CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT.

RENAL FUNCTION pt. 1 Parts of the Nephron Loop:

 Descending Loop of Henle
 Ascending Loop of Henle

Two Kinds of Nephron: (based on loc.)

 Cortical Nephron (cortex)
 Juxtamedullary Nephron
Major organs of the urinary system: (medulla)
 Kidneys
 Ureter RENAL FUNCTIONS
 Urinary bladder  Filters blood
 Urethra  Excretes wastes and excess water
and ions through the urine
MALE vs. FEMALE URINARY  Regulates:
SYSTEM o Blood pressure
 Male: urinary and o Blood volume
reproductive are joined o Blood pH
(urogenital system) o Salts
 Female: separate orifice for
urinary and reproductive
3 Major Processes:
 Tubular Secretion
KIDNEYS  Tubular Reabsorption
- Located at the retroperitoneal area  Glomerular Filtration
of the abdominal cavity
- Lower back of the body RENAL BLOOD FLOW
- Before the 3 major processes
- Rate of the flow of the blood from
systemic circulation that will pass
through glomeruli for filtration
- Affects the 3 major processes

GLOMERULAR FILTRATION
- Separation of solutes or dissolved
substances
NEPHRON - Can only filter those with lower
- Functional unit of the kidney molecular weights
- Process of selective permeability
Major Parts of the Nephron - Filtration of blood
- in order from afferent arterioles:
 Bowman’s/Glomerular capsule Glomerular Filtration Rate
 Proximal tubule - Volume of blood filtrated per minute
 Loop of Henle (Nephron loop)
 Distal tubule  Kidney filters out 1,200-1,500mL of
 Collecting duct blood each minute
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 1

 Filters out 125-130mL of Renal Function Tests

glomerular filtrate - Also known as Renal/Kidney Panel
- Under pud diri ang Lipid Profile/Panel
Renal threshold o Total Cholesterol
- Maximum capacity of the glomeruli o Triglycerides
to hold the filtered substances o High Density Lipoprotein
- May specific threshold for each o Low Density Lipoprotein
substances
 If concentration of substance in SUMMARY AND REVIEW
blood exceeds the threshold, the
excess substances cannot proceed to
the next step (tubular reabsorption)
therefore ma excrete siya sa urine
Dalton
- Unit of measurement for molecular
weight

TUBULAR REABSORPTION
- Reabsorption of essential Glomerular Filtration
substances for the body’s usage - Water and solutes smaller than
- Ibalik ang ultrafiltrate sa blood proteins are forced through the
circulation capillary walls and pores of the
- Not all mareabsorb glomerular capsule into the renal
 Depends on concentration of tubule
substance din, if too much na ang
nareabsorb, it will be excreted to Tubular Reabsorption
the urine
- Water, glucose, amino acids, and
Two transport mechanisms: needed ions are transported out of
 Active Transport the filtrate into the tubule cells and
- Requires ATP/energy enter the capillary blood
- Usually substances that are highly Tubular Secretion
regulated/not permeable
- Uses gated channels - H+, K+, creatinine and drugs are
removed from the peritubular cells
 Passive Transport
- Does not require ATP
- Area with higher concentration to SPECIFIC LOCATION SA PROCESSES
area with lower concentration
Ex. Diffusion, osmosis Renal Corpuscle
- Production of filtrate
TUBULAR SECRETION - Where filtration exclusively occurs
- Secretion of waste products from Proximal Convoluted Tubule
metabolism that are uneeded by
the body - Reabsorption of water, ions, and
- Not just waste, pati na din ang all organic nutrients
mga over na ang concentration sa Descending Loop of Henle
blood
- Regulated by hormones - Further reabsorption of water

Ascending Loop of Henle

Urine
- Reabsorption of sodium and
- Ultrafiltrate of the plasma
chloride ions
- Product of the 3 processes
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 1

o Renin is secreted & produced

Distal Convoluted Tubule
by juxtaglomerular cells of the
- Secretion of ions, acids, drugs, and kidneys
toxins
- Variable reabsorption of water,
sodium ions, and calcium ions
(under hormonal control)

Collecting Duct
- Variable reabsorption of water or
sodium
- Secretion of sodium, potassium,
hydrogen, and bicarbonate ions

Papillary Duct
- Delivery of urine to minor calyx

Urinary Bladder
- Where urine is temporarily stored

URINE REGULATORY HORMONES

1. Anti-diuretic Hormone (ADH)

- Increased reabsorption of water
- By hypothalamus but stored in
posterior pituitary gland

2. Aldosterone
- Increase sodium reabsorption
- Increase blood pressure and volume
since nag retain and sodium ug
water
- Produced by the adrenal cortex

3. Atrial Natriuretic
Hormone/Peptide (ANP)
- Produced specifically in the atrium
- Opposes aldosterone
- Stimulates excretion of sodium in
urine
- Results to lowered blood pressure

4. Angiotensin II
- Increases sodium reabsorption
- Results to vasoconstriction
- Precursor is Angiotensin I
o Precursor nasad ni Angiotensin
I is si angiotensinogen
which is stimulated by renin
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 2

A. UREA CLEARANCE
RENAL FUNCTION pt. 2
- Present in highest
RENAL FUNCTION TESTS concentration in the blood
among the NPN (non-protein
nitrogen)
GLOMERULAR FILTRATION RATES - Major excretory product of
- Standard test to measure the protein metabolism
filtering capacity of the glomeruli - Formed in the liver from
- Measures the rate at which the amino groups and free
kidneys are able to remove a ammonia generated during
filterable substance from the blood protein catabolism
- Is the rate in milliliters per minute
at which substances in plasma are Urea Cycle
filtered through the glomerulus - Protein catabolism
- Synonymous to the term
clearance
o The ability to remove
substances from blood

How fast does kidneys filter blood?

 90-120 mL/min

 Glomeruli has 3 filtration barriers

GFR Biomarkers

- Substances or indicators that are

used to assess GFR Final product of protein
catabolism:
Criteria for biomarkers:  Carbon dioxide
1. Should appear endogenously in the  Ammonia
plasma at a constant rate
2. Should be freely filtered at the  Carbon dioxide and Ammonia will
glomerulus undergo further catabolism by
3. Can neither be reabsorbed or carbamoyl phosphate synthase into
secreted by the renal tubule carbamoyl phosphate and now
4. Should not undergo extra-renal enter the urea cycle
elimination  Forms into (sunod sunod na ang ff):
5. Some substances can be excreted  Citrulline
through sweat or GI tract  Arginosuccinate
 L-Arginine
 Mag-focus ra ta sa first two  L-ornithene
criterias
Substrates/Amino acids in the Kreb’s cycle:
 L-Aspartate
CLEARANCE TESTS  L-Fumarate
 Urea Clearance  Urea
 Creatinine Clearance  Utilized in the Urea Cycle
 Cystatin C Clearance
 Inulin Clearance
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 2

UREA CREATININE
- Freely filtered by the glomeruli - Freely filtered by the glomeruli
- Variably reabsorbed by the renal - Reabsorbed by the renal tubules
peritubular cells (minute amount)
o Dependent on urine flow - Secreted by PCT (only about 10%)
o High urine flow = less urea - Routinely used clearance test since it
o Low urine flow = more urea is more accurate
- Less than 10% are excreted through
GI tract and skin Specimens:
- Widely used however not plausible  24-hour urine
 Serum/Plasma (non-fasting)
B. CREATININE CLEARANCE
24-hour Urine Instructions
- Formed from creatine and 1. First-voided urine (Ex. 7AM) should
creatine phosphate in muscle be discarded since urine is formed
- Major excretory product of protein prior sa time.
metabolism 2. First-voided urine after the cut-off
time should be collected.
3. Urine container should be
disposable, dry, and wide-
mouthed.
4. Use smaller container first then
iipon sa bigger container (at least
1 liter).
5. Bigger container should be
transparent, refrigerated, and
labeled with:
o “Urine”
o Name
o Date and time of collection

- After creatine undergoes What to do with the urine?

degradation, it will be utilized in the  Iinvert invert para mamix before
interconversion or interformation of aliquoting
creatine and creatine phosphate
- Catalyzed by the enzyme creatine  Pag abot ni patient with his/her 24-
kinase hour urine, kuhaan siyag dugo
o Reversible siya so meaning,
pwede from creatine to CP or GFR formula:
CP to creatine through the
process of phosphorylation

Phosphorylation
- One phosphate from the ATP is
transferred to creatine to create
creatinine phosphate or…
- One phosphate from creatine
phosphate transfers to ADP to create
ATP and creatine GFR
Us = concentration of substance in urine
 More creatinine formed from Vu = volume of urine within the 24-hour
creatine phosphate than creatine PSt = concentration of substance in plasma
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 2

Some EGFR (Estimated Glomerular

Filtration Rate):
 Cockcroft-Gault
 Jelliffe
 Normalized Jelliffe
 Wright
 Corcoran-Salazar

 Consider age, sex, and weight

CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

CYSTATIN C CLEARANCE Elimination:

- Freely filtered by glomeruli
Properties:
- Reabsorbed completely by the PCT
- Mol. Weight approx. 13,000
Daltons
- Inhibitor of Cysteine protease *can assess GFR but it is not accurate;
- Produced by all nucleated cells at same with cystatin C
constant rate
RENAL FUNCTION TESTS
Elimination: - Group of tests that assess renal
function in general
- Freely filtered by glomeruli
HOWEVER, it is completely
Biomarkers:
reabsorbed in proximal convoluted
tubule (PCT)
- Normally not seen in the urine 1. BIOMARKER: UREA
o Main waste product of
*not commonly used as a clearance test
nitrogen-containing chemical
in the body
 Major excretory
INULIN CLEARANCE product of CHON
metabolism
- Inert (chemically inactive) o Synthesized in the liver from
polysaccharide CO2 and ammonia from the
- A polymer of fructose deamination of amino acids
- Inulin is an exogenous source o Widely used as a measure of
- Our body cannot synthesize inulin renal dysfunction but its value
- Cannot be used in our body as a measure of GFR is not
- GOLD STANDARD among the plausible
clearances (MOST ACCURATE)  Dependent to the rate
- HOWEVER, it is not routinely of urea production
performed GFR (inulin is and not solely to renal
exogenous; invasive) function
 Freely filtered by the
Elimination: glomerulus but is
reabsorbed
- Freely filtered by glomeruli
(approximately 40%) in
- Not reabsorbed
some parts of the
- Not secreted
nephron
- 100% present in urine
- PLASMA: 0% in blood circulation
*the faster the urine flow,
the lesser urea is
reabsorbed; the slower,
BETA 2-MICROGLOBULIN more urea is reabsorbed
CLEARANCE
o In renal dysfunction, urea is
Properties: the first biomarker to
- Mol weight of approx. 11,800 increase its concentration
Daltons o Highest concentration in a
- Dissociates from human leukocyte normal condition
antigens at a constant rate
- Endogenous substance
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

DISEASE CORRELATION:  Severe Vomiting and

Diarrhea
o AZOTEMIA  Severe dehydration
 A condition characterized by an  Maapil ang urea pag
increase NPNs (Non-Protein vomit/excrete
nitrogen) in blood
 Pregnancy
 PRE-RENAL AZOTEMIA  Transient decrease of urea
 Caused by reduced renal  Only during pregnancy
blood flow  After preg, will normalize
 Mataas lang ang urea sa again
blood circulation  multifactorial
 Kidneys are normal
 Congestive heart failure, ANALYTIC/LABORATORY
Shock, Hemorrhage, METHODS:
Dehydration o measurements were
originally performed on a
 RENAL AZOTEMIA PFF (Protein Free Filtrate)
 Caused by decreased renal (through precipitation) of
function whole blood based on
o Increases blood urea measurement of nitrogen
(BUN) o Current: Nitrogen
 Kidneys itself have concentration
dysfunction o Very important conversion
 Acute renal failure, Chronic factors:
renal failure, glomerular  UREA NITROGEN x 2.14
nephritis, Tubular necrosis, = UREA (mg/dL)
Intrinsic renal diseases  UREA NITROGEN
(mg/dl) x 0.36 = UREA
 POST RENAL AZOTEMIA (mmol/L)
 Due to obstruction of the
urine flow (bladder, ureter, o 3 METHODS:
urethra)  CONVENTIONAL
 Renal calculi, tumors of the METHOD
bladder or prostate, severe  KINETIC METHOD
infections  CHEMICAL METHOD

o UREMIA or UREMIC SYNDROME o ISOTOPIC DILUTION MASS

 A condition characterized by SPECTROMETRY
an increase NPNs but  Gold Standard;
accompanied by renal failure Reference method
 Not commonly utilized
o LOW UREA CONCENTRATION in laboratories

 Decreased Protein intake

 Urea is byproduct of protein
metabolism; lower protein,
lesser urea concentration

 Severe Liver Diseases

 Majority of proteins are
synthesized in the liver
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

1. CONVENTIONAL METHOD 3. CHEMICAL METHOD

o Hydrolysis of Urea by Urease (to o Diacetyl Monoxime (Direct
release ammonium ion) Method)
o Quantification of NH4+ (to  Urea reacts directly with
measure nitrogen because NH4+ diacetyl monoxime in strong
have ammonia) acidic condition to form a
 Nessler’s Reagent yellow diazine deriviative
 Berthelot’s  Not BUN
Reaction with  Absorbance of the yellow
Nitruprusside diazine derivative is directly
proportional to the
concentration of urea
2. KINETIC METHOD (Coupled-
 Ferric iron and
Enzyme)
thiosemicarbazide
o Urease and GLDH (Glutamate  Stabilizes color
Dehydrogenase)  Intensifies reaction
o Measures the disappearance of
NADH at 340 nanometer o o-Pthalaldehyde Method
o Used on common automated (Direct Method)
machines  o-Pthalaldehyde
o Reference method  Naphthylethylenediamine
*almost same with diacetyl monoxime
but using the two reagents above

