CLINICAL CHEMISTRY 2
FUNCTIONS OF THE LIVER
• Receives, processes and stores materials
absorbed from the digestive tract (e.g. amino
acids, CHO, fatty acids, cholesterol)
• Synthesis: multiple plasma proteins
o Contribute to body’s immune system
• Main organ of detoxification
• Metabolic conversion of endogenous and
exogenous compounds
• Synthesis of bile acids from cholesterol
• Major site of catabolism of hormones
o Participates in regulation of plasma
hormone levels
o Responds to hormonal and neural
stimuli to regulate blood glucose
concentration
ANATOMY OF THE LIVER
• Largest organ (~1.2 – 1.5 kg in adult)
• Located in the upper right quadrant of the
abdomen
• Dual Blood Supply:
o Portal Vein: carries blood from
alimentary tract
o Hepatic Artery: carries oxygenated
blood to the liver
MICROSCOPIC ANATOMY
• Composed of lobules demarcated by CT sepat
and vascular and biliary vessels
• Central Hepatic vein: found in the center of the
lobule
• Parenchymal epithelial cells radiate from the vein
• Sinusoids: blood-carrying vascular channels on
either side of the liver cell plates
• Lining cells: either endothelial or Kupffer cells
(largest group of fixed macrophages)
• Space of Disse: tissue space between the
edothelila cells and hepatocytes
• Filled with interstitial fluid
• Transfer of nutrients and waste products from
blood occurs
• Hepatocytes: polygonal cells that constitute ~ • Rough ER
60% of the liver mass o Lamellar profile with
ribosomes
o Synthesis of specific proteins
SPECIALIZED SURFACE OF THE CELL (e.g. albumin, coagulation
• Sinusoidal surface factors)
• Intercellular surface
• Canalicular surface 4. GOLGI COMPLEX
• Produces lipoproteins (VLDL)
ASSESSMENET OF LIVER FUNCTION • Glycoprotein synthesis
Indications: • Albumin secretion (“packaging
1. Screening for abnormalities in liver function process”)
2. Documenting an abnormality
3. Identifying the type and site of injury 5. MICROTUBULES AND MICROFILAMENTS
4. Prognostication and follow-up of patients with • Maintain cell shape and provide
hepatic disease contractile force
Factors to consider: CLINICAL MANIFESTATIONS OF LVIER DISEASE
1. Diagnostic SENSITIVITY in screening for hepatic 1. JAUNDICE – “ICTERUS”
dysfunction • Yellow discoloration of the plasma,
2. SPECIFICITY for liver disease skin, sclerae and mucous membranes
3. SELECTIVITY in differentiating these disorders caused by bilirubin accumulation
• Not specific symptom
ULTRASTRUCTURE • May be the 1st and often the only
- Hepatic parenchymal cells have a well-developed manifestation of liver disease
organellar substructure: • Clinically apparent when serum Total
Bilirubin reaches 2-3 mg/dL
1. MITOCHONDRIA • Other signs of disturbances in bilirubin
• Energy generation (oxidative metabolism: dark urine, acholic stool
phosphorylation and fatty acid 2. PORTAL HYPERTENSION (>20 mmHG)
oxidation) • Portal vein: influx from the abdominal
portion of the digestive tract, pancreas
2. LYSOSOMES and spleen (Pressure: ~5-10 mmHg)
• Degradative functions (hydrolytic • Increased: sinusoidal infiltration,
enzymes) scarring or hepatic vein obstruction
• Major complications:
3. ENDOPLASMIC RETICULUM Hepatosplenomegaly, Ascites and
• Site of many functions: drug Hepatorenal syndrome
metabolism and bile acid synthesis • Ascites – accumulation of fluid within
• Smooth ER the peritoneal cavity
o Tubules and vesicles 3. HEPATIC FAILURE AND ENCEPHALOPATHY
o Bilirubin conjunction, drug • Metabolic abnormalities occur in the
detoxification and cholesterol presence of chronic liver disease
synthesis
 • Accumulation of substance (e.g. NH3)
that are normally metabolized by the
liver
• Cerebrotoxic effect: Neuropsychiatric
disturbances
• Major finding:
o Hyperammonemia
o Accumulation of NH3,
glutamine and alpha-
ketoglutarate in brain and
CSF
o Increase in short- and
medium-chain fatty acids and
amino acids in plasma
o Mercaptans: account fir the
characteristic odor “fetor
hepaticus”
4. ALTERED DRUG METABOLISM
• In hepatic dysfunction: drugs undergo
limited or aberrant transformation,
accumulate and exert unwanted effects
5. ENDOCRINE ABNORMALITIES
• Hormone imbalance
• E.g. Among men, “Feminization” (i.e.
