ENZYMOLOGY Metalloenzyme- enzyme whose metal ions are

intrinsically part of the molecule such as catalases and

cytochrome oxidase
Proenzyme- inactive precursor of enzymes, also
ENZYMES
referred to as zymogens
-Biological proteins that catalyze biochemical reactions
Substrate- substances acted upon by the enzyme which
without altering the equilibrium point
are specific for each of their particular enzyme
Nomenclature:
Cofactors- these are non-protein substance/compounds
-name of the substrate with the addition of the suffix
needed by an enzyme before enzymatic activity can be
‘ase’
manifested. Cofactors are thermostable and dialyzable
Example:
Transferase- transfer of amino group from other COFACTORS
substrate to another
Kinase- transfer to phosphate group from a high - organic molecule; it hastens enzymatic reaction that
energy phosphate compound to its substrate undergoes a change or is consumed to another product
Phosphatase- effect if hydrolysis in phosphate esters - metal ion; serve as a bridge to hold the substrate
Dehydrogenase- removal of hydrogen atoms from its and enzyme together the primary catalytic center
substrate stabilizing agent in the conformation for catalytic activity
(Sysytemic name) ENZYME KINETICS
-According to the numerical designation given by the - An enzyme catalyzes a reaction by combining with
Enzyme Commission (E.C) its substrate to create an enzyme-substrate
-first number defines the class to which the enzyme complex
belongs, while the next two numbers indicate subclass - Michaelis Menten Method: gives the means to
to which the enzyme is assigned. The last number is a determine total enzyme concentration in serum and
specific serial number to each enzyme other body fluids; accurately describes virtually all
single substrate enzyme catalyzed reactions and
GENERAL CLASSIFICATION OF ENZYMES many bisubstrate
 Oxidoreductase- removal or addition of electrons TYPES OF SPECIFITY
Example: LDH, MDH, ICD (Isocitrate Absolute Specificity- enzymes combine with only one
dehydrogenase)
substrate and catalyzes only one corresponding reaction
 Transferase- catalyze the transfer of a chemical Group Specificity- enzymes combining with all
group from one substrate to another substrates containing a particular chemical group
Example: AST, ALT, CK, CPK, GGI, OCT Bond Specificity- enzymes are specific to chemical
 Hydrolase- hydrolyze the splitting of a bond by the bonds
addition of water Stereoisomeric Specificity- enzymes that
Example: ACP, ALP, CLS, LPS, PTS, PPS, LAP predominantly combine with only one optical isomer of a
 Lyases- remove groups from substrate without certain compound
hydrolysis, leaving only double bonds in the ENZYME SPECIFICITY
molecular structure of the product  Emil Fisher’s LOCK AND KEY THEORY- based on
Example: Glutamate decarboxylase, Pyruvate
the rigid enzyme molecule into which the substrate
decarboxylate, Tryptophan decarboxylase fits
 Isomerase- catalyze the intramolecular  Koshland’s INDUCED FIT THEORY- based on the
rearrangement of the substrate compound
attachment of a substrate to the active site of an
Example: Glucose phosphate isomerase, Ribose
enzyme, which then causes conformational
phosphate isomerase
changes in the enzyme
 Ligases- joins two substrate molecules together
TYPES OF REACTION ORDER
using energy released from hydrolyzing a
1. Zero Order Reaction- is the rate of reaction
pyrophosphate bind to a high energy phosphate
linear with time, independent of concentration of
compound
substrate and directly proportional to enzyme
concentration
TERMINOLOGIES:
2. First Order Reaction- the rate of reaction is
Holoenzyme- an active substance formed by
determined by the concentration of substrate as
combination
well as of enzyme
Apoenzyme- protein portion subject to denaturation, in
which the enzyme loses its activity. Catalytically FACTORS AFFECTING ENZYME REACTIONS:
inactive protein when cofactor is removed. They are  Enzyme Concentration- if the amount of enzyme
heat labile and dialyzable. is doubled, the reaction proceeds twice as fast
Isoenzyme- enzymes present in an individual with  Substrate Concentration- increase in the
similar enzymatic activity but differ in their physical concentration of substrate produces also an
biochemical and immunologic characteristics. increase in the rate of reaction, provided all other
conditions are kept constant
  Temperature- rate of any chemical reaction is QUALITY CONTROL PROGRAM FOR
usually increased 2-3 times for every 10C in ENZYME ASSAY
temperature - strict adherence to zero order kinetics
 Hydrogen Ion Concentration or pH- enzymatic - proportionality with increments to sample
reactions proceed at their fastest rate at an optimum - use of pooled frozen serum or stable reference
pH and are considerably slowed or even stopped at materials as controls
higher or lower pH value - replicate measurements to evaluate precision
ENZYME INHIBITION to assays
 Competitive Inhibitor- substances that compete SPECIFIC ENZYMES
with the substrate for enzyme binding because they
are chemically analogous to the substrate and bind PHOSPHATASES
to the active sites of enzyme
 Non competitive Inhibitor- substances that do not - Characterized by its ability to hydrolyze a large
resemble the substrate and bind to the enzyme in variety of organic phosphate esters with the
areas other than the active site formation of an alcohol and a phosphate ion
 Uncompetitive Inhibition- inhibits enzyme by
binding to the enzyme substrate complex ALKALINE PHOSPHATASES (EC3.1.3.1)
ENZYME INDUCTION - Alkaline Orthophosphoric Monoester
- This phenomenon states that a certain enzyme Phosphohydrolase
has the ability to adapt to their biochemical - An enzyme involved in the cleavage of phosphate
systems containing compounds in alkaline pH. It facilitates
movement of substances across cell membranes
TYPES OF ENZYME ASSAYS
Reference value: 30-90U/L
Endpoint Analysis
- reaction is initiated by addition of substrate
- reaction is allowed to proceed for a period of
time
- measurement is done at the end of reaction
Disadvantage: underestimation of the “true”
enzyme activity and linearity of reaction cannot
be observed
Multi-point and Kinetic Assay
-change in concentration of the indicator
substance at several intervals
-continuous measurement of change in CARCINO-PLACENTAL ALP
concentration as function of time  Regan ALP- lung, breast, ovarian, and
Use of Coupled Reactions gynecological cancers; bone ALP co-migrator;
-enzymatic activity is measured by coupling the most heat stable, inhibited by phenylalanine
activity with colorimetric reaction reagent
UNITS FOR EXPRESSING ENZYME ACTIVITY  Nagao ALP- adenocarcinoma of the pancreas
International Unit (I.U or U) and bile duct, pleural cancer, variant of Regan;
- Equivalent to the amount of enzyme that inhibited by L-leucine and phenylalanine
catalyzes the conversion of 1 micromole of  Kasahara ALP- hepatoma/hepatocellular
substrate per minute under controlled conditions carcinoma
Katal Unit (K.U)
- Equivalent to the amount of enzyme that
catalyzes the conversion of 1 mole of substrate
per second under controlled conditions
PITFALLS IN ENZYME ASSAYS
- Hemolysis cause falsely elevated values due to
the release of enzymes from red blood cells
- Serum rather than plasma is the preferred
specimen due to the adverse effects of
anticoagulants on enzyme activity
- Lactescent or milky serum causes variable
absorption by the spectrophotometer
Storage:
-most enzyme are stable at 6C for at least 24
hours
-few enzymes are inactivated at refrigerator
temperature (LD 4 and 5)
 TARTRATE-INHIBITED ACP
Measurement: - Prostatic cancer
-assays to measure ALP activity use - Benign prostatic hyperplasia
p-nitrophenyl phosphate substrate an alkaline pH - Prostatic infarction
-Activators: zinc, magnesium and other cations - Urinary tract obstruction
-chelators can falsely lower activity - Medico legal cases (implicated in suspected
Activity of an enzyme increases slightly on rape cases)
storage due to loss of inhibitors TARTRATE-RESISTANT ACP
-ALP is relatively stable at 4C for up to 1 week - Hairy cell leukemia
Optimum pH: 8.6-10 - Active osteoclast mediated bone resorption
- Gaucher’s cells