SPECIMEN REQUIREMENT &

INTERFERING SUBSTANCES

1. PLASMA
o Not with ammonium ions,
Sodium citrate and Sodium
fluoride (inhibit urease)
o EDTA can be used
*The concentration of the o Fasting is not required
disappearance of NADH is correlated
to the concentration of Urea 2. SERUM
o Best specimen to measure
*The disappearance of NADH is urea content
being measured (340 nanometer); o There should be no
Absorbance is decreasing because hemolysis
NADH is naging NAD+
3. URINE
*Hydrolysis of urea forming o Should be refrigerated if
ammonium ion and carbonate by the cannot be analyzed within an
enzyme urease hour
 UREA is susceptible to
*Oxidation-reduction reaction of bacterial decomposition
ammonium ion (NADH to form (there are urease +
NAD+) catalyzes by Glutamate bacteria which breaks
dehydrogenase down urea if present)

*final product = Glutamate and

Water
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

o CREATININE
2. BIOMARKER: CREATININE
 Associated with abnormal
renal function
o CREATINE  Smaller amount is secreted
 Synthesized primarily in the liver and reabsorbed
from the arginine, glycine, and
methionine
 Transported to tissues and is
converted to phosphocreatinine, ANALYTICAL/ LAB METHODS
which serves as a high-energy
source 1. JAFFE REACTION (1886)
o Creatinine reacts with picric
acid (trinitrophenol) in alkaline
solution (usually sodium
hydroxide) to form a red-
orange chromogen
o Jaffe Reagent
 Picric acid
 Sodium hydroxide
= to form alkaline
picrate
o The absorbance of red-orange
chromogen in the
spectrophotometer is directly
proportional to the
o CREATININE concentration of creatinine
 Endogenous substance
 With a mol. Weight of 113 Name of method applying
Daltons Jaffe reation:
 Waste product of muscle
metabolism o Folin-Wu Method (1919)
 Produced by the muscle from  Non-specific; not react
creatine and creatine to creatinine
phosphate thru non-  Interference/reagent
enzymatic dehydration will react to:
process acetoacetate,
 Most widely used marker for acetone, ascorbate,
GFR glucose, and
 Advantages: pyruvate
 Endogenous substance with o Fuller’s Earth
constant rate of production  Adsorbent
 Not bound to plasma  Remove other
proteins substance in the
 Not reabsorbed by the PFF
tubules  Aluminum
Magnesium
DISEASE CORRELATION: Silicate
o CREATINE o Method of Hare (addition to
 Muscle diseases Lloyd’s Reagent)
 Muscle dystrophy,  PFF + Sodium
poliomyelitis, aluminum silicate
hyperthyroidism, trauma  Eluted and
reacted with
alkaline picrate
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

2. COUPLED-ENZYMATIC
3. BIOMARKER: URIC ACID
METHOD
o CREATININASE-CK
METHOD
 Commonly utilized
 Creatininase
 CK, Pyruvate Kinase,
LDH
 NADH to NAD+
(340 nm)
o CREATININASE-HYDROGEN
PEROXIDE METHOD
 Creatininase, Creatinase
 Sarcosine oxidase,
Peroxidase
o REFERENCE STANDARD
 Isotope dilution-mass
spectrometry

SPECIMEN REQUIREMENT o Metabolic waste product of

o Plasma, Serum purine metabolism
 Hemolyzed and icteric o Purines (A and G) from the
samples should be ingested nucleic acids or from
avoided tissue destruction
 Lipemic samples  Converted to uric acid in
produce the liver
erroneous results  Filtered by the
 Fasting is not required glomerulus
o Urine  98-100%
 Urine specimens should reabsorbed by
be refrigerated or PCT
frozen if longer than 4  Small amount are
days secreted by DCT
o Present as monosodium urate
INTERFERING SUBSTANCES in the plasma
o Ascorbic Acid o Relatively insoluble (blood pH)
 Interfere peroxidase  At concentrations
o Glucose greater than 6.4 mg/dL,
o Protein, Urea the plasma is saturated
o Alpha-keto acids/ ketones  Resulting to urate
o Cephalosporins, Dopamine, crystals formations
Lidocaine  Precipitation in the
 False increase tissues and joints
o Hemoglobin and bilirubin –
false decrease DISEASE CORRELATION
o Uric acid
 Decreased in kinetic o Elevated Uric Acid
methods  Gout Arthritis
 Characterized by increased
uric acid; formation of uric
acid crystals in joints that
can cause arthritis
 Precipitation of sodium
urates in the joints
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

 Cause hyperuricemia ANALYTICAL METHOD

o Overproduction of uric
acids
1. CARAWAY METHOD
o Purine rich diet, drugs,
or alcohols o Based on the oxidation of UA in PFF,
 Prone to renal calculi with subsequent reduction of
phosphotungstic acid which will
 Increased catabolism of nucleic react to uric acid to form tungsten
acids blue
 Uric acid is the waste  Sodium carbonate – provides
product of purine catabolism alkaline pH for color
 In patients with development
proliferative disorders on o Lacks specificity
chemotherapy (Leukemia,
Lymphoma, Multiple *the reagent will not react to uric acid
myeloma, Polycythemia) only; not commonly used
 Increased Metabolism of Cell
Nuclei
 Hemolytic or megaloblastic
anemia 2. URICASE METHOD
 Glycogen Storage Disease o Uricase catalyzes the oxidation of
 G-6-PD Deficiency UA to allantoin
 Lesch-Nyhan Syndrome  Differential absorption of uric
o X-linked genetic acid and allantoin at 293 nm
disorder
o Completely deficiency of o More specific
hypocanthine
guanine
phosphoribosyltransf
erase (synthesis of 3. COUPLE ENZYMATIC METHODS
purines) o Measures hydrogen peroxide
o Prevents reutilization of produced as UA is converted to
purine base resulting to allantoin
increased UA  Peroxidase or catalase
 Chronic Renal Diseases  Catalyze a chemical
 Filtration and secretion are indicator reaction
impaired o Color produced is directly
proportional to UA in the specimen
 Hyperuricemia
 Toxemia of pregnancy and
lactic acidosis
 Ingestion of Purine-rich diet
o Liver, kidney, shellfish,
legumes
 Hypouricemia
 Secondary to liver disease
 Defective tubular *Urease catalyzes the hydrolysis of uric
reabsorption acid to form allantoin, carbon dioxide and
o Fanconi’s Syndrome hydrogen peroxide
(disorder in which the
proximal tubular function *the hydrogen peroxide formed will be
of the kidney is catalyzed (oxidation-reduction reaction)
impaired) using peroxidase or catalase
 Chemotherapy
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 3

*the final product will be correlated to the

concentration of the uric acid

SPECIMEN REQUIREMENTS

- Heparinized Plasma, Serum, or

urine
o Serum
 Should be removed
from cells ASAP to
prevent dilution by
intracellular contents
 Fasting is NOT required
 Avoid lipemic specimens
(rich in serum that is
turbid due to
chylomicrons that may
interfere in the
absorbance)
 Hemolysis decreases
value because of
glutathione release
 May be refrigerated for
3-5 days

INTERFERING SUBSTANCES
- High bilirubin concentration
o Falsely decrease results to
peroxidase
- Drugs
o Salicylates
o Thiazides
 False increase
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

LIVER FUNCTION - Directly connected with the

hepatocytes
ANATOMY OF THE LIVER - Secretions and excretions of
hepatocytes will flow onto the
canaliculi

B. Sinusoids
- Pathway for blood cells and other
cells

 There are cases where fluid will

impede the flow of fluids on the 2
mentioned pathways
 There are enzymes synthesized in
these areas that are essential in
LIVER diagnosis
- Located on the abdominal region of
the body PHYSIOLOGY OF THE LIVER
- Occupies the superior part of the - Liver is like a factory since it will
abdomen synthesize a lot of substances in the
- Right side is larger than the left side body
since the left also occupies the heart
and stomach  Synthesis

 Carbohydrates are synthesized

during the
glycogenesis/glycogenolysis.
 Proteins are also synthesized in the
liver to be distributed to other cells
in the body.
 Lipids are also synthesized in the
liver through transport of lipids to
and from cells and liver.

 Detoxification
Falciform ligament
- Connects right lobe with left lobe  All compounds not needed by the
body will be cleared by the liver.
Gallbladder  Metabolized by the liver cells and
- Locate under the right lobe hepatocytes will remove the toxic
products
Common bile duct
- Meets with the pancreatic duct  Transport Substances
- Terminates on the duodenum
Hepatocytes  Lipids and fats transported by the
- Cells of the liver liver (Ex. Triglycerides) will be
- Functional cell units of the liver transported to tissues

There are different pathways of secretions  Store substances

in the liver:  Excrete Substances
 Synthesize Substances
A. Canaliculi
- Very small canals that will terminate  Liver dysfunctions can create great
into the bile duct impact on metabolism
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

SECRETORY FUNCTIONS OF THE LIVER BILIRUBIN METABOLISM

BILE 1. Red blood cells only live for about

- Major secretory substance of the 80-120 days (average of 100). When
liver they reach the senescent age, they
- Incorporated with other secretions of will undergo hemolysis. Remnants
the liver and temporarily stored in of the RBC will be phagocytized by
the gallbladder to be released in the the phagocytes (macrophages).
duodenum for the emulsification of
fats 2. Hemoglobin will undergo
- Emulsifying agent degradation and form heme (iron
o Bile will convert large globules portion) and globin (protein
of fat molecules to small portion).
molecules
o Easier catabolic activity for the 3. Globin Portion
enzymes - Will undergo catabolism releasing
amino acids of the protein portion
Components of Bile - Amino acids will be reused for
another protein synthesis
1. Bile Acids
 Cholic Acids 4. Heme Portion
 Chenodeoxycholic acid - Will undergo further catabolism
 Iron portion
2. Bile Salts  Protoporphyrin IX
3. Bile Pigments - In a ferrous state ang iron in
 Bilirubin hemoglobin
4. Cholesterol - In a ferric state ang iron pag na
bulag na kay heme
 Some of these compounds are - Heme (ferric) is not soluble in the
insoluble that is why they can form blood therefore it will be transported
stones in the canaliculi, sinusoids, by transferrin (transport protein)
gallbladder, or even in the common
bile duct 5. Transferrin will transport ferric
 Stones can impede the bile flow heme to liver cells.
causing complications
6. Heme will be stored in a form of
ferritin.
o Main storage form of iron in
the liver

7. Ferritin will transport ferric state of

heme to the bone marrow for
erythropoiesis.

8. Erythropoiesis will undergo in the

red bone marrow with the following
components:
o Ferric state of iron
How bilirubin is formed and how does o Globin
the liver eliminate the bilirubin? o Vitamin B12 (stimulant)
o Erythropoietin
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

9. The protoporphyrin IX will undergo Erythropoiesis

further degradation to form biliverdin.
Biliverdin will be converted to bilirubin I. - Production and maturation of RBCs
o This process occurs inside the in the red bone marrow and blood
macrophage in the spleen or circulation
bone marrow or liver. Hemoglobin
- major compound in RBCs
10. Bilirubin I will be released to the - transports oxygen since heme has
blood circulation. Since it is not soluble, it high affinity to oxygen
will be carried by a transport protein called
albumin.
o Main transporter of bilirubin I CONJUGATION OF BILIRUBIN

11. Bilirubin I will be eliminated via the

liver. The liver will conjugate bilirubin I and
form the water-soluble bilirubin II.
o Waste-product of heme
o Will be incorporated with the
bile secretions of the liver
o Temporarily stored in the
gallbladder
o Released in the duodenum
1. Heme (protoporphyrin IX portion)
12. Upon the arrival of bilirubin II in the will be converted to bilirubin I and
duodenum, because of the bacterial will be carried by albumin through
activity, it will be converted to the sinusoids of the liver to undergo
urobilinogen. Some urobilinogen will conjugation via facilitated
proceed to the kidney and some will diffusion.
proceed to the large intestines. 2. Bilirubin I will bind to transporter
protein ligandin (red crescent) to
13. Urobilinogen that will be metabolized facilitate the entry of bilirubin I.
in the kidneys to form urobilin. This 3. Bilirubin I will undergo conjugation
urobilin will be excreted during urination. with UDP-Gu.
o Uridyl Diphosphate glucoronic
14. Urobilinogen that proceed to the acid
large intestines will be converted to 4. Bilirubin I + UDP-Gu will form
stercobilin which will be incorporated in bilirubin diglucuronide (Bilirubin
the fecal material (mao yellow-brown). II).