gynecomastia and testicular atrophy)
due to increased estrogen effect
6. NUTRITIONAL AND METABOLIC ABNORMALITIES
• Example: alternations in glucose
metabolism due to insulin resistance
7. IMMUNOGLOBULIN ABNORMALITIES
• The liver efficiently sequesters dimeric
IgA and secrets it into the bile
• Liver disease: defects in uptake
increases serum IgA
8. DISORDERED HEMOSTATIS
• Alterations in hemostasis and a
generalized hemorrhagic tendency
• Due to: decreased synthesis of clotting
factors
: production of qualitatively
abnormal proteins
• Altered platelet count METHODS
• Presence of endogenous • American Association for Clinical Chemistry
anticoagulants (AACC) and National Bureau of Standards (NBS)
• Aberrant fibrinolytic systems • Published a candidate reference method for Total
• Intravascular coagulation Bilirubin: Modified Jendrassik-Grof
BIOCHEMICAL ASSESSMENT OF LIVER FUNCTION • Jendrassik-Grof & Evelyn-Malloy procedures:
o Both have acceptable precision
HEPATIC EXCRETORY FUNCTION
o Adapted by many automated
• Organic anions of both endogenous and instruments
exogenous origin are removed from the o Most frequently used methods to
sinusoidal blood and excreted into the bile measure Bilirubin
BILIRUBIN METABOLISM
Daily bilirubin production ADVANTAGES OF J-G OVER E-M
•
• (average 250 – 300/350*Henry’s mg) Not affected by pH changes
• 85%: derived from heme of the hemoglobin • Insensitive to a 50-fold variation in protein
concentration of the sample
• 15%: erythrocyte precursors destroyed in the
• Maintains optical sensitivity even at low bilirubin
BM; catabolism of other heme-containing
concentrations
proteins (Myoglobin, Cytochromes and
• Has minimal turbidity & a relatively constant
Peroxidases)
serum blank
FORMS OF BILIRUBIN
• Not affected by Hgb up to 750 mg/dL
• Unconjugated: bound by albumin and
transported to the liver; water insoluble MALLOY – EVELYN METHOD
• Conjugated: formed from the reaction of UCB
with glucuronic acid; aka Bilirubin diglucuronide
• Delta: conjugated bilirubin bound to albumin
LIVER FUNCTION TESTS
History – Analysis of Bilirubin
• (1883) Ehrlich: introduced the Diazo reaction for
the detection of Bilirubin using urine; reaction of
bilirubin with a diazotized sulfanilic acid
• (1913) Van den Bergh and Snapper: applied the
Diazo reaction in serum in the presence of an
accelerator (solubilizer)
• (1916) Van den Bergh and Muller: discovered the JENDRASSIK – GROF METHOD
polar (direct reading) and non-polar (indirect
reading) types of bilirubin in serum
• (1937) Evelyn and Malloy: 50% CH3OH as the non-
polar solvent; first useful quantitative method for
bilirubin
• (1938) Jendrassik and Grof: used the Diazo
reaction with caffeine-benzoate- acetate as an
accelerator Ascorbic acid – terminates the DIAZO reaction
REFERENCE RANGES - Reduced conjugating - Liver can conjugate bilirubin.