Decreased ALP
ACP Elevation
- After blood transfusion or cardiopulmonary - Prostatic cancer: ACP is inferior to
bypass prostate-specific antigen
- Malnutrition
- Prostatic hyperplasia and prostatic
- Hypophosphatemia
infarction
- Zinc deficiency (necessary cofactor in ALP - Urinary tract obstruction, carcinoid tumors
activity) of rectum and prostatic massage
ACID PHOPHATASE (3.1.2.3)
AMINOTRANFERASES
- Catalyzes same reaction made by ALP
- Catalyzes the transfer of amino group of one
- Active at pH 5.0
amino acid to a hydrocarbon to form a different
Diagnostic significance:
amino acid
- Detection of prostatic carcinoma
Specimen Stability
- Evaluation of rape case
- AST is stable in room temp for up to 3 weeks,
Tissue sources:
indefinitely if frozen
- Prostate (major source)
- ALT has the same stability but markedly
- RBC
decreases with freezing
- Platelets
- Specimens for AST and ALT are stable in whole
- Bone
blood for up to 12 to 24 hours, but increases with
Reference value: 2.5-11.7 (total ACP male)
time due to release from RBC
0-3.5ng/ml (prostatic ACP)
Optmum pH: 7.4
Half-life: AST- 17+5 hours
ACP ISOENZYME ALT – 47+ 10 hours
Band 1- major source is prostate gland; inhibited by
ASPARTATE AMINOTRANSFERASE
tartrate
Band 2 and 4- isoenzymes are from granulocytes -involved in the transfer of an amino group between
Band 3- major form present in plasm; derived from aspartate and a-ketoacids with the formation of
oxaloacetate and glutamate
platelets, erythrocytes, and monocytes
Band 5- mainly found in osteoclast; resistant to tartrate 2 Isoenzyme: Cytoplasm, Mitochondrial
Major tissue source: Cardiac tissue, liver and
inhibition
skeletal muscle
 Other sources: kidney, pancreas, RBC CREATINE KINASE (EC 2.7.3.2)
Reference value: 5-37U/L -involved in the reversible phosphorylation of creatine by
ATP
-an enzyme involved in energy storage of tissues
-during period of rest, ATP is converted to creatine
phosphate by CK to serve as energy reservoir
Cofactor: Magnesium
Isoenzymes:
 CK1 or CK-BB- brain and smooth muscle; most
rapidly moving isoenzyme
 CK2 or CK-MB- normal muscle contains 14% to
20% of CK-MB; hybrid
 CK3 or CK-MM- skeletal muscle; slowest and
most common form
 MACRO CK- oligomer present in mitochondria
and is seldom released into circulation
6% of total CK = CK-MB
Reference value: 15-160U/L (male)
15-130U/L (female)