Other things mentioned: 5. Bilirubin II will exit the hepatocyte

Protoporphyrinogen to be incorporated with the bile then
- Precursor compound of exit via canaliculus to the gallbladder
protoporphyrin IX then common bile duct then lastly
the duodenum.
Vitamin B12 Note:
- Required in the maturation of RBCs Bilirubin I: Bilirubin monoglucuronide
specifically the nucleus of young RBCs
Bilirubin II: Bilirubin diglucuronide
Erythropoietin UDP-glucuronyl tranferase
- Hormone that will stimulate RBC
production and maturation - Transfers glucuronyl to bilirubin I
- Synthesized in the kidneys
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

REACTIONS DURING THE

CONJUGATION

LIVER FUNCTION TESTS

 The conventional/old tests are based

Heme-oxygenase on the concentration of bilirubin.
- Enzyme that catalyzes heme to
biliverdin Total Bilirubin: Bilirubin I + Bilirubin II
- oxidation  If concentration of bilirubin exceeds
the normal limit, the plasma
Biliverdin reductase becomes darker. Skin and sclera will
- Enzyme that catalyzes biliverdin to also have yellow discoloration.
bilirubin
- Reduction JAUNDICE
- Also known as Icterus
- Condition characterized by yellow
discoloration of the skin and sclera
- Caused by hyperbilirubinemia

Icteric serum/plasma
- Description of the plasma pag dark
yellowish due to increased bilirubin

TYPES OF JAUNDICE

1. Prehepatic jaundice
- Excessive amount of biliurubin that is
UDP glucuronyl transferase presented/transported by albumin to
- Enzyme that catalyzes the transfer of the liver for metabolism
glucuronyl to bilirubin I - B1 is in excessive amount causing
- Forms bilirubin II (bilirubin hyperbilirubinemia
diglucuronide)
- transferase Causes:
 Hemolysis
Remember:  Hemolytic anemia
Bilirubin monoglucuronide o Increased rate of red blood
cell hemolysis causing more
- Water insoluble
production of biliverdin
- Aka Bilirubin I
o Can happen even before
Bilirubin diglucuronide
senescent age
- Water soluble
 Malaria
- Aka Bilirubin II
o Increased Bilirubin I kay ang
malarial parasite may
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

erythrocytic cycle wherein - Elevated B2

RBCs are hemolyzed
o Magrupture ang RBCs due to D.) Rotor’s Syndrome
the ring-forms of malaria - Same symptoms with Dubin-Johnson
(schizont) na mugawas Syndrome
- Elevated B1
2. Hepatic jaundice - Liver cells are not pigmented
- result from impaired cellular uptake, - No formation of B2, retaining B1
defective conjugation, or abnormal
secretion of bilirubin by the liver 3. Posthepatic jaundice
- due to abnormality of the - Elevated B2
hepatocytes - impaired bilirubin excretion
- best example are gallstones
 Cellular uptake refers to the uptake - will impede the flow of bile from t he
of B1 by the hepatocyte liver to the gallbladder or from
 Defective conjugation refers to the gallbladder to duodenum
defective conjugation of B1 and UDP-
Gu  Tumors can also be cause of
 Abnormal secretion refers to posthepatic jaundice since it will
defective secretion of B2 to the cause blockage sa bile flow.
canaliculus  Parasites like schistosoma can also
cause posthepatic jaundice because
Diseases: of the migration of the parasite in
 Gilbert Syndrome the bile duct.
 Crigler-Najjar Syndrome
 Rotor’s Syndrome  Ascaris lumbricoides can also cause
 Dubin-Johnson Syndrome posthepatic hyperbilirubinemia since
erratic ang larvae and adult worm
A.) Gilbert Syndrome tapos mag migrate siya.
- Characterized by impaired cellular
uptake of bilirubin ANALYSIS OF BILIRUBIN
- B1 will not enter hepatocyte and not
undergo conjugation A.Ehrlich’s Reaction (1883)
- B1 now will go to blood circulation - Urine bilirubin reacted to diazotized
- Elevated B1 sulfanilic acid to form a complex

B.) Crigler-Najjar Syndrome B.Van den Bergh (1913)

- Characterized by deficiency of - Using serum bilirubin
enzyme UDPGT - Used alcohol accelerator for the
- Therefore no formation of B2 coupling of bilirubin to diazotized
- Elevated B1 sulfanilic acid
- Can either be:
 Type 1: Coupling accelerator
- complete absence of UDPGT  serum bilirubin will be coupled to
 Type 2: reagent
- less severe deficiency UDPGT  only B1 needs this because insoluble
siya
C.) Dubin-Johnson Syndrome
- An autosomal recessive disease Note:
which presents shortly after birth  Urine bilirubin = B2
with an increase of conjugated  Serum bilirubin = B1
bilirubin without elevation of liver  Kaya need ng accelerator ni
enzymes (ALT, AST) serum bilirubin kay insoluble pa
- Defective excretion by the liver cells siya
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 4

- Measurement of degree of
Two methods under Van den Bergh: yellowishness of serum/plasma
- Icompare sa 0.01% potassium
a.) Malloy-Evelyn Method (1937) dichromate
- Uses serum bilirubin - If darker, then there is
- Uses 50% methanol as coupling hyperbilirubinemia
accelerator - Not specific, sensitive

b.) Jendrassik and Grof (1938) LIVER ENZYMES

- uses serum bilirubin
- uses caffeine-benzoate-acetate as ALP: Alkaline Phosphatase
coupling accelerator ALT: Alanine Aminotransferase
- uses ascorbic acid which AST: Aspartate Aminotransferase
terminates the reaction 5’N: 5-nucleotidase
GGT: gamma-glutamyl transferase
 If mag gamit kag accelerator LAP: Leucyls Aminopeptidase
o Gameasure kag total LDH: Lactate Dehydrogenase
bilirubin (B1 + B2)
 If di ka mag gamit accelerator
o Gameasure kag B2 ra

So therefore, mag gamit tag two tubes

(one with accelerator; one without) para
mameasure nato ang three values which
are:
 Total bilirubin (w/ accelerator)
 Bilirubin I (TB-B2)
 Bilirubin II (w/o accelerator)
Direct Bilirubin: B2
Indirect Bilirubin: B1

 Mas ginagamit si Jendrassik and Grof

since ang 50% methanol ni Malloy-
Evelyn kay toxic.
 Mas gwapo si Malloy-Evelyn tho if sa
reaction mag base

REFERENCE VALUES OF BILIRUBIN

 Normal ra na si infant mas higher

ang value kay mas rapid ilang
hemolysis kay tungod sa ilang rapid
na RBC production

C. Icterus Index
- Oldest method
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

ENZYMES (Part 1)
2. Reaction it catalyzes
ENZYMOLOGY  Oxidation = oxidase
- Branch of biochemistry  Reduction = reductase
- Study of enzymes:  Hydrolysis = hydrolase
 Activity of enzymes  Remove carboxyl groups
 Properties of enzymes = decarboxylase
 Chemical reactions it catalyze  Remove hydrogen atoms,
 Clinical use transferring them to a
 Laboratory tests coenzyme, or dehydration
 Laboratory methods = dehydrogenase
 Diagnosis
Enzymes Ex. Glucose oxidase
- Biologic catalyst Substrate: Glucose
- Compounds that hasten chemical
reactions (speed up the rate) Note: Not all enzymes are named based
- Not consumed during the reactions on this nomenclature
- Not undergo chemical change after
the reactions 3. Enzyme Commission
Nomenclature (E.C.)
Substrate
- Substance acted by the enzyme Ex. E.C. 1.1.1.21

 1st digit: class

 2nd digit: subclass
 3rd and 4th: serial number
Note:
 Function of this nomenclature is for
enzyme only, not for substrates.

IUB Nomenclature
- International Union of Biochemistry
- Composes of:
a.) E.C. Nomenclature
 Substrate will bind to the active site
b.) Chemical Name
of the enzyme to form the enzyme-
c.) Enzyme naming itself
substrate complex
 Enzyme will speed up the reaction of
CLASSIFICATION OF ENZYMES
the substrate, forming prooducts.
- Basis for the 1st digit (class)
 1 second ang maconsume sa pag
bind with the enzyme
1. Oxidoreductases
 1.001 seconds ang state na maging
2. Transferases
ES complex sila
3. Hydrolases
 1.002 seconds ang formation of
4. Lyases
products.
5. Isomerases
6. Ligases
Note: Not all reactions have enzymes
A. OXIDOREDUCTASES
NOMENCLATURE OF ENZYMES
- oxidation and reduction
- hand on hand sila; buy 1 take 1
1. Substrate + -ase
 Lipid = lipase (lipidase noon)
Oxidation: removal of H ion
 Ester = esterase
Reduction: accept H ion
 Protein = protease
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

Ex. Lactate Dehydrogenase  L-alanine’s (substrate) amino group

 E.C. 1.1.1.27: L-lactate NAD+ is transferred to alpha-ketoglutarate
Oxidoreductase forming pyruvate and glutamate

 L-glutamate’s amino group can also

be transferred to pyruvate to form L-
alanine and alpha-ketoglutarate

C. HYDROLASES
 Hydrogen on the second carbon of - Hydrolysis of various bonds
the lactate is being removed and - Addition of water resulting to
accepted by the coenzyme NAD+ to breaking of bonds
form NADH
Ex. Alpha-Amylase
 Another hydrogen is being removed  E.C. 3.2.1.1: 1,4-D-Glucan
to balance the carbon and releases a Glucanohydrolase
free hydrogen (kanang last na H+)  Substrate: Glucan group

 L-lactate becomes pyruvate with

the help of enzyme lactate
dehydrogenase

 Oxidation si L-lactate; Reduction si

NAD+

 Oxidation si NADH + H; Reduction si  Lipase catalyzes the breaking of

pyruvate bonds of triglycerides
 Fused with 3 bonds of fatty acids
Note: Oxidoreductase reactions are (pink) that is covalently
reversible. bonded/linked to glycerol (blue)
 Addition of 3H2O will break the 3
B. TRANSFERASES bonds forming 3 fatty acids and 1
- Transfer of functional groups other glycerol
than hydrogen from one substrate to
another D. LYASES
- About 8 functional groups - Addition of a group to a double bond
or the removal of a group to form a
Ex. Aspartate Aminotransferase double bond
 E.C. 2.6.1.1: L-Aspartate: 2-
Oxaloglutarate Aminotransferase Ex. Carbonic Anhydrase
 L-aspartate transferred to Citrate Lyase
oxaloglutarate
 Amino group ang itransfer mao
aminotransferase

 Water will not break bonds but

rather form bonds
 Water (pink) will be part of the
product
 Alanine aminotransferase dapat
ang enzyme sa figure
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

E. ISOMERASE TERMS ASSOCIATED WITH ENZYMES

- From the root word isomere (equal)
- Rearrange the functional groups Substrate
within a molecule and catalyze the - Acted upon by the enzyme
conversion of one isomer into - Specific (like the enzyme)
another o For each other ra jud sila sa
specific enzyme
Ex. Phosphoglycerate mutase
Isoenzyme
- Different form but with the same
action
- Ex. Lactate dehydrogenase
 Produced and released by
different cells in the body
 Catalyzes the interconversion
of lactate to pyruvate
 may 5 different forms
 Tissue location is important
 3-phosphoglycerate becomes 2-
Phosphoglycerate Cofactor
 Same parin ang number of molecules - Non-protein molecule of any
iba lang ang arrangement enzymatic reaction
 Mura rag nirotate - Ex. Creatine Kinase
 Catalyzes the transfer of
F. LIGASES phosphate group from
- From the root word ligation (cut) creatine phosphate to ADP
- Catalyze a reaction in which the  ADP = cofactor
following bonds are made or broken:
 C-C Two types of cofactors:
 C-S
 C-O  Inorganic cofactor (activator)
 C-N - Main function is to activate enzyme
- Accompanied by an ATP-ADP activity
interconversion - If activator is absent, there would be
no ES complex
Ex. DNA Ligase - non-protein part
- Ex. ADP, NADH, NAD+

 Organic cofactor (coenzyme)

Apoenzyme
- The protein/polypeptide part of the
enzyme

Holoenzyme
 Introns are cut out of the long strand - Apoenzyme combined with the
of DNA and joined coenzyme (organic cofactor)
 DNA ligase will catalyze the joining

 Substrate: Lactate
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

 Coenzyme: NAD+ - Inactive form of enzyme

 Enzyme: Lactate dehydrogenase - Converted (usually by proteolysis),
 Product: Pyruvate, NADH, H+ to the active form when it has
reached the site of its activity
- Encountered usually in
synthesization of enzymes
 Substrate: Creatine
 Coenzyme: ATP ENZYME KINETICS
 Enzyme: Creatine Kinase
 Product: Creatine phosphate, ADP A. Michaelis-Menten Theory
- Pioneering theory
- By Leonor Michaelis and Maud
Menten
- Substrate will bind to active site of
the enzyme forming ES complex
then resulting to product
- Enzyme is still compact in the end

Active site
 If inorganic factor is absent, no ES - Where the substrate will form a bond
complex formed (taas na illustration) with the enzyme

 Activator will bind first to active site Allosteric site

to allow binding of substrate to - The site of the substrate where
enzyme (below na illustration) enzymes cannot bind

Note: Almost all kay naga require ng

inorganic cofactor

LOCK AND KEY THEORY

1. The substrate, sucrose, consists of

glucose and fructose bonded
together
2. The substrate binds to the enzyme,
 Holoenzyme is where the forming an enzyme-substrate
apoenzyme and coenzyme are linked complex
together = reaction occurs 3. The bonding of the substrate and
enzyme places stress on the glucose-
Proenzyme fructose bond, and the bond breaks.
- pro means precursor 4. Products are released, and the
- Also known as zymogen enzyme is free to bind other
substrates
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

PROPORTIONAL to
the rate of
disappearance of A or
the appearance of P

o Second-order reaction
 The rate is proportional
to the product of the
concentration of two
reactants
 A+BP
 Rate of reaction is
INDUCED FIT THEORY
EXACLTY
PROPORTIONAL to
Example: The Enzyme Lysozyme
the rate of
disappearance of A or
the appearance of P

o Zero-order reaction
 The reactions are zero-
order with respect to
the reactants
 Rate of reaction
DEPENDS on the
LYSOZYME concentration of the
- helps kill bacteria by binding to the molecular species
polysaccharide coating of the (enzyme) undergoing
bacteria. reaction
- The fit is not perfect, so the shape of
the active site changes to fit the First & Second order depends on the
polysaccharide substrate (induced SUBSTRATE; Zero order depends on
fit) the ENZYME

Induced Fit
- Change of shape of the active site
- To initiate, the enzyme breaks the
polysaccharide, ultimately helping
kill the bacteria.

