structure. *Alpha n *C-Reactive *IgG, IgM, IgA,
Feto *Alpha 2- Protein IgD, IgE
CLASSIFICATION Protein Macroglobulin *Fibrinogen
1. SIMPLE PROTEINS *HDL *Hemopexin
contain peptide chains only (puro peptides lang, puro amino acids lang) *Alpha 1- *LDL
Acid *Beta 2-
2. CONJUGATED PROTEINS *Glycoprot Macroglobulin
● proteins coupled with a prosthetic group (CHO, Lipids or ein
Metals) *Alpha 1-
○ there’s an additional, such as metals etc. Antichymot
a. metalloproteins- proteins complexed with metals rypsin
○ ferritin, ceruloplasmin, hemoglobin, flavoproteins
b. lipoproteins- proteins complexed with lipids ● Ang gamma, sila yung naiiwan or yung malapit lang sa
○ HDL, LDL, VLDL, CM point of origin.
○ the alpha here is the leader in the marathon (yung ● Alpha 1, si HDL the followed by nung mga may Alpha 1
pinaka mabilis), which is yung pinaka lean/ pinaka sa pangalan and lastly AFP. Sikat na sikat si AFP kasi
protein → HDL part ito ng cancer marker.
○ yung dulo naman is yung pinakamataba → ● Alpha 2 are the haptoglobin, ceruloplasmin and alpha
Chylomicrons 2-macroglobulin.
c. glycoproteins- CHO (5-10% or <40%) attached to ● For Beta, transferrin, complement, LDL, beta
CHON residue 2-macroglobulin etc.
○ haptoglobin, ceruloplasmin, alpha1-antitrypsin,
transcortin, TBG
d. mucoproteins/proteoglycans/mucopolysaccharides/
mucin- has higher CHO (carbohydrate) than CHON;
>40% CHO
○ connective tissue
e. nucleoproteins- chromatin (combine with nucleic acid)

Negative Albumin
Acute Phase Transthyretin (Prealbumin)
Reactants Transferrin
Alpha-1 Antitrypsin ● If you take a look in the schematic pattern, ang levels na
Haptoglobin mataas normally ay si albumin. There’s always the
albumin spike, but all the other components (Alpha 1,
Ceruloplasmin
Acute Phase Alpha 2, Beta and Gamma) have relatively mababa lang
Fibrinogen yung concentration.
Reactants
Complement PLASMA PROTEINS
C-Reactive Protein Prealbumin (Transthyretin)
Alpha-1 Acid Glycoprotein ● transthyretin & retinol-binding protein are transport
proteins that migrate together
● Mas i-emphasize daw ni sir itong negative acute phase ● acts as a transport mechanism for thyroid hormones
reactants and acute phase reactants than the structures. ● migrates ahead of albumin on high resolution
● Mas marami ang acute phase reactants. electrophoresis
● ACUTE PHASE REACTANTS- these are the proteins ● has half-life of only 2 days
that are elevated in inflammation; kapag may ● if decreased: indicates poor nutritional status
inflammatory processes, mataas ito. ● used as a marker for CSF
● NEGATIVE ACUTE PHASE REACTANTS- kapag may ● If increased: alcoholism,chronic renal failure,steroid
inflammatory processes, mababa ito. treatment,NSAID therapy,Hodgkin disease
● Ano yung test sa hema na non-specific indicator for ● REFERENCE VALUE: 18-45 mg/dL (0.1-0.4 g/L)
inflammation? ANSWER: ESR Albumin
● Kaya tumataas ang ESR dahil sa presence ng acute ● small globular protein that is present in highest
phase reactants. concentration in plasma
● So ano ang acute phase reactants ang typical na ● major protein component of most extravascular body fluid
tumataas kaya elevated ang ESR? Ano ang notorious ● maintains colloidal osmotic pressure
kapag ang ESR ay elevated? ANSWER: FIBRINOGEN ● serves as indicator of nutritional status
● Fibrinogen is actually the determinant of ESR. ● serves as reservoir of amino acids
● So how do we group the proteins according to ● major transport protein carrier for free fatty
electrophoresis? So same with the others, may mabibilis acids,phospholipids,metallic ions,drugs, hormones &
and mababagal. (Alpha 1, Alpha 2, Beta and Gamma). bilirubin
● INCREASED: dehydration (Hemoconcentration)
● DECREASED: Acute inflammation,Malnutrition,Liver
disease(decreased synthesis),Renal diseases (increased
urinary loss; ex. nephrotic syndrome, GI disorders (peptic
ulcer or colitis)
● REFERENCE VALUE: 3.5-5.0 g/dL (35-55 g/L)