Adults CB 0.0 – 0.2 mg/dL capability of the liver Transporter protein transport
UCB 0.2 – 0.8 mg/dL - Most common cause of CB to the bile ducts
TB 0.2 – 1.0 mg/dL hepatic jaundice but does not - If there is no transporter
carry morbidity or mortality protein, the CB conjugated by
- From 100% conjugation liver cells will go back to the
Premature Infants TB at 24 hrs 1 – 6 mg/dL
capability, 30% chance our circulation
TB at 48 hrs 6 – 8 mg/dL
liver will conjugate - Defective removal of
TB at 3 – 5 days 10 – 12 mg/dL - Total serum bilirubin conjugated bilirubin from the
fluctuates between 20 – 50 liver cell & the excretion into
Full-term Infants TB at 24 hrs 2 – 6 mg/dL um/L the bile
TB at 48 hrs 6 – 7 mg/dL - No medication - Obstructive in nature; much
TB at 3 – 5 days 4 – 6 mg/dL of the CB circulates bound to
b) Crigler- Najjar Syndrome (1952) albumin (delta bilirubin: non
CLINICAL SIGNIFICANCE - Chronic unconjugated toxic)
1. PRE-HEPATIC JAUNDICE hyperbilirubinemia - Appearance of dark-stained
• Problem causing the jaundice occurs - Molecular defect w/in the granules on a liver biopsy
prior to liver metabolism gene involved with bilirubin yielding to
• Caused by an increased amount of conjugation hyperbilirubinemia and
bilirubin being presented to the liver - Rare & more serious disorder bilirubinuria
(e.g. Acute & Chronic Hemolytic - Type I – complete absence of - Delta bilirubin may interfere
Anemia: Increase concentration of RBC enzymatic bilirubin because it acts like C/D
production thus there will be a conjugation and considered bilirubin causing false
subsequent release amount of bilirubin to be inherited autosomal increase
from hemolyzed RBCs then liver will recessive disorder. Most of
respond by functioning in its maximum) the patients die within the b) Rotor Syndrome
first year of life. - Clinically same with dubin
• Unconjugated hyperbilirubinemia
* kenicterus: but:
*Pre Hepatic and Post Hepatic Jaundice
deposition of bilirubin • Relatively less
into the brain common
- Abnormality is outside the liver (either before or
after the processing of bilirubin)
*early liver • No dark stained
transplantation granules
- Liver cells are NORMAL: working at its maximum
- Type II – mutation causing a - Hypothesized to be due to a
because it wants to compensate for the
severe deficiency of the enzyme for reduction in the
abnormality elsewhere
conjugation concentration or activity of
2. HEPATIC JAUNDICE – Intrinsic (within) liver intracellular binding proteins
B. Transport Defects: Conjugated (e.g. Ligandin)
disease
Hyperbilirubinemia - Liver biopsy does NOT show
Causes:
a) Dubin – Johnson Syndrome dark pigmented granules
A. Disorders of Bilirubin Metabolism
- Carrier; autosomal recessive
a) Gilbert’s Syndrome
disorder which is caused by 3. PHYSIOLOGIC JAUNDICE of the NEWBORN
- Intermittent
hyperbilirubinemia
deficiency of the canalicular • Deficiency in the enzyme Glucuronyl
transporter protein (MDR2: transferase (one of the last liver
- Reduction in the expression
multidrug resistance functions to be activated in pre-natal
of UGT1A1 gene (isoform of
2/cMOAT: multispecific life)
most important enzyme in
bilirubin conjugation: uridine
organic anionin transporter • Unconjugated hyperbilirubinemia
protein)
diphosphoglucose
glucoronyltransferase)
 • Kernicterus – deposition of bilirubin in
the nuclei of brain & nerve cells; cell
damage & death of newborn; 3 weeks
✓ RBCs are shorten and also has
intravascular hemolysis
✓ Lack of glucoronyltransferase at
birth
✓ Breastfeeding is not advisable
because there are inhibitors of
conjugation present in the
breastmilk acting as an antibody
preventing conjugation
✓ Babies are treated with UV light at
450nm destroying bilirubin
present at the capillaries making it
water soluble to be easily pass out
urine
4. POST-HEPATIC JAUNDICE
• Usually results from biliary obstructive
disease (e.g. gallstones, tumors)
• Prevents the flow of conjugated
bilirubin into the bile canaliculi
• Clay-colored stool; the stool will lose
it’s natural source of pigmentation
(stercobilinogen)
• Abnormality is outside the liver (either
before or after the processing of
bilirubin)
CLINICAL ENZYMOLOGY
- A field of laboratory medicine which focuses on
the study of enzymes and their significance to the
diagnosis and treatment of disease.