FACTORS INFLUENCING ENZYMATIC

REACTIONS

1. SUBSTRATE CONCENTRATION - All enzyme present will form a

complex with a substrate
o First-order reaction - If reaches the limit, any excess
 Those which proceed at substrates will NOT INCREASES the
a rate exactly rate of reaction
proportional to the - Why? because all enzymes already
concentration of one formed a complex with the
reactant substrates (Zero-order or reaches
 A-P the maximum enzyme reaction
 Rate of reaction is velocity)
EXACTLY
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

reaction does not

2. ENZYME CONCENTRATION occur
o The higher the enzyme level, - Inhibitor and
the faster the reaction will substrate both
proceed compete for the
active site.
3. pH
o pH= 7.0 – 8.0
o changes in pH may denature  Non-competitive
the enzyme
o Protein in nature

4. TEMPERATURE
o 37 degrees Celsius (Normal)
o Denaturation at 40-50
degrees Celsius
o Assay temperatures:
 At 25, 30, or 37
degrees C

5. COFACTORS
o Non-protein entities that must
bind to particular enzymes
before a reaction occurs
o Metallic or Non-metallic - The inhibitor will bind to
o Calcium, ferrous, the Allosteric
magnesium, manganese, Enzyme/Regulatory
zinc, potassium ions site of the enzyme,
allowing the change of
the shape of the active
site.

 Uncompetitive
6. INHIBITORS
o Interfere with enzyme
reactions
 Competitive

- Will not compete

- Rather, the inhibitor will
bind AFTER the substrate
will form a complex with
the enzyme
- Able to fit at the
active site. Inhibition is said to be a form of Negative
- When it is there, the Feedback, because an increase in the
substrate is unable to product, forces a decrease in the enzyme
bind to the enzyme reaction.
and the desired
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

ENZYMATIC ASSAYS
MEASUREMENT OF ENZYME ACTIVITY
1. COUPLED-ENZYME ASSAY
(First 3 are common) o Substances other than
 Increase in production substrate or coenzyme are
concentration necessary and must be
o The more enzyme present, present in excess
more E-S complex will be o NAD+ or NADH
formed
o Therefore more product will be 2. FIXED-TIME METHOD (End-point)
formed Process:
o More product formed=higher o Reactants are combined
enzyme concentration  Substrate, cofactors,
o Higher enzyme concentration etc.
= faster rate of the reaction o Reaction proceeds for a
designated time
 Decrease in substrate  e.g. incubate for 5 mins
concentration o Reaction is stopped
o If more enzyme, the lower the  to terminate the
substrate concentration activity/catalysis, add
because it is consumed during another reagent
the reaction forming products o Measurement is made of the
o the rate of disappearance of amount of reaction has
the substrate is inversely occurred
proportional to the rate of the
reaction 3. CONTINUOUS-MONITORING
o if more substrate consumed, METHOD (Kinetic Assay)
the higher the enzyme o Most commonly used
concentration method
o Multiple measurements
 Decrease or increase in o Total of 5 readings
coenzyme concentration o Absorbance change
o Depends on the location of  Increasing in
the NADH and NAD+ absorbance
o NADH can absorb light than  Decreasing in
NAD+ absorbance
o If NADH is at the product side, o Time intervals
increase in concentration o Change of absorbance will
o If NADH is at the substrate depict the rate of reaction
side, decrease in
concentration
CALCUALTION OF ENZYME ACTIVITY
 An increase in the concentration
of the altered enzyme (rare case 1. INTERNATIONAL UNIT (IU)
to measure enzyme) o Amount of enzyme that will
catalyze the reaction of 1
GOAL: TO REACH ZERO ORDER micromole of substrate per
REACTION minute under specified
conditions (per minute
therefore continuous
monitoring method siya)
o Universally used
CLINICAL CHEMISTRY 2 PRELIMS: RENAL FUNCTION PT. 5

2. KATAL UNIT
o Amount of enzyme that will
catalyze the reaction of 1 mole
of substrate per second under
specified conditions
o 1.0 IU = 16.7 nkat
ENZYMES (part 2) ➢ Fallopian tube

AMYLASE (AMY) ➔ Both are metalloenzymes

- E.C.3.2.1.1 • Containing calcium ion
- 1,4-D-Glucan Glucanohydrolase
- Breakdown of starch and glycogen to Activators:
monosaccharides ➢ Bromide
- Hydrolysis ➢ Iodide
- Catalyzes only the a-1,4-glycosidic
bonds to produce degradation
products

Degradation products:
➢ Glucose
➢ Maltose
➢ Dextrins
• Intermediate chains which
contain a, 1-6 branching
linkages

a-1,4-glycosidic bonds
➔ Pancreatic duct is attached with the
- Found in starch and glycogen
duodeum
➔ Liver’s common bile duct is also
Activators:
attached with the duodenum
➢ Calcium ions
➢ Chloride
Sphincter of Oddi
- will control the flow of the pancreas
➔ Therefore if we are going to have in
and the liver’s secretions
vitro testing, the activators will be
- surround the end portion of the
present in the reagents
common bile duct and the pancreatic
duct

Properties:
✓ Molecular weight: 50,000-55,000 D
✓ Readily filtered by the renal
glomerulus (appears in the urine)
✓ Mouth-starch initial digestion by
salivary amylase
• Inactivated in the stomach
because of the pH
Tissue Sources: • ang amylase man gud kay
➢ Acinar cells of the pancreas slightly alkaline, tapos ang
• Exocrine portion stomach ay 1-2 ang pH
• Pancreatic amylase ✓ Final digestion by the pancreatic
➢ Salivary glands amylase happens in small intestine
• Salivary amylase ✓ Has the smallest/lowest molecular
• Ptyalin weight that is why it can be readily
filtered by the glomeruli
2 types of Isoenzymes ✓ Can normally appear in the urine

A. P-type isoenzyme Diagostic Significance:

➢ Pancreas
1. Acute pancreatitis
B. S-type isoamylase • Rise: 6-48 hours/2-12
➢ Salivary gland hours after onset of an attack
➢ Lungs • Peak: 24 hours
• Normalize: 3-5 days
➔ The ideal time to collect blood is ❖ The decrease in color is proportional
during the onset of the attack to amylase concentration.
➔ Attacks refers to signs and
symptoms (e.g. pancreatic pain, left 2. Saccharogenic
quadrant pain) - Uses a starch substrate that is
hydrolyzed by the action of amylase
2. Salivary gland involvement to its constituent carbohydrate
molecules that have reducing
3. Macroamylasemia properties
- results to when the amylase - Classic reference method
molecule combines with - Measured in Somogyi units
immunoglobulins to form a complex - Old name is Nelson Somogyi
that is too large to be filtered across method
the glomerulus - Oldest method
- usually IgG - Lacks specificity and and sensitivity

Increased activity of AMY can be due to: Principle:

➢ Mumps ➔ The amount of reducing sugars is
• Self-limiting measured where the concentration is
• Caused by Mumps Virus directly proportional to amylase
• Children should have MMR activity
vaccine ➔ The more starch hydrolyzed, the
• May lead to partial deafness or higher the concentration of the
may reach testis and cause reducing carbohydrates formed
infertility
➢ Parotitis/Parotiditis 3. Chromogenic
• Same with mumps pero ibang - Uses a starch substrate to which a
organism chromogenic dye has been attached,
➢ Perforated peptic ulcer forming an insoluble dye-substrate
➢ Intestinal obstruction (duodenum) complex
➢ Cholecystitis - Lack specificity and sensitivity
• Stone formation in gallbladder
➢ Ruptured ectopic pregnancy Principle:
➢ Mesenteric infarction ➔ As amylase hydrolyzes the starch
• In the large intestine substrate, smaller dye-substrate
➢ Acute appendicitis fragments are produced, and these
are water-soluble
Assay for Enzyme Activity ➔ The increase in color intensity of the
soluble dye-substrate solution is
1. Amyloclastic directly proportional to amylase
- Amylase is allowed to act on a starch activity
substrate to which iodine has been
attached Sensitivity: ability of the method to detect
- Conventional type even the smallest amount
- Lacks specificity and sensitivity
Specificity: ability of the method to detect
Principle: or measure the specific analyte
➔ Serum/Plasma then add starch
solution to specimen then add iodine 4. Coupled-Enzyme
➔ Initial color is bluish to black - Have been used to determine
➔ As amylase hydrolyzes the starch, amylase activity by a continuous-
the iodine is released and a decrease monitoring technique in which the
in the initial dark-blue color intensity change in absorbance of NAD+ at
of the starch-iodine complex occurs 340nm is measured
➔ Pag marelease ang iodine, mawala - Optimum pH: 6.9 pH
na bit by bit ang bluish to black color
Sources of error
✓ Amylase in serum and urine is stable
✓ Little loss of activity occurs at room
temperature for 1 week or at 4C for
2 months or 6 months
• pero rule of thumb, dapat as
soonest as possible jud itest
✓ Amylase values may be normal in
acute pancreatitis with hyperlipemia
➔ Maltopentose is the main substrate • triglycerides suppress or
incorporated in the reagent inhibit serum amylase activity
➔ AMS/AMY is the unknown activity ✓ Morphine and other opiates
from the serum sample • can cause falsely elevated
➔ Maltopentose is hydrolyzed to form serum amylase levels
maltrotriose and maltose • can cause constriction of
➔ Maltrotriose and maltose undergoes Oddi’s sphincter and
oxidation acted upon by alpha- pancreatic ducts
glucosidase to form 5-glucose • with consequent elevation of
➔ 5-Glucose with 5 ATP coenzyme, are inarticulate pressure causing
catalyzed by hexokinase to form 5- regurgitation of amylase into
Glucose-6-phosphate + 5 ADP the serum
➔ 5-Glucose-6-phosphate and 5 NAD ✓ Citrate or oxalate as anticoagulant
ion is catalyzed by glucose-6- • Falsely low activity
phosphate dehydrogenase forming • Amylase is a calcium-
5,6-phosphogluconolactone and 5 containing enzyme
NADH • Heparin does not interfere
with its activity so kani dapat
So therefore si NADH ginameasure sa gamiton
coupled-enzyme/continuous monitoring kay ✓ Contamination of saliva
maka absorb man siyag light at 340nm • Approximately 700 times that
of serum
Continuous monitoring: • Dapat no talking, closed din
- Measure the absorbance of the ang tube
reaction tube per minute • Cause falsely high level
✓ Red cells contain no AMS, so
Summary of Enzyme Activity Assays hemolysis generally presents no
problem with most methods except
Assay Principle those coupled-enzyme methods

Amyloclastic Measures the LIPASE (LPS)

disappearance of - E.C. 3.1.1.3
starch substrate - Triacylglycerol Acylhydrolase
Saccharogenic Measures the - Hydrolase
appearance of the - Hydrolyzes the ester linkages of fats
product to produce alcohols and fatty acids
Chromogenic Measures the - Catalyzes the partial hydrolysis of
increasing color dietary triglyceride in the intestine to
from production of the 2-monoglyceride and fatty acids
product coupled
with a
chromogenic dye
Continuous Coupling of
monitoring several enzyme
systems to
monitor amylase
activity
➔ It should be “3H2O” not 2H2O Intra-abdominal conditions that could
➔ 3 molecules of water will break the elevate LPS:
bonds of the chain releasing 2- ➢ Duodenal Ulcers
monoglyceride and 2 fatty acids ➢ Perforated peptic ulcers
➢ Intestinal Obstruction
Note: ➢ Acute cholecystitis
✓ Enzymatic activity is specific for the
fatty acid residues at positions 1 and Assay
3 of the triglyceride molecules
✓ Substrate must be an emulsion for A. Estimation of liberated fatty
activity to occur acids and turbidimetric methods
✓ Reaction rate is accelerated by the
presence of colipase and bile salt

What inhibits serum lipase?