33
 NOTE Haptoglobin
● albumin is known to maintain osmotic pressure it used ● binds free hemoglobin
to pull the osmotic pressure from cell para lumabas ● it prevents loss of hemoglobin in the urine
● natural bacteriostatic agent for iron-requiring bacteria
yung water kasi pag excess water mag-mamanas
such as E.coli
● so if walang albumin walang mag-mamaintain ng ● evaluates the degree of intravascular hemolysis
osmotic pressure example sa liver poor synthesis of ● INCREASED: corticosteroid hormones & many NSAIDS
albumin, then sa renal failure inihi nang inihi or ● DECREASED: hemolysis,estrogens
inilabas ng inilabas si albumin so mas lalong walang ● REFERENCE VALUE: 26-185 mg/dL
mag mamaintain kaya prone sa pag mamanas Ceruloplasmin
● copper-containing protein which also has peroxidase
Alpha-1 antitrypsin (AAT) activity
● marker for Wilson’s disease
● Is a serpin (serine protease inhibitor); it neutralizes ● INCREASED: inflammation,cancer,pregnancy
trypsin-like enzymes ● DECREASED: Wilson’s disease,malnutrition,
● other serpins are alpha-1 antichymotrypsin,alpha-2 malabsorption, Menke’s kinky hair syndrome
antiplasmin, antithrombin III, C1 inhibitor ● REFERENCE VALUE: 150-240 mg/dL
● acute phase reactant
● it neutralizes the enzyme neutrophil elastase released by Alpha-2 macroglobulin (AMG)
WBC’s ● major component of the alpha-2 band in the SPE
● present in highest concentration of alpha-1 plasma ● inhibits proteases such as trypsin,pepsin & plasmin
proteins ● forms a complex with prostate-specific antigen (PSA)
● INCREASED: inflammation, pregnancy, Oral ● INCREASED: nephrotic syndrome,diabetes & liver
Contraceptive use disease,estrogen, children
● DECREASED: Emphysema, Juvenile hepatic ● DECREASED: severe acute pancreatitis,advanced
cirrhosis/AAT deficiency prostate carcinoma
● REFERENCE VALUE: 145-270 mg/dL ● REFERENCE VALUE : 150-420 mg/dL
Hemopexin
Alpha-1 Fetoprotein (AFP) ● it binds the heme released by degradation of
● During our fetal development we have AFP, which hemoglobin
disappears as we grow old. ● helps in early dx of hemolysis
● kapag meron tayo neto tas matanda na tayo it is ● INCREASED: inflammation,DM,DMD & some
associated with liver cancer. malignancies
● one of the first a-2 globulin to appear in mammalian sera ● DECREASED: hemolytic anemia
during development of the embryo & is the dominant ● REFERENCE VALUE: 50-115 mg/dL
serum protein in early embryonic life Transferrin (Siderophilin)
● major protein in fetal serum,synthesized primarily by the
● Transports iron
yolk sac & liver
● AFP reappears in the adult serum during certain ● Saturated with iron
pathological states ○ If there's no saturation, there will be abundance.
● migrates between albumin & AAT on electrophoresis of That's why it is increased in IDA = hindi siya
fetal or newborn serum saturated
● INCREASED: Spina bifida/Neural tube defects, multiple ● Used for differential diagnosis of anemias
fetuses,fetal demise,feto-maternal blood, incorrect ● Increased:
estimation of fetal age, Hepatocellular carcinoma
○ Iron Deficiency Anemia (IDA)
● DECREASED: Fetal trisomy 18; Trisomy 21
Alpha-1 acid glycoprotein/orosomucoid (AAG) ● Decreased:
● implicated in the formation of cell membrane ○ Inflammation
● negative charge in acid solutions ○ Liver disease
● binds & inactivates basic & lipophilic hormones,including ○ Malnutrition
progesterone & progesterone antagonist RU 486 ● Reference Value:
(abortion drug); binds and reduces bioavailability of many
○ Male: 215 - 365 mg/d
drugs
● INCREASED: inflammatory disease,malignant ○ Female : 250 - 380 mg/dL
neoplasms
● DECREASED: estrogen (from pregnancy & OCP), Fibrinogen
nephrotic syndromes ● Most abundant coagulation factor
● REFERENCE VALUE: 55-140 mg/dL ● Acute phase reactant
Alpha-1 anti-chymotrypsin ○ Markedly increased in inflammatory process
● migrate between a1&a2 one ● High levels in plasma
● serine protease inhibitor-inhibits cathepsin G,pancreatic
○ May cause elevated ESR
elastase, mast cell chymase & chymotrypsin
● binds & inactivates PSA ■ Coats the cells and allows them to sediment in
● associated with the pathogenesis of Aleimer’s disease - it faster clumps
is a vital component of the amyloid deposits found in ● Increased:
persons with this disorder ○ Inflammatory disorders
● INCREASED: infection,malignancy,burns AMI & ○ Pregnancy
Alzheimer’s disease ○ OCP
● DECREASED: liver disease
● Decreased:
● REFERENCE VALUE: 30-60 mg/dL
○ Coagulation