ENZYMES- substances that catalyzes a given chemical
reaction; serological binding catalysts (substances that
speed up the metabolic reactions in our body)
Characteristics
1. Complicated type of protein
- They have specific amino acid sequence
2. Easily denatured with varying MW
- Their integrity, the way they function
depends on the structure
3. Amphoteric
- Capable of ionizing either acids or
bases
4. Reaction catalyzed are frequently reversible
- They can both synthesize and
decompose at the same time; the
operation done or reaction catalyzes by
this enzyme
5. Operates with assistance of a cofactor
- The operation done or reaction
catalyzes by this enzyme is usually
assisted by renal protein cofactor
- Cofactor can either be organic or
inorganic molecule (activator)
6. Synthesized in an inactive state
- We call them zymogen/proenzyme
- Ex: Digestive enzymes are produced in
different organs and sometimes pass
thru intestine wherein they will confirm
or change their molecular structure to
be in activated form because digestive
enzyme when active after synthesizing
in organ of synthesis, the problem is
they can dissolve materials where they
are produced and we do not want that
to happen
*Changes in enzyme concentration in tissue cells reflects
changes in states of health and disease of the tissue:
enzyme are present in all tissues of the body meaning all
cells of the body contain particular enzymes
Ex: There is an increase in the enzyme concentration, there
is a problem. When we do enzyme concentration analysis,
we do not want a hemolyzed RBC because there are
enzymes present inside of the cells
DEFINITION OF TERMS
1. Coenzyme: non protein organic biochemical;
essential to the catalytic activity as a co-substrate
- Nicotinamide adenine dinucleotide
(NADH): common electron acceptor
present in redox reaction and help
enzymes to fulfill their actions and
catalytic activity
- Pyridoxal phosphate: this coenzyme are
essential to catalytic activity as it
compensates
2. Activators: inorganic ionic cofactors (usually
cations); usually metals; form ionic bridges to
orient the substrate to the active site of an
enzyme at the correct configuration
- Ex: Mg, Na, K, Zn (they are electrolytes)
help enzyme increase the catalytic
activity
3. Holoenzyme: refers to the combined enzyme and
coenzyme
4. Apoenzyme: enzyme without a cofactor
5. Prosthetic group: a coenzyme that cannot be
removed from its attachment with an enzyme
- Ex: Pyridoxal phosphate in
transaminase reaction/transamination:
usually incorporated in the synthesis of
amino acids
Difference of Holoenzyme and Apoenzyme
Importance of Activator
- Inorganic molecule
- It make the binding of substrate to enzyme
stronger
6. Metalloenzyme: inorganic activators existing as a
part of the enzyme molecule; most often metal
ions
7. Substrate: substances which are acted upon by
an enzyme and is converted into a new substance
(product)
8. Product: substance derived from a transformed
substrate
9. Active Site: the area or portion of an enzyme with
which the substrate is attached to the enzyme
molecule
- Only contains 5% of total amino acid in
entire enzyme
*Allosteric site: allows molecules to either activate or
inhibit/turn off catalytic activity of enzyme; serves as on
and off switch for enzyme activity

Protein Stucture
a) Primary structure
- Sequence of AA joined by peptide
bonds: Peptide bonds to form
polypeptide chains
b) Secondary structure
- Conformation of segments: Hydrogen
bond
c) Tertiary structure
- Maintenance of Covalent disulfide bond
- Beta polypeptide is important because
primary and secondary structures are
the most important configuration
d) Quaternary structure
- Separate, bended, folded structures
put together to form functional unit
- Ex: Lactate dehydrogenase; Creatine
Kinase
CLASSIFICATION OF ENZYMES
• Based on the catalytic activity of an enzyme
• The International Union of Biochemistry (IUB)
Enzyme Commission categorized all enzymes into
six (6) classes
1. Oxidoreductases
- Catalyze oxidation and reduction reactions
between 2 substrate
- There is a transfer of electrons in one
molecule to another molecule
- Assayed for the investigation of cardiac and
liver problems
- Ex: Glucose oxidase; Lactate dehydrogenase
2. Transferases
- Enzymes that move an intact group of atoms
(amine or phosphate group) from one
molecule to another
3. Hydrolases
- Enzymes involved in the splitting of
molecules with water as a part of the
reaction process
- Split of esters will yield to alcohol and acids
- Enzymes will only be functional if there is
H20 in inhibition
- 3 groups of hydrolases
a) Estarases
➢ Acid phosphatase (ACP)
➢ Alkaline phosphatase (ALP)
➢ Lipase
b) Peptidases
➢ Leucineaminopeptidase
➢ trypsin
c) Glycosidases
➢ Amylase
➢ Amylo-1,6-glucosidase
4. Lyases
- Enzymes responsible for splitting of
molecules or breaking bonds (bonds
between carbon atoms and oxygen
- Single substrate + lysase = 2 new subs
- Assayed in disorders of skeletal muscles
- Ex: Aldolase; Glutamate decarboxylase
5. Isomerases
- Enzymes responsible for the conversion of
one isomer to another
- All reactions are reversible
- Ex: cis and trans- all of the atoms in the
molecule will be arranged
- Ex: Glucose-PO4 Isomerase; Ribose-PO4
Isomerase
6. Ligases
- Enzymes that cause bond formation
between two molecules to form a larger
molecule
- Ex: Amino acyl t-RNA synthetase