➢ Proteins
➢ Bile Acids ➔ Trigylceride with 2 molecules of
➢ Phospholipids water at 8.6-9.0 pH forms 2-
Monoglyceride + 2 fatty acids
➔ So if may hyperproteinemia, high ➔ Not commonly requested in a clinical
bile acids and phospholipids most set-up
likely lower ang lipase results ➔ Lacks sensitivity and specificity

What reverses the inhibition? B. Classic Cherry-Crandall Method

➢ Colipases - Substrate: olive oil
• Highest concentration of
Calcium triglyceride
- necessary for maximal lipase activity - Measured the liberated fatty acids by
titration after a 24-hour incubation
Heavy metals and Wuinine - Oldest method
- inhibit lipase activity - Obsolete since wala nang titration sa
lab
Tissue Sources
➢ Primarily in the pancreas C. Turbidimetric Methods
➢ Present in small amounts sa: - As the fats are hydrolyzed by LPS,
• Stomach the particles disperse
• Small intestine - The rate of clearing can be measured
as an estimation of LPS activity
➔ If acute pancreatitis ang idiagnose, - hydrolysis of LPS is not on an
mas better si lipase kaysa kay optimal level
amylase
➔ Most accurate indicator for acute D. Colorimetric Methods
pancreatitis - Most commonly used
- Based on coupled reactions with
Diagnostic Significance enzymes such as peroxidase or
glycerol kinase as enzyme indicator
✓ Almost exclusively to the diagnosis
of acute pancreatitis Sources of Error
• Rise: 2-12 hours after onset
of an attack ✓ LPS is stable in serum
• Peak: 24 hours ✓ Loss in activity:
• Persists for approximately 5 ▪ Room temperature: 1 week
days or 7-10 days ▪ 4C: 3 weeks
✓ Extent of LPS elevations does not ✓ Hemolysis should be avoided
correlate with severity of the disease because hemoglobin inhibits serum
✓ Normal in conditions of salivary LPS
gland involvement
ASPARTATE AMINOTRANSFERASE Diagnostic Significance
(AST) ✓ Limited mainly to the evaluation of
- E.C. 2.6.1.1 hepatocellular disorders and skeletal
- L-Aspartate: 2-oxaloglutarate muscle involvement
Aminotransferase ✓ Used for diagnosis of acute
- Transferase myocardial infarction (AMI)
- Transfer of an amino group between • Rise: 6-8 hours
aspartate and alpha-keto acids to ▪ Best time
form oxaloacetate and glutamate • Peak: 24 hours
- Former name : serum glutamic- • Normalized: within 5 days
oxaloacetic transaminase ▪ This stage is not useful
(SGOT/GOT) in diagnosis of AMI

Pyridoxal phosphate (Vitamin B6) Acute myocardial infarction

- Cofactor of AST - Most common symptom is chest pain
or pectoralis angina

High AST can be due to:

➢ Pulmonary embolism (lungs)
➢ Chronic heart failure
➢ Acute hepatocellular disorders
➢ Viral hepatitis
➢ Liver cirrhosis
➢ Muscular dystrophies
➢ Inflammatory conditions

➔ NH2 is transferred from aspartate to Assays

a-keto-glutarate forming
oxaloacetate and glutamate A. Karmen Method
- Most commonly used
Note: - Incorporates a coupled enzymatic
✓ Transamination reaction is important reaction using malate
in intermediary metabolism because dehydrogenase as the indicator
of its function in the synthesis and reaction
degradation of amino acids - Monitors the change in absorbance
✓ Ketoacids formed are ultimately at 340nm continuously as NADH is
oxidized by the tricarboxylic acid oxidized to NAD+
cycle to provide a source of energy - Optimal pH: 7.3-7.8 pH

Tissue sources
➔ Widely distributed in human tissue:
(in decreasing order)
➢ Cardiac tissue – highest concen. ➔ Aspartate (from sample) plus alpha-
➢ Liver ketoglutarate (from reagent)
➢ Skeletal muscles becomes oxaloacetate and glutamate
➢ Kidney ➔ Oxaloacetate and NADH will be
➢ Pancreas reduced to malate and NAD+ ion
➢ Erythrocytes ➔ Since NADH can absorb light, it is
used to measure AST
Isoenzyme Fractions (from the ff)
B. Dinitrophenylhydrazine
A. Cell cytoplasm - Couples color reagent with keto acid
- Predominant from occurring in serum product

B. Mitochondria C. Diazonium salts

- Increases in disorders producing - Couples the salt with the keto acid
cellular necrosis product and forms a color
Sources of Error - Catalyzes the reduction of pyruvate
to lactate with simultaneously
✓ Hemolysis should be avoided oxidation of NADH (340nm)
✓ AST activity is stable in serum for 3- - Optimal pH: 7.3-7.8 pH
4 days at refrigerated temperature

ALANINE AMINOTRANSFERASE (ALT)

- E.C. 2.6.1.2 Sources of Error
- L-Alanine: 2-oxaloglutarate ✓ ALT is stable for 3-4 days at 4C
Aminotransferase ✓ Relatively unaffected by hemolysis
- Transferase
- Same enzymatic activity with AST ALKALINE PHOSPHATASE (ALP)
but different substrate - E.C. 3.1.3.1
- Transfer of an amino group between - Orthophosphoric Monoester
alanine and alpha-keto acids to form Phosphohydrolase (Alkaline
glutamate and pyruvate Optimum)
- Former name: serum glutamic- - Hydrolysis
pyruvic transaminase (SGPT//GPT) - Catalyze the hydrolysis of various
phosphomonoesters at an alkaline
Pyridoxal phosphate pH
- coenzyme - Liberate inorganic phosphate from an
organic phosphate ester with the
concomitant production of an alcohol
- Main substrate:
phosphomonoester
- Optimal pH: 9-10 pH
▪ varies with the
substrate used

Magnesium and Zinc ions

- activators

Tissue Sources
✓ Widely distributed in human tissue
➢ Liver – highest concentration
• Considered the more liver-
specific enzyme of the
transferase ➔ Phosphomonester will form a
compound of alcohol and
Diagnostic Significance phosphate ion
✓ Confined mainly to evaluation of
hepatic disorders Tissue Sources
✓ Higher elevations are found in ✓ Present on cell surfaces in most
hepatocellular disorders than in tissues (descending order):
extrahepatic or intrahepatic ➢ Intestine – highest concentration
obstructive disorders ➢ Liver
✓ In acute inflammatory conditions of • Specifically in sinusoids and
the liver, ALT is higher than AST bile canalicular membranes
➢ Bone
Assay • Specifically osteoblasts since it
is involved in production of
A. Coupled Enzymatic Reaction bone matrix
- Uses lactate dehydrogenase as ➢ Spleen
the indicator enzyme ➢ Placenta
➢ Kidney
➔ Though mas daghan sa intestine, b. Intestinal ALP
mas significant parin sa liver and
bone when it comes to diagnosis c. Liver ALP
- If it is greater than 20%
Isoenzymes (Electrophoresis)
- Descending order: d. Bone ALP
➢ Liver ALP: migrates fastest to anode - if the residual activity before heating
➢ Bone ALP is less than 20% of the total
➢ Placental ALP activity before heating
➢ Intestinal ALP
2. Chemical Inhibition
1. LIVER ALP
Phenylalanine
2 Fractions of Liver ALP - Inhibits intestinal ALP and placental
A. Major liver fraction ALP to a much greater extent than
• Useful for diagnosis for liver and bone ALP
hepatobiliary conditions - Impossible to differentiate the four
▪ Liver and bile ducts fractions
▪ Gallbladder - Most commonly used inhibitor

B. Fast liver fraction Levamisole

• Alpha1 liver - Inhibits bone ALP and liver ALP
• Diagnostic of metastatic
carcinoma of the liver and Abnormal Fractions associated with
hepatobiliary diseases Neoplasms:
• As a valuable indicator of
obstructive liver disease Carcinoplacental Alkaline
Phosphatases
2. BONE ALP - Similarities to the placental
- Increases due to osteoblastic activity isoenzyme
- Normally elevated in children during - Increased to 3% to 15% in cancer
periods of growth and in adults older patients
than age 50 - Two kinds:
1. Regan Isoenzyme
3. INTESTINAL ALP 2. Nagao Isoenzyme
- Depends on the blood group and
secretor status of the individual (“B” A. Regan Isoenzyme
and “O”) - Has been characterized as an
▪ Mas high ang concentration sa example of an ectopic production of
mga “B” and “O” an enzyme by malignant tissue
- Bound by erythrocytes of “A” group - Detected in various carcinomas:
- Increases after consumption of fatty ▪ Lung
meal ▪ Breast
- High with diseases of the digestive ▪ Ovarian (highest incidence)
tract and cirrhosis ▪ Colon
- Migrates as the bone ALP
How do we differentiate these isoenzymes? - Most heat stable of all ALP
- Resists denaturation (65C for 30
1. Heat Stability mins)
- ALP activity is measured before and - Inhibited by phenylalanine
after heating the serum at 56C for
10 minutes B. Nagao Isoenzyme
- Considered as a variant of regan
a. Placental ALP isoenzyme
- Most heat stable - Identical properties with regan
- Resist heat denaturation at 65C for fraction:
30 minutes ▪ Electrophoreticity
▪ Heat-stability Decreased ALP
▪ Phenylalanine-inhibition - Inherited condition of
properties hypophosphatasia
- can also be inhibited by L-leucine - Inadequate bone calcification
- detected in metastatic carcinomas of
pleural surfaces and adenocarcinoma Assay
of the pancreas and bile duct
A. Bowers and McComb method
Diagnostic Significance - Continuous-monitoring technique
✓ Evaluation of hepatobiliary and bone - Calculation of ALP activity based on
disorders the molar absorptivity of p-
nitrophenol
Hepatobiliary disorders
- Obstructive conditions/disorders Principle of the method
- Opposite of hepatocellular disorders

Bone disorders
- Involvement of the osteoblasts

Biliary Tract Obstruction

- ALP levels range from 3-10 times
the upper limit of normal
- Primarily as a result of increased
synthesis of the enzyme induced by
cholestasis ➔ P-nitrophenylphosphate
▪ Kay may pressure na sa ducts (colorless) is hydrolyzed to p-
nitrophenol (yellow)
Hepatocellular Disorders ➔ Optimum pH: 10.2 pH
- Hepatitis and cirrhosis ➔ Increases in p-nitrophenol at 405nm
- Usually less than 3 times the upper is directly proportional to ALP activity
limit of normal
B. Bessy-Lowry-Brock Method
Bone Disorders - Conventional metod
➢ Paget’s Disease (osteitis deformans) - Also uses the principle (up above)
➢ Osteomalacia
➢ Rickets Sources of Error
➢ Hyperparathyroidism
➢ Osteogenic carcinoma ✓ Hemolysis can cause slight
➢ Healing bone fractures elevations because ALP is 6 times
➢ During periods of physiologic bone more concentrated in RBC than in
growth serum
✓ Should be run ASAP
Normal Pregnancy ▪ Activity increases 3-10% on
- Increased ALP standing at room temperature
- Average approximately 1 ½ times or 4C for several hours
the upper limit of normal ✓ Diet may induce elevations to “B”
- Can be detected between weeks 16 and “O” individuals who are
and 20 secretors
- Persists until the onset of labor ▪ 25% higher following fat
- Activity returns to normal within 3-6 meal
days after labor

ALP may also be elevated in:

➢ Hypertension
➢ Preeclampsia
➢ Eclampsia
➢ Threathened abortion
ENZYMES OF CLINICAL SIGNIFICANCE - Tartrate
PART 3 o Commonly used chemical
inhibitor of prostatic ACP (to
make the test/assay more
ACID PHOSPHATASE (ACP) specific)
- E.C. 3.1.3.2 o Not entirely specific for
- Orthophosphoric Monoester prostatic ACP (because it will
Phosphohydrolase (Acid inhibit prostatic ACP)
Optimum) - ACP has two major fractions in
- Same with ALP but in acid medium serum (high concentration):
(pH 5.0) o Prostatic ACP (males both)
o RBC (female)

- How to determine if tartrate/tartaric

acid will inhibit prostatic ACP?
o Measure the total ACP
(without the inhibitor)
o Measure ACP after the
addition of tartrate inhibitor
- Hydrolysis of Phosphomonoester to o TOTAL ACP – ACP after
form Alcohol and Phosphate ion tartrate inhibition =
prostatic ACP