34
 ○ DIC
○ Liver disease Troponins (Troponin T, Troponin I, Troponin C)
■ Too much transfusion of FFP, plasma products ● Complex of three proteins that bind to the thin filaments
● Reference Value: of cardiac muscles
○ 200 - 400 mg/dL ● Regulators of actin & myosin
Complement ● Troponin T, Troponin I & Troponin C are found in cardiac
● Membrane Attack Complex (MAC) & skeletal muscles
● Acts as an opsonin ● Troponin T & Troponin I are undetectable in many
○ Facilitating phagocytosis and cytolysis healthy individuals
○ Acute phase reactant produced by the liver ● They serve as the most marker for AMI
● Increased: ● Their levels may elevate after AMI attack in the absence
○ Inflammation of CK-MB elevations
● Decreased: ○ Troponin T
○ DIC ■ valuable for AMI diagnosis
○ Hemolysis ■ Useful for the assessment of early & late AMI;
○ Malnutrition also in renal & muscle diseases
C - Reactive Protein (CRP) ■ Sensitive marker for unstable angina
● Non - specific indicator for inflammation ■ Useful in monitoring effectiveness of
● Not a driving force for ESR, it is Fibrinogen thrombolytic
● Used as a separate test for inflammation (a) rises in 3-4 hours after MI
○ More practical test for inflammation (b) peak level is at 10-24 hours
■ Because ESR requires larger volume of blood (c) returns to normal in 7 days (but may
● It's not a problem if its an adult, the remain elevated for 10-14 days)
problem comes when the patient is a child ■ ≥ 1.5 ng/mL is suggestive of AMI
and hard to extract blood from ○ Troponin I
■ Only microliters of sample needed, so CRP is ■ Only found in the myocardium
much practical ■ Highly specific for AMI
○ Sensitive indicator ■ 13x more abundant in the myocardium than
● Cardiac marker CK-MB on a weight basis
○ Used as an early warning test for people at risk with ■ Very sensitive indicator of even minor amount
coronary artery disease of cardiac necrosis
○ No one uses CRP to investigate AMI ■ In AMI
● Opsonin (a) levels begin to rise in 3-6 hours,
● Inflammatory marker that appeared to reflect the severity (b) peak in 12-18 hours,
of CHD and may contribute to its pathogenesis (c) returns to normal in 5-10 days
● Used as a rapid test for presumptive diagnosis between (d) Faster than CK-MB (levels begin to rise
bacterial infection VS. infection at 4-8 hours
● CRP test is very helpful during COVID times to indicate ● Mabilis maa-assay
inflammation
Lipoproteins B - Natriuretic Peptide (BNP)
Immunoglobulins ● Marker for congestive heart failure
● Increases in response to peptide ventricular systolic &
MISCELLANEOUS PROTEINS diastolic & is diagnostic of congestive heart failure
MYOGLOBIN
● Found in skeletal and cardiac muscles Cystatin - C
● Transports and stores oxygen from Hemoglobin to ● For kidney function test
intracellular respiratory enzymes of contractile cells ● Low molecular weight & a cysteine proteinase inhibitor
○ Protein that binds oxygen for muscles ● Freely filtered in the glomerulus & completely reabsorbed
● Higher affinity for oxygen than does Hemoglobin & catabolized by the PCT
● Has small MW (18 kDa) thus leaks from damaged cells ● Used as an endogenous renal marker
more easily ● Increased: Renal Disease
○ If skeletal or cardiac muscle cells has damage, it
easily leaks Fibronectin
● Potential nephrotoxin ● Glycoprotein used to help predict the short-term risk of
● One of the early protein markers for MI premature delivery
○ Onset 1 - 3 hours ● Produced at the boundary between the amniotic sac &
○ Peak 5 - 12 hours the decidua (the lining of the uterus) and functions to
○ Normalizes in 18 - 30 hours maintain the adherence of the placenta to the uterus
○ Marker for Angina ● Cystatin-C and Fibronectin are good to know proteins, na
● Assay for Myoglobin is difficult and not readily available hindi typically pinapa-assay sa laboratory
in laboratories