Tissue Sources: - Proven useful in forensic clinical

 Prostate chemistry, particularly in the
o Richest source among males investigation of rape
o Diagnostic tool for ACP in o Vaginal washings are
serum among males for the examined for seminal fluid-
diagnosis of Prostatitis and ACP activity, which can persist
Prostatic carcinoma for up to 4 days
 Bone o Presumptive evidence of rape
 Liver o Not specific
 Spleen
 Kidney - Other conditions: Increase ACP
 RBC o Bone disease
 Platelets  Associated with the
osteoclasts
Diagnostic Significance: o Paget’s Disease
- Been used as an aid in the detection  Chronic bone disorder
of prostatic carcinoma, particularly that is due to irregular
metastatic CA of the prostate breakdown and
o Prostatic Specific Antigen formation of bone tissue
(PSA) o Breast Cancer with bone
 More specific laboratory metastases
test (serological) than o Gaucher’s Disease
serum ACP  Caused by a deficiency
 May elevate in benign of the enzyme
prostatic hypertrophy glucocerebrosidase
and prostatitis  Carbohydrate metabolic
abnormality
- Thymolphthalein Monophosphate o Platelet destruction
o Substrate  Thrombocytopenia –
o Exogenous compound resulting from excessive
incorporated in the reagent platelet destruction
from Idiopathic
Thrombocytopenic
Purpura (ITP)
 RIA procedures for measurement of
- Other substrates than can be used: prostatic ACP require nonacidified
o Thymolphthalein serum samples
monophosphate o Stable for 2 days at 4 degrees
 Substrate of choice for celsius
quantitative endpoint
reactions
o Alpha-naphthyl phosphate CREATINE KINASE (CK)
 Substrate of choice for - E.C. 2.7.3.2
continuous monitoring - ATP: Creatine N-
methods phosphotransferase
- Transferase
- Associated with ATP regeneration in
contractile or transport system
o Predominant in muscle cells
- Reaction is reversible
- To make the ACP assay more
specific, it can be done through
immunochemical techniques:
o Radioimmuno Assay (RIA) - Creatine plus ATP in which one
o Counterimmunoelectrophor phosphate group from ATP is
esis transferred to creatine forming
o Immunoprecipitation Creatine phosphate (CP) and ATP
o Immunoenzymatic assay becomes ADP
(Tandem E)
 Incubation with an Tissue Sources:
antibody to prostatic  Skeletal muscle (M subunit)
ACP followed by  Heart muscle (MB subunit)
washing and incubation  Brain Tissues (B subunit)
with p-NPP  Low concentrations:
 p-NP formed is o Bladder
proportional to the o Placenta
prostatic ACP in the o GIT
sample o Thyroid
o Uterus
Sources of Error: o Kidney
 Serum should be separated from the o Lung
red cells ASAP to prevent leakage of o Prostate
RBC and platelet ACP o Spleen
o Formaldehyde/Formalin – o Liver
commonly used inhibitor to o Pancreas
inhibit RBC ACP
 Activity decreases within 1-2 hours if Isoenzymes:
the sample is left at room  CK-MM/CK-3
temperature o Skeletal muscles (highest in
o RULE OF THUMB: If enzyme normal serum)
assays, process immediately o Condition:
 Decrease activity is a result of a loss  Skeletal muscle disorder
of CO2 from the serum (increase pH)  Muscular dystrophy
 If not assayed immediately, serum  Polymyositis
should be frozen or acidified to a pH  Hypothyroidism
lower than 6.5  Malignant hyperthermia
 With acidification, APC is stable for 2  Physical activity
days at room temperature  Intramuscular injection
 Hemolysis should be avoided
because of contamination from
erythrocyte ACP
 CK-MB/CK-2 Other isoenzymes:
o Heart muscle
o Condition:  CK-Mi
 Myocardial infarction o Bound to exterior surface of
 Rise: within 4-8 the inner mitochondrial
hours after the onset membranes of muscle, brain,
 Peak: 12-24 hours and liver
 Return to normal: o Mi stands for mitochondria
within 48-72 hours
 Myocardial injury  MACRO CK
 Ischemia o Largely comprises CK-BB
 Angina complexed with Ig (IgG)
 Inflammatory heart o CK-MM + lipoproteins
disease o Molecular weight is higher
 Cardiac surgery than its usual molecular
 Duchenne-type weight
muscular dystrophy o Cannot be filtered by the
 Polymyositis glomerulus
 Malignant hyperthermia
 Reye’s syndrome Assay:
 Rocky mountain  Tanzer-Gilvarg Method
 Spotted fever o Make use of the forward
 Carbon monoxide reaction
poisoning o Continuous monitoring
method
 CK-BB/CK-1 o Catalyzes the transfer of
o Brain tissues (highest phosphate group from ATP to
concentration) creatine forming creatine
 CNS shock phosphate and ADP
o Bladder o ADP formed in the first
 Anoxic encephalopathy reaction will be utilized for
o Lung oxidation and reduction
 Cerebrovascular o ADP will react with
accident phosphoenolpyruvate and
o Prostate catalyzes by pyruvate kinase
 Seizure o Transfer phosphate group
o Uterus from phosphoenolpyruvate to
 Placental or uterine ADP forming pyruvate and ATP
trauma o Pyruvate will be utilized in the
o Colon third reaction to undergo
 Carcinoma oxidation-reduction catalyzes
o Stomach by lactate dehydrogenase
 Reye’s syndrome with the coenzyme NADH
o Thyroid
 Carbon monoxide - NADH will absorb more of the light in
poisoning the spectrophotometer at 340 nm
 Malignant hyperthermia - Since NADH is part of the substrate
 Acute and chronic renal side, the rate of disappearance of
failure NADH to form NAD+ is correlated
to the concentration/activity of
Creatine Kinase
- The faster NADH to from NAD+, will
be correlated to the activity of CK
- EXPECT A DECREASING IN
ABSORBANCE
for 1 month when the assay
is conducted using a
sulfhydryl activator
o Muscle activity prior the blood
collection affects CK activity

 Oliver-Rosalki Method
LACTATE DEHYDROGENASE (LD)
o Reverse reaction of CK
- E.C. 1.1.1.27
o Continuous monitoring
- L-Lactate: NAD+ Oxidoreductase
method
- Catalyzes the interconversion of
o MOST COMMONLY
lactic and pyruvic acids
PERFORMED METHOD
- Hydrogen-transfer enzyme that uses
o Proceeds 2-6 times faster than
the coenzyme NAD+
the forward reaction
- Least tissue specific
o Optimal pH (backward):
6.8
o Optimal pH (forward): 9.0
o Creatine phosphate plus ADP
to form creatine and ADP
o ADP plus the glucose
catalyzed by hexokinase
(HK) the transfer of one
phosphate group to form ADP - Forward: Lactate to Pyruvate
and Glucose-6-phosphate - Backward: Pyruvate to lactate
o G6P will be utilized in the third - Reversible
reaction for the oxidation-
reduction with the coenzyme Tissue Sources:
NADP+ catalyzed by Glucose- - Widely distributed in the body
6-phosphate dehydrogenase o Heart
forming 6- o Liver
phosphogluconate and o Skeletal muscle
NADH o Kidney
o Erythrocytes
- NADPH is in the product side o Lunch
- The rate of the appearance of o Smooth muscle
NADPH will be correlated to o Brain
creatine kinase enzymatic
activity Implication during assay/diagnosis:
- EXPECT INCREASING IN - If there are conditions in the body,
ABSORBANCE LD ALONE cannot be utilized to
diagnose diseases.
- Should be partnered with other
enzymes

Isoenzymes:
 LD-1 (HHHH)
o 14-26% - third highest
concentration
Sources of Error: o Heart
- Hemolyzed samples elevate CK o RBC
activity  Myocardial infarction
- Serum should be stored in dark  Hemolytic anemia
place
o Activity can be restored after  LD-2 (HHHM)
storage in the dark at 4 o 29-39% - HIGHEST
degrees Celsius for 7 days CONCENTRATION in normal
or at -20 degrees Celsius serum; widely used in
diagnosis of myocardial - Second is AST
infarction - Last is LD
 Rise: within 12-24
hours after onset
 Peak: 48-72 hours
 Remain elevated: 10
days

o Renal cortex
 Megaloblastic anemia
 Acute renal infarction
 Hemolyzed specimen

<side comment>
 LD FLIPPED PATTERN ! Non-enzyme analyte that is MORE
o LD1 has a greater SPECIFIC for AMI than CK-MB and the rest
concentration compared of the heart enzyme: TROPONIN
to LD2
o Suggestive of AMI Why? Because Troponins will rise
immediately during the onset of the
attack/chest pains
 LD-3 (HHMM)
o 20-26% - Second Highest 1. TROPONIN C
concentation 2. TROPONIN T
o Lung, Lymphocytes, Spleen, 3. TROPONIN I
Pancreas o Exhibits higher specificity
 Pulmonary embolism compared to troponin c
 Extensive pulmonary
pneumonia In clinical set-up right now, TROPONIN I
 Lymphocytosis & CK-MB are best tandem for the
 Acute pancreatitis diagnosis of Acute Myocardial
 Carcinoma Infarction

 LD-4 (HMMM) Analysis of LD:

o 8-16% - fourth highest  Electrophoresis
concentration o Isoenzymes can be detected
o Liver either by flurometry or
 Hepatic injury or colorimetry
inflammation o Substrate: alpha-
hydroxybutyrate (with
 LD-5 (MMMM) increase affinity with H
o 6-16% - lowest/least subunit than M subunit)
concentration  For LD-1 activity
o Skeletal muscle
 Skeletal muscle injury

Review: There are 3 major heart

enzymes for the diagnosis of AMI:
1. EST
2. CK-MB
3. LD-2
- Catalyzes by alpha-hydroxybutyrate
- The first isoenzyme/enzyme that
dehydrogenase
will rise during the onset of chest
pain is CREATINE KINASE (CK-MB
is the highest among the creatine
kinase)
Assay: - Regulation of tissue glutathione
levels
 Conversion of Lactate to - Transport of amino acids across cell
Pyruvate membranes
o Most commonly utilized assay
o Rate of the reverse reaction Tissue sources:
(pyruvate to lactate) is  Kidney
approximately 3 times  Brain
faster but more susceptible to  Prostate
substrate exhaustion and loss  Pancreas
of linearity  Liver
o Optimal pH (forward): 8.3 – o Highest concentration
8.9 o Clinical applications are
o Optimal pH (Backward): 7.1 – confined mainly to evaluation
7.4 of liver and biliary system
o Lactate + NAD+ <-> disorders
pyruvate + NADH + H+
Diagnostic Significance
Sources of Error: - Located in the canaliculi of the
- RBC contains LD concentration 100- hepatic cells and particularly in the
150 times than found in serum (LD epithelial cells lining the biliary
is very susceptible to hemolysis) ductules
- LD activity is unstable in serum o Elevated in all hepatobiliary
regardless of the temperature disorders
o Should be stored at room  Best diagnostic tool for
temperature and analyzed Biliary tract
within 48 hours obstruction
o LD-5 is the most labile (loss - Common liver enzymes include:
of activity more quickly at 4 o Alkaline phosphatase (ALP)
degrees Celsius at room temp)  For obstructive liver
o LD isoenzyme analysis should diseases
be stored at room  Produced in the
temperature and analyzed sinusoids
within 24 hours of collection  Hepatocellular
involvement: HIGH (if
bone ALP is normal)
GAMMA-GLUTAMYLTRANSFERASE o Alanine transaminase
(GGT) (ALT)
- E.C. 2.3.2.2  Highest specificity with
- (5-Glutamyl) Peptide: Amino regards to liver however
Acid-5-Glutamyl-Transferase it is confined to
- Involved in the transfer of the hepatocellular damage
gamma-glutamyl residue from o Aspartate transaminase
gamma-glutamyl peptides to amino (AST)
acids, H20, and other small peptides o Gamma-glutamyl
- Glutathione serve as the glutamyl transferase (GGT).
donor  For obstructive liver
- Substrate: Glutathione & amino diseases
acid  Hepatocellular
- Glutathione + amino acid > involvement: NORMAL
glutamyl – peptide + L-
cysteinylglycine - Within the hepatic parenchyma, GGT
exists to a large extent in the
Physiology: smooth ER, subject to hepatic
- Has not been clearly established microsomal induction
- Involved in peptide and protein o GGT is increased in patients
synthesis receiving enzyme-inducing
drugs (warfarin, Tissue Sources:
phenobarbital, phenytoin)  Adrenal Cortex
 Spleen
- Used in diagnosis of Chronic  Thymus
Alcoholism  Lymph nodes
- Unknown etiology  Lactating mammary gland
o Acute pancreatitis  Erythrocytes
o DM and MI o Most commonly utilized
diagnostic tool
Assay:
 Szasz Assay Diagnostic significance
 RBC
o Maintain NADPH in reduced
form
 Required to regenerate sulfhydryl-
containing proteins (e.g. glutathione)
from the oxidized to the reduced
state
o Glutathione in the reduced
form protects Hb from
oxidation
 G-6-PD deficiency
o Inherited sex-linked trait
- Substrate: Gamma-Glutamyl-p- o Can cause hemolytic anemia
nitroanilide to form p-Nitroaniline
- Para-nitroaniline is measured in
the spectrophotometry because it Assay:
can absorb light at specific
wavelength at pH 8.2 and correlated
to the activity of GGT
- Continuous monitoring assay =
INCREASING ABSORBANCE

Sources of Error:
- Forward reaction
- GGT activity is stable, with no
loss activity for 1 week at 4
degrees Celsius
ANGIOTENSIN-CONVERTING ENZYME
- Hemolysis does not interfere with
(ACE)
GGT level
- E.C. 3.4.15.1
- Angiotensin I-Converting
Enzyme
GLUCOSE-6-PHOSPHATE
- Kininase II
DEHYDROGENASE (G-6-PD)
- Peptidyl-dipeptidase A
- E.C. 1.1.1.49
- Reaction: Hydrolysis of peptide
- D-Glucose-6-Phosphate: NADP+
bonds at a free C-terminus
1-Oxidoreductase
- Releasing a dipeptide in the
- Catalyzes the oxidation of
reaction
glucose-6-phosphate to 6-
- Act as endopeptidase or an
phosphogluconate or the
aminopeptidase
corresponding lactone
- Conversion of angiotensin I to
- Reaction is important in the pentose-
angiotensin II (RAAS)
phosphate shunt of glucose
- Inactivation of BRADYKININ
metabolism with the ultimate
(Kallikrein-Kinin System)
production of NADPH
- Modulates peripheral vascular
- Substrate: Glucose-6-phosphate
resistance as well as renal and
- Coenzyme: NADP+
cardiovascular function
- Renal and cardiovascular function
- Zinc-metalloprotease Causes of abnormal results:
o Requires Zinc (Activator) - The most common reason for
ordering ACE levels in in diagnosis
and monitoring of Sarcoidosis
- SARCOIDOSIS
o Commonly referred to simply
as “sarcoid”, areas of
inflammation called
“granulomas” may appear on
the body
o Any part of the body can be
affected
o most commonly affected
areas:
 lungs
 skin
- LIVER is producing the  eyes
angiotensinogen  lymph nodes
- Angiotensinogen is converted to
angiotensin I
- LUNGS will produces the CHOLINESTERASE (ChE)
Angiotensin converting enzyme
(ACE) which catalyzes the 2 types:
conversion of Angiotensin I and - E.C. 3.1.1.7
Angiotensin II - Acetylcholine Acylhydrolase
- Which now stimulate the adrenal - Commonly called as:
gland to produce Aldosterone Acetylcholinesterase
(Zona glomerulosa) - “TRUE” AChE
- Aldosterone target tissue: - Why true? Because this is the type of
o Kidney cholinesterase that hydrolyzes the
 Sodium and water hydrolysis of acetylcholine to form
retention (Increased acetic acid and choline
BP) o Found in the synapse of the
o Heart nerve and RBC
 Increased heart beat o Not normally found in amniotic
and BP fluid
o Arteries o Hydrolyzes acetyl-beta-
 Constriction of blood methylcholine
vessels to increase BP

- E.C. 3.1.1.8
Tissue Location: - Cholinesterase
 Tissue-bound, with much lower - Pseudo-acetylcholinesterase
levels circulating in plasma (PCHE)
 Predominantly found in endothelial o Found in the serum
cell membranes throughout the o Production occurs primarily
body in the liver
 Lungs & Testes – rich in ACE o For cleavage of
succinylcholine and
mivacurium
Measurement: o Hydrolyzes butyryl- and
- Measured by its ability to cleave benzoylcholine
synthetic peptides, releasing
hippuric acid (measured to be Measurement:
correlated to the concentration or - Ellman’s Method
activity of ACE) - Substrate: Acylthiocholine ester
- After hydrolysis it will release 5’-NUCLEOTIDASE (5’N)
thiocholine and react with - E.C. 3.1.3.5
dithiobisnitrobenzoic acid - 5’-Ribonucleotide
- FINAL PRODUCT (YELLOW COLOR): Phosphohydrolase
o 5-thio-2-nitro benzoic acid
(measured correlated to the
activity of cholinesterase)

 AChE
o Useful in organophosphate
exposure and poisoning
o Qualitative analysis in
amniotic fluid may be useful
in the diagnosis of neural
tube defects - Hydrolysis of 5’-ribonucleotide to
form ribonucleoside and phosphate
 PseudoChE - Important in metabolism of nucleic
o Serum acids
o To monitor exposure to - A cytoplasmic membrane-bound
cholinesterase inhibitors phosphatase
(most common) - Acts only on nucelotides
- Function in extracellular adenosine
 Organophosphate
Insecticides production, nutrient absorption, and
 Irreversible cell proliferation
inhibitors of both - A metalloenzyme (zinc)
AChE and PChE - Widely distributed in the body,
 PChE activity predominantly attached to cell
(serum) falls membranes (similarly to ALP and
before AChE GGT)
activity (RBC) - Predominantly derived from the
 Reflects liver synthetic LIVER
function rather than
hepatocyte injury Measurement:
 Decreased: Acute - Made difficult because other
hepatitis: Cirrhosis, and phosphatases are capable of cleaving
CA metastatic to liver the substrate
- Uses ALP inhibitors
o As a liver function - Chelating agents inhibit activity
o For diagnosis of genetic
variants Clinical Significance
- Commonly used to determine if the
 True ChE source of an elevated ALP is from
o Hemolysate of washed RBC liver or bone
- Cholestatic disorders
- Ovarian CA, rheumatoid arthritis
CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Water

• There should be a balance in water content and

solute between the ECF and ICF via Osmosis for
water and diffusion for solute in general passive
transport and active transport

• Sodium ion is the major cation in the ECF

• Potassium is the main cation for ICF
• Chloride is the major anion in ECF
• Any changes of concentration have an
implication in the status of the fluid distribution

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Electrolytes Functions: (Introduction page 356-bishop)

• Sodium • Volume and osmotic regulation (Na, Cl, K)

• Potassium • Myocardial rhythm and contractility (K, Mg, Ca)
• Calcium • Cofactors in enzyme activation (Mg, Ca, Zn)
• Chloride • Regulation of ATPase ion pumps (Mg)
• Magnesium • Acid-base balance (HCO3, K, Cl)
• Lactate • Blood coagulation (Ca, Mg)
• Phosphate • Neuromuscular excitability (K, Ca, Mg)
• Bicarbonate • Production and use of ATP from Glucose (Mg,
PO4)
• Routinely requested by physician to monitor
cases like myocardial infraction

Sodium

• Monovalent cation
• Most abundant cation in the ECF
o Accounts 90% of all the ECF cations
o Large determinant of plasma osmolality

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Sodium ion

• Normally, 60-75% of filtered Na+ is reabsorbed

in the PCT
• Sites of reabsorption
o Loop of Henle
o DCT
o Connecting segment and cortical
collecting tubule to
▪ Exchange for K (ANP)

Regulation

a) Intake o water in response to thirst

b) Excretion of water (affected by ADH in response
to changes in either blood volume or osmolality
Aldosterone is also involved)
c) Blood volume status (affects sodium excretion
through aldosterone, angiotensin II, and atrial
natriuretic peptide)
d) Primary active transport Clinical Significance
a. Na-K ATPase pump
b. NA-K leak channels • Hyponatremia
o <136 mEq/L
o One of the most common electrolyte
disorders
o Probable cause
▪ Increase Sodium loss
▪ Increase water retention
▪ Water imbalance
• Hypernatremia
o >142 or >145 mEq/L
o Less common
o Probable cause:
▪ Excess water loss
▪ Decrease water intake
▪ Increase sodium intake or
retention

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Hyponatremia Determination of Sodium

• Increase water retention 1. Chemical methods

o Renal failure 2. FES (flame emission spectrophotometry)
o Nephrotic syndrome 3. AAS (
o Hepatic cirrhosis 4. Ion Selective Electrode (glass ion exchange
o CHF membrane) most sensitive most used
• Increase Sodium loss • Reference ranges
o Hyperthyroidism o Serum: 136 – 145 mmol/L
o K+ deficiency o Urine (24-hour) 40-220 mmol/day,
o Diuretic use varies with diet
o Salt-losing nephropathy o CSF: 136-150 mmol/L
o Prolonged vomiting
Chemical Method
o Diarrhea
o Severe burns Albanese Lein Method (oldest)
• Water imbalance
• Principle:
o Excess water intake
o Sodium is made to react with zinc
o SIADH (syndrome of inappropriate ADH)
uranyl acetate to produce a sodium
o Pseudohyponatremia
uranyl acetate precipitate after the
addition of polyvinyl alcohol
o With the addition of water, a yellow
Hypernatremia
solution is formed which is then
• Increase water loss measured spectrophotometrically
o Profuse sweating o Protein precipitant = Trichloroacetic
o Diarrhea acid
o Severe burns
Magnesium-Uranyl Method
o DI ()
o Renal Tubular disorder • Principle
• Increase sodium intake/retention o Sodium is precipitated with magnesium-
o Hyperaldosteronism uranyl acetate; the uranyl ions
o Excess Sodium bicarbonate remaining in suspension from yellow-
o Excess hypertonic dialysis fluid brown complex with thioglycolic
▪ Neonates o The difference between reagent blank
• Decrease water intake and analysis is proportional to the
o Geriatric patients sodium concentration
o Infants o Protein precipitant = Uranyl acetate and
o Mental impairment magnesium acetate
▪ 1L water/day-insensible H2O
loss
4

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Functions

• Regulation of neuromuscular excitability

• Contraction of the Heart
• ICF volume
• Hydrogen Ion concentration
o Low serum K
o Sodium and H ions move into the cell

3 factors that influence the distribution of potassium

between cells and ECF

• The electrode containing a specific electrode for • Potassium loss frequently occurs whenever the
specific ion Na-K ATPase pump is inhibited by conditions
• These electrodes will only attract specific ion (hypoxia)
• Highly specific to an ion • Insulin promotes acute entry of K ions into
skeletal muscle and liver by increasing Na-K
ATPase activity
Potassium • Catecholamines (beta-stimulator) promote
cellular entry of K, where areas propranolol
• Opposite of sodium ion in distribution (beta-blocker) impairs cellular entry of K
• Major Intracellular cation in the body
• Concentration 20 times greater inside the cells
than outside Exercise
• “House” Within the Cell
• K is released from cells during exercise
Regulation • Increases K by 0.3-1.2 mmol/L
• Primary active transport • Reversed after several minutes of rest
o Na-K ATPase pump • Forearm exercise during venipuncture can
o Na-K leak channels cause erroneous high plasma K concentrations
• PCT reabsorbed nearly all the potassium Cellular Breakdown
• Secreted into the urine in exchange for sodium
in both the SCT and the collecting duct • Releases K into the ECF
• Under the influence of aldosterone • Severe trauma, tumor lysis syndrome, and
massive blood transfusions

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Clinical Significance

Hypokalemia • Artifactual
o Sample hemolysis
• GI Loss
o Thrombocytosis
o Vomiting, Diarrhea, Gastric Suction,
o Prolonged tourniquet
Intestinal Tumor,
application/excessive fist clenching
o Malabsorption, Cancer Therapy, Large
doses of laxatives
• Renal Loss
o Diuretics
o Nephritis
o Renal Tubular Acidosis
o Hyperaldosteronism
o Cushing Syndrome
o Hypomagnesemia
• Cellular Shift
o Alkalosis
o Insulin dose
• Decreased Intake
o K-rich food: banana, beans, fish,
peanut, potatoes, tomatoes, meats,
milk Collection of specimens & Specimen handling

• Increase Platelet count = increase K+

o Use heparin
Hyperkalemia • Prolonged tourniquet application or fist
• Decreases renal excretion clenching = increases K+ shift
o Acute/Chronic RF o Proper patient care
o Hypoaldosteronism • In vitro hemolysis
o Addison’s disease • Whole blood samples should be stored at room
o Diuretics temperature and analyzed ASAP
• Cellular Shift
o Acidosis
o Muscle/cellular injury
o Chemotherapy
o Leukemia
o Hemolysis
• Increased intake
o Oral or IV potassium replacement
therapy (Potassium Chloride)

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Colorimetric methods Electrical Neutrality

• Lockhead and Purcell Method • Act s rate-limiting component

o Potassium is reacted with sodium o Limits the amount of Na+ reabsorbed in
cobaltinitrite to produce sodium the kidney by the amount of Cl-
potassium cobaltinitrite available
o With the addition of phenol (color • Chloride shift
developer), a blue color is produced and o CL- diffuses into the red blood cell in
determined spectrophotometrically exchange of bicarbonate ions and
sodium ions
ISE (Ion selective electrode)

• Uses valinomycin membrane and KCl as inner

electrolyte solution

Chloride

• Major extracellular anion

• Functions
o Involved in maintaining osmolality,
blood volume, and electric neutrality
o Chloride shifts secondarily to a
movement of sodium or bicarbonate

Physiology and regulation

• Main source: DIET to GI absorption

• Kidney filtration and reabsorption in PCT
• Excess chloride is excreted in the urine and
sweat
o Excessive sweating stimulate
aldosterone secretion→ sweat glands
(conserve sodium and chloride)
Note: baliktad na ang hyper sa left then hypo sa right

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

▪ Free thiocyanate reacts with

ferric ions present in ferric
Determination of Chloride
nitrate to form a reddish-brown
• Specimen: Serum or plasma complex of ferric thiocyanate
• Anticoagulant of choice: lithium heparin ▪ Reference ranges
o Hemolysis does not cause a significant • Plasma, serum: 98 –
change in serum or plasma values 107 mmol/L
o However, with marked hemolysis, levels • 24-hour urine 110-250
may be decreased as a result of mmol/24-hour
dilutional effect
• Methods
o ISE (ion-exchange membrane to
selectively bind Cl-)
Calcium
o Amperometric-coulometric titration
▪ Silver ions + chloride = AgCl2 Regulation
▪ Excess Ag → endpt. Of titration
o Mercurimetric titration (not used) • 3 hormones which regulate calcium secretion
▪ Schales and Schales Method o Parathyroid hormone
▪ PFF preparation using tungstic ▪ Increase calcium ion in the
acid as precipitating agent of blood by bone resorption,
protein increases calcium ion retention
▪ PFF is titrated with standard in the kidney, increase calcium
solution of mercuric ions absorption in intestine and
(mercuric nitrate) to form a activate vitamin D so that it will
soluble compound of mercuric stimulate calcium ion
chloride which does not reabsorption and bone
dissociate to mercuric ions resorption
▪ Excess mercuric ions combine o Vitamin D
with s-diphenyl carbazone to o Calcitonin
form a blue-violet colored ▪ Opposite action
complex ▪ Inhibit PTH an inactivate
o Colorimetry vitamin D
▪ Mercuric Thiocyanate Distribution
(Whitehorn titration) method
▪ Specimen is mixed with a • 99% → bone
solution of mercuric • 1% → circulation (blood) + ECF
thiocyanate • ECF
▪ Mercuric chloride – final o 15% → bound to anions
product o 40% → bound to protein (albumin)
o 45% → free/ionized calcium ion

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

▪ 50-60% is reabsorbed in
ascending loop of Henle
Clinical Significance
▪ 2-5% is reabsorbed in DCT
o Renal threshold: 0.60-0.85 mmol/L
o Only about 6% of filtered Mg is excreted
in the urine per day