35
 TOTAL PROTEIN ABNORMALITIES Acid precipitation (TCA or tungstic acid) of protein with
HYPONATREMIA measurement of total nitrogen
● Excessive loss by excretion in the urine in renal disease ● Kjeldahlization- conversion of nitrogen to ammonia
● Ammonia measurement
● Leakage into the gastrointestinal tract in inflammation of
○ Nessler's reaction (double iodide of Hg and K) (pic)
the digestive system ○ Berthelot reaction (pic)
● Loss of blood in open wounds, internal bleeding, or
extensive burns 2. Biuret Method
● Decreased intake either because of malnutrition or ● Most widely used method; recommended method
through intestinal malabsorption as seen in sprue ● Depends upon the presence of 2 or more peptide
● Decreased synthesis: linkages
i) liver disease ● Principle: Cupric ions complex the groups involved in the
ii) immunodeficiency states peptide bond forming a violet-colored chelate which is
proportional to the number of peptide bonds & reflects
● Mas madalas na mas mababa ang protein levels,
the TP levels at 545 nm
mababa ang protein levels pwedeng dahil renal, liver ● False elevations: icterus, lipemia, hemolysis
disease, maraming excretion like blood loss,etc ● Cannot be used to directly measure urine protein
HYPERNATREMIA ● Reagents: alkaline copper sulfate, Rochelle salt (NaK
● Dehydration tartrate), NaOH & KI
● Excessive production of gamma-globulins ● COMPOSITION:
○ Cupric ions- breaks the peptide bonds
○ Tartrate salt- keeps copper in solution
○ Potassium iodide- stabilizes cupric ions
● POSITIVE RESULT; Violet color

3. Folin- Ciocaulteau
● Highest analytical sensitivity
● Principle: oxidation of phenolic compounds such as
tyrosine, tryptophan and histidine to give a deep blue
color
● Color is not proportional to concentration
● Albumin should be the only one which is elevated here. ● Used to measure small amounts of proteins (enzymes,
Ag/Ab reaction)
METHODS ● Reagent: phenol reagent or phototungstic-molybdic acid
Specimen precautions: ● Color enhancer: Biuret reagent
● Serum is preferred over plasma
● Fasting or non-fasting specimen 4. Ultraviolet Absorption Method
○ Depends on the protein to be analyzed ● Principle: the absorbance of proteins at 210 nm is due to
○ If total proteins,globulin,and albumin,it requires the absorbance of peptide bonds at specific wavelengths.
fasting. Proteins absorb light at 280 nm and at 210 nm.
○ Except for these three, all are non-fasting ● Absorption at 280 nm is due to tryptophan, tyrosine,and
specimens. phenylalanine
● Hemolysis may falsely elevate total protein
5. Serum Protein Electrophoresis
● Principle: migration of charged particles in an electrical
TOTAL PROTEIN
field
● Used primarily to assess the nutritional status, functional ● The single most important clinical application is for the
capacity of the liver, kidneys and the bone marrow identification of monoclonal spike of immunoglobulin and
● Reference values: 6.5-8.3 g/dL differentiating them from polyclonal
hypergammaglobulinemia
METHOD PRINCIPLE ● Demonstrates monoclonal gammopathies
● Reference method.
Kjeldahl ● Not done routinely
● Assume average nitrogen content of 16%
● Measurement of refractive index due to
● Albumin (fastest), alpha-1 (2nd), alpha-2 (3rd), beta (4th),
Refractometry & gamma (5th)
solutes in serum
● Formation of violet-colored chelate ● Reference values for each fractions:
Biuret ○ Albumin- 53-65% (3.5-5.0 g/dL)
between Cu2+ ions and peptide bonds
● Protein binds to dye and causes a spectral ○ Alpha-1 - 2.5-5% (0.1-0.3 g/dL)
Dye binding
shift in the absorbance maximum of dye ○ Alpha-2 - 7-13% (0.6-1.0 g/dL)
○ Beta- 8-14% (0.7-1.1 g/dL)
1. Kjeldahl Method ○ Gamma- 12-22% (0.8-1.6 g/dL)
● Classic/reference method ● Abnormal patterns
● Principle: oxidation with heat & strong acid (tungstic acid) ○ Gamma spike- multiple myeloma
to form protein-free filtrate ○ Beta-Gamma bridge- hepatic cirrhosis ("tau")
● Based on the measurement of nitrogen content of protein ○ Alpha-2 spike- nephrotic syndrome
● N is converted to ammonia ○ Alpha-1 flat curve- juvenile cirrhosis (AAT
● Reagent; sulfuric acid (digesting agent) deficiency)
● According to this method : 1 g N= 6.54 g proteins ○ Spikes at Alpha-1 , Alpha-2 & Beta regions-
inflammation

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● Dyes used: used to make colors visible ● It’s used for urine (specific gravity) but not used for
○ Bromphenol blue (for paper medium) proteins
○ Ponceau S (gel and acetate) 7. Turbidimetry/ Nephelometric Methods
○ Amino Black (gel and acetate) ● Uses SSA or Trichloroacetic Acid
○ Coomassie Brilliant Blue ● SSA: prone to contamination
● Measurement depends on the formation of a uniform fine
ELECTROPHORESIS precipitate which scatters incident light in suspension
● proteins separated based on electric charge densities (nephelometry) or block light (turbidimetry)
● Cellulose acetate/agarose gel (support media) ● Confirmation of presence of protein in urine:
○ Urine strip will have a color change (green if positive
sa protein)
○ When you drop salicylic acid sa urine na may
protein, magiging turbid yun
● Hindi na dapat ginagawa ito now, since our urine strips
are now very sensitive
● Recommended: Urine strips
8. Salt Retraction
● Globulins can be separated from albumin by salting-out
procedures using sodium salts
● Reagents: Sodium Sulfate Salt

ALBUMIN
● Concentration is inversely proportional to the severity of
liver disease
● Plasma levels decline when severe hepatocellular
disease lasts for more than 3 weeks
● Decreased serum albumin concentration may be due to
decreased synthesis
● Decreased in kidney diseases
● Results to edema since wala nang nag memaintain ng
pressure
After separation, protein fractions are immersed in acid ● Requires Fasting:6-8 hours
solution then stained by dyes.
METHODS
SALT PRECIPITATION
● Globulins are precipitated, albumin in supernatant is
quantitated by biuret reaction

DYE BINDING TECHNIQUE

● Uses only dyes or indicators that bind very tightly to
albumin
● Ideally 100% of dye will be bound to albumin none to
other fractions
● Dissociation-association technique influenced by
temperature
● Serum is the sample of choice
● Heparin yields falsely high results because it enhances
dye binding to albumin
The medium is placed in scanning densitometer which ● Binding should not be affected by small changes in ionic
compute the area under the absorbance strength & pH
○ Bromocresol Green (BCG)
■ most commonly used method
■ sensitive; overestimates low albumin levels
■ used extensively in automatic analyzers in parallel
with Biuret reagent for TP
○ Methyl Orange
■ non-specific for albumin
○ Hydroxyazobenzene Benzoic Acid (HABA)
■ many interferences (salicylates, bilirubin)
○ Bromocresol Purple (BCP)
■ most sensitive & specific method
■ accurate, gives overview of relative changes in
● Dapat may umbok-umbok kaso nag-isa nalang sila so different protein fractions
nawala ang beta-gamma, nagkaroon ng TAU bridge ■ measured through spectrophotometry
6. Refractometry
● Based on the refractive index of solutes in serum —-----------------------------------END--------------------------------------
● Alternative to chemical analyses
● Measurement of Refractive index (velocity of light in air
and water) due to solutes in serum.

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