Regulation

• PTH
o Increases the renal reabsorption and
intestinal absorption
• Aldosterone and Thyroxine
o Increases the renal excretion of
magnesium
Magnesium

• Fourth most abundant cation in the body and

second most abundant intracellular ion Specimens
o 53% - bone • Non-hemolyzed serum
o 46% - Muscle and other organs and soft • Lithium Heparin Plasma (recommended)
tissue o Oxalate, EDTA, and citrate will bind with
o Less than 1% - serum and erythrocytes magnesium
▪ Protein-bound (primarily
• 24-hour urine
albumin
o Preferred for analysis because of
▪ Free or ionized form
diurnal variation in excretion
▪ In complexed with other ions
o Must be acidified with HCl to avoid
Regulation precipitation

• Richest source
o Raw nuts, dry cereal, and “hard”
drinking water
o Vegetables
• Small intestine may absorb 20-65% of the
dietary magnesium
• Controlled largely by the kidney
o Nonprotein-bound are filtered by the
glomerulus
▪ 25-30% is reabsorbed by the
PCT

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Colorimetric Methods In alkalosis

• Calmagite Method o With relative increase in bicarbonate

o Mg binds with calmagite to form a ion compared to carbon dioxide
reddish-violet complex that may be • Kidneys increase excretion of bicarbonate into
read at 532 nm the urine, carrying along a cation (Na+)
• Formazen Dye Method • Loss of this ion from the body helps correct pH
o Mg binds with the dye to form colored
complex that may be read at 660 nm
• Methylthymol Blue Method In acidosis
o Mg binds with the chromogen to form a
colored complex • With a relative increase carbo dioxide
• Atomic Absorption Spectrophotometry (most • Kidneys increase excretion of hydrogen ion into
preferred) the urine
o Reference method • Bicarbonate ion reabsorption is virtually
complete (90%) in the PCT and the remainder in
the DCT
Bicarbonate

• Second most abundant anion in the ECF Determination of Carbon Dioxide

• Total CO2 = HCO3- + H2CO3 + dCO2
o More than 90% HCO3- at pH 7.35 – 7.45 • Specimen
o Thus, total CO2 measurement is o Venous serum or heparinized plasma
indicative of HCO3- measurement o Anaerobic collection
▪ If the sample is left uncapped
• Major buffer system in the blood
before analysis, CO2 escapes
• Carbonic anhydrase
(decrease by 6mmol/L per hour)
• Hydrogen ion can lead to acidosis
• Increase CO2 led to acidosis Methods

• ISE
o Uses an acid to convert all the forms of
CO2 to CO2 gas and is measured by
pCO2 electrode

10

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Phosphate

• Found everywhere in living cells • Vitamin D acts to increase phosphate in the

• Participate in many of the most important blood
biochemical processes o Increases both phosphate absorption in
the intestine and phosphate
reabsorption in the kidney
• GH
o Increase secretion or administration,
phosphate concentrations in the blood
may increase because of decreased
renal excretion of phosphate

Distribution

• Total phosphorus
o About 12 mg/dL (3.9 mmol/L)
o 3-4 mg/dL inorganic phosphate
• Predominant Intracellular anion
• Reservoirs of biochemical energy o 80% is in the bone
o ATP, creatine phosphate, and PEP o 20% in soft tissues
• 2,3-DPG in red blood cells (lowers affinity of o Less than 1% is active in serum/plasma
oxygen)

Regulation
Determination of Inorganic phosphate
• Phosphate may be absorbed in the intestine
from dietary sources • Specimen
• Released from cells into the blood and lost from o Serum (best choice)
bone o Lithium heparin plasma
o Oxalate, citrate, or EDTA can interfere
• Hormones/ conditions affecting PO4 levels:
with the analysis
(almost the same with calcium)
o Hemolysis should be avoided
o Vitamin D
o Circulating phosphate levels are subject
o Calcitonin
to circadian rhythm
o GH, and acid-base status
o Highest in late morning and lowest in
o PTH – which overall lowers blood
the evening
concentration by increasing renal
o 24-hour urine
excretion

11

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Method

• Involve the formation of an ammonium

phosphomolybdate complex
• Measured by UV at 340nm
• Or can be reduced to molybdenum blue, which
is read between 600 and 700nm

Lactate
• Is a by-product of an emergency mechanism
that produces a small amount of ATP when
oxygen delivery is severely diminished
• Pyruvate is the normal end product of glucose
metabolism (glycolysis)
• Conversion to lactate is activated when a
deficiency of oxygen leads to an accumulation
of excess NADH

Regulation

• Lactate is a by-product of anaerobic

metabolism, it is not specifically regulated
• As oxygen delivery decreases below a critical
level, blood lactate concentrations rise rapidly
and indicate tissue hypoxia earlier than pH
• Liver is the major for removing lactate by
converting lactate back to glucose by
gluconeogenesis

12

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ELECTROLYTES

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Clinical Application • Enzymatic method

o Uses lactate oxidase to produce
• For metabolic monitoring in critically ill patients,
pyruvate and hydrogen peroxide
for indicating the severity of illness, and for
o Addition of chromogen and catalyzed
objectively determining patient prognosis
by peroxidase to form a colored
complex

Type A lactic Acidosis

• Associated with hypoxia conditions, such as

shock, MI, severe CHF, pulmonary edema, or
Anion Gap
severe blood loss
• Concentration difference between commonly
measured cations (Na + K) and commonly
Type B lactic acidosis measured anions (Cl + HCO3)
• Cations are mostly measured
• Metabolic Origin
• Diabetes mellitus
• Severe infection
• Leukemia
• Liver or renal disease
• Toxins (ethanol, methanol, or salicylate
poisoning)

• Useful in indicating an increase in one or more

Determination of lactate
of the unmeasured anions in the serum
• Specimen handling • As a form of quality control for the analyzer
o Tourniquet should not be used used to measure electrolytes
o After sample collection, anaerobic o Consistently abnormal AG serum from
glycolysis will occur healthy persons may indicate an
o Heparinized plasma but must be instrument problem
delivered on ice and the plasma must
be separated quickly
o Fluoride inhibits glycolysis but the
specific method directions must be
consulted

13

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

The Endocrine System

• Hypothalamus and pituitary gland

• Thyroid gland
• Parathyroid glands
• Pancreatic islets
• Adrenal glands
• Testes
• Ovaries
Hormone Classifications and Actions

Introduction • Target Cells that have the specific protein

receptors to bind a given hormone
• The endocrine glands include the pituitary,
thyroid, parathyroid, adrenal, and pineal glands
• Functions of Hormones
• Types of Hormones (based on chemical
o Help regulate- Chemical composition
composition)
and volume of internal environment;
• Peptide Hormones – Proteins (at least 3 amino
metabolism and energy balance;
acids)
contraction of smooth and cardiac
• Steroid Hormones – derived from cholesterol
muscle fibers; Glandular secretions;
• Amine Hormones – derived from tyrosine amino
immune activities
acid
o Control growth and development
o Regulate operation of reproductive
systems
o Help establish circadian rhythms Peptide Hormones

• Water soluble
• Most abundant
• No need for transport protein
• Stored in vesicles
• Bind to cell surface receptors in the target cells
• Action: activation of messenger enzymes

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Peptide Hormones (classification and action) Amine Hormones (classification and actions)

• Hypothalamus • Catecholamines (adrenal medulla)

o Releasing (stimulate production and o Epinephrine and Norepinephrine
release of hormone in anterior pituitary ▪ Behave like protein hormones
gland) and inhibiting Hormones • Thyroid hormones (thyroid gland)
(opposite) o T3 and T4
• Pituitary gland ▪ Behave like steroid hormones
o Anterior lobe (tropic cells produce and
release tropic hormones)
o Posterior lobe (temporary storage area CONCEPTS TO REMEMBER!!
of the two major hormone from
hypothalamus) • Cell membrane are Bilipid-Layer (Hydrophobic)
• Parathyroid gland • Blood is more than 90% water
o Located posterior in thyroid glands o Any water-soluble substance cannot
• Pancreas enter but a hydrophobic substance can
o Islets of Langerhans easily enter a cell
o Hydrophobic substances require
• Pineal Gland
transport proteins
o Example: Glucose (water soluble)
cannot easily enter the cells. It requires
Steroid Hormones (classification and Action)
channel for it to enter. On the other
• Water- insoluble (lipid-soluble) hand, cholesterol (hydrophobic) can
• Require transport protein easily diffuse into the cell
• Bind to intracellular receptors in the target cells
• Action: protein synthesis

Steroid Hormones (classification and action)

• Adrenal cortex
• Testis
• Ovaries

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Peptide Hormone Feeback Mechanisms Regulating Hormone release

• Homeostatic – negative feedback

o The final hormone produced regulates
its own secretion by inhibiting the
secretion of one or more of the
precursor hormones (prohormones-
inactivated HORMONES)

• Metabolic processes

Feedback loops
Steroid Hormone
• Rule: Hormones elicit their own shut off
mechanism

• Production of new protein

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Disorders of the Endocrine system

Hypersecretion and Hyposecretion (two defects)

• Primary
o Dysfunction may be caused by a defect
in the target gland itself
• Secondary
o Dysfunction may be caused by a defect
in the pituitary gland, which releases
hormone to stimulate the target gland
to secrete its hormone
• Tertiary
o Dysfunction may be caused by a defect
in the hypothalamus, which produces
releasing factors, which in turn
stimulate the pituitary to secrete its
hormone

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Hypothalamus and Pituitary gland Structure of the Pituitary gland

• Hypothalamus has two communication Two distinct lobes

pathways the posterior pituitary gland via
• Anterior pituitary
neurons and then the anterior pituitary gland
o Adenohypophysis; pas distalis
via blood circulation
▪ True endocrine tissue
• Anterior pituitary gland has five endocrine cells
▪ Secretes classic hormones
• Posterior pituitary
o Neurohypophysis; pars nervosa
▪ Neural tissue
▪ Secretes neurohormones

Hormones of the anterior Pituitary

5 Hormone-synthesizing-secreting Cells:

1. Somatotrophs (a) – GH
2. Lactotrophs (a) – PRL
3. Thyrotrophs (b) – TSH
4. Gonadotrophs (b) FSH and LH
5. Corticotrophs (b) – POMC to ACTH

Posterior Pituitary Gland

• Hormones made in the hypothalamus and

Hypothalamus: Releasing & Inhibiting Hormones released and stored in the posterior pituitary
• Thyrotropin releasing hormone (TRH) • Oxytocin
• Corticotrophin releasing Hormone (CRH) o Secretion is stimulated by uterine
• Gonadotropin releasing Hormone (GnRH) stretching and by suckling of nipple
during nursing
• Growth hormone releasing hormone (GHRH)
o Stimulates milk ejection
• Growth hormone inhibiting hormone (GHIH)
• ADH/AVP
• Prolactin releasing Hormone (PRH)
o Secretion is controlled by the osmotic
• Prolactin inhibiting hormone (PIH; dopamine)
pressure of the blood and blood volume
o Stimulates reabsorption of water in the
collecting duct
o Increases blood pressure (constriction
of blood vessels)
5

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Thyroid Gland • Follicular cells

o Thyroxine (T4) and Triiodothyronine
• Butterfly-shaped and is located just below the
(T3)
larynx (voice box)
• Parafollicular cells
o Calcitonin

• Has two lobes right and left lobe

• Consist of functional unit called thyroid follicles
which consist a simple cuboidal cell which is
known as the follicular cells which is responsible
of the release of T3 and T4

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

• Thyroid Hormone (T3 and T4)

o Regulate oxygen use and metabolic
rate, cellular metabolism, and growth
and development
• Calcitonin
o Can lower the blood level of calcium
o Its secretion is controlled by the level of
calcium in the blood

Parathyroid Glands

• The Parathyroid glands are embedded on the

posterior surfaces of the thyroid
• Parathyroid Hormone (PTH)
o Produced by chief cells
o Regulates the homeostasis of calcium,
magnesium, and phosphate by
increasing blood calcium and
magnesium levels and decreasing blood
phosphate level
o Is controlled by the level of calcium in
the blood Pancreatic Islets (islets of Langerhans)
o PTH and calcitonin are the primary
• The pancreas is a flattened organ located in the
hormones for calcium regulation in the
curve of the duodenum.
blood
• It has both endocrine and exocrine functions
• The endocrine portion consists of pancreatic
islets
o Alpha cells
▪ Secretes glucagon
o Beta cells
▪ Secretes insulin
o Delta cells
▪ Secretes somatostatin
o

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY

CLINICAL CHEMISTRY 2 MTPC12
LESSON : ENDOCRINE SYSTEM

Prof. Geromil J. Lara, RMT, MSMT

Prelim: 2nd semester

Adrenal Glands (supernal gland)

two adrenal glands

• Outer cortex
o Zona glomerulosa (outer)
▪ Secretes mineralocorticoids,
mainly aldosterone (sodium
reabsorption in kidneys)
o Zona fasciculata (middle)
▪ Secretes glucocorticoids, mainly
cortisol (increases glucose level)
o Zona reticularis (inner)
▪ Secretes androgen
▪ DHEA and DHEAS
▪ Secondary sex characteristics
• Adrenal medulla (innermost layer)
o Chromaffin cells secrete epinephrine
and norepinephrine (NE)
catecholamines
o Neurotransmitters
o Glucose metabolism

PEREZ, A.J.L BSMT 3-1 LPU-D MEDICAL TECHNOLOGY