ALANINE AMINOTRANSFERASE
-has enzymatic activity similar to AST
-highest concentration in the liver
Other sources: kidney, pancreas, RBC, heart, skeletal
muscle, lungs
Reference value: 6-37U/L

Measurement:
Measurement: - Electrophoresis is the method of choice. All
- Aminotransferase is done by coupled enzymatic isoenzymes can be measured at one time
reactions, using NADH as the final reaction because of technical difficulties, it has been
product seldom used
- Reagents with NH4 will give falsely increased - Immune-inhibition assays for CK-MB uses
antibodies against the CK-M subunit
ALT and AST owing to the conversion of NADH
to NAD by the ammonium ion - Mass immunoassay is the most commonly used
- Internation Federation of Clinical Chemistry method for measuring CK-MB. It may use 2
(IFCC) recommended the methods should antibodies or by using “conan” monoclonal
include P-5’-P in the reagents antibody
Diagnostic Significance: Consideration in CK Assays:
-light sensitive; exercise and IM injections causes
-evaluation of hepatic disorders
CK elevations
-monitors the course of hepatitis treatment and effect of
drug therapy GAMMA-GLUTAMYL TRANSFERASE (EC 2.3.2.1)
Aminotransferase levels are altered in: -catalyze the transfer of glutamyl moiety from peptides to
- Hepatocyte injury amino acids, other peptides, or water molecules
- Muscle injury (increase) -plasma membrane bound on cells that has high
- Kidney infarcts (increased) secretory or absorptive properties
- Renal failure (falsely lowered) Half-life: 7-10 days
 Increases to 28 days (alcoholic liver disease)
Measurement:
- Szasz assay
- Measured by cleavage of chromogen o-carboxyl
p-nitroaniline from a glutamate modified form of
the compound
GGT Elevations:
- Liver damage is the major source of GGT
release
- Medication increase GGT levels up to 5x
(ethanol, phenytoin, barbiturates, AMYLASE/DIASTASE (EC 3.2.1.1)
carbamazepine, valproic acid) - Catalyzes the breakdown of starch; smallest
GGT Decreases enzyme
- Pregnancy - Earliest pancreatic marker
- Oral contraceptives Isoenzyme: S-type (ptyalin), P-type (amylopsin)
Uses of GGT: Reference value: 60- 180SU/dl (Somogyi units),
- Evaluation of liver injury 95-290U/L
- Test for alcoholic abuse Optimum pH: 6.9-7.0
LACTATE DEHYDROGENASE (EG 1.1.1.27) Diagnostic Significance:
-zinc containing enzyme and its activity is part of the -Acute pancreatitis
glycolytic pathway -Rise: 2-12 hours
-all bands of isoenzyme are low -Peak: 24 hours
-subunits: H (heart) and M (muscle) -Normalize: withing 3-5 days
Isoenzymes: -AMS in urine is elevated up to 7 days
Methods of Determination:
 Saccharogenic- measures the amount of
reducing sugars produced by the hydrolysis of
starch
 Amyloclastic- amylase activity is evaluated by
following the decrease in substrate
concentration
-LD2 is always higher than LD1  Chronometric- measure the time required for
Tissue sources: amylase to be completely hydrolyzed
LD1 and LD2 – heart, RBC, kidney  Amylometric- measures the amount of starch
LD3 – lungs, pancreas, spleen hydrolyzed in a fixed period of time
LD4 and LD5 – skeletal muscle, liver, intestine LIPASE
Reference value: 100-225U/L (forward reaction) - Enzyme that hydrolyzes the ester linkage of fats
80-280U/L (reverse reaction) to produce alcohol and fatty acids
- Most specific pancreatic marker: secreted
exclusively in the pancreas, not affected by renal
disorders
Reference value: 0-1.0U/mL
Lipase Determination:
-Cherry Crandall Method
Principle: hydrolysis of olive oil after incubation
for 24 hours at 370C and titration of fatty acids
using NaOH
Clinical Significance: Substrate: 50% olive oil/ triolein
- Highest levels are seen in pernicious anemia End product: Fatty acid
and hemolytic disorder Diagnostic Significance:
- Hepatic carcinoma and toxic hepatitis (10fold -acute pancreatitis: lipase levels rise 6 hours
increase) after onset of attack; peak at 24 hours, remain
- Viral hepatitis and cirrhosis (2-3x increased) elevated at 7 days, and normalize in 8-14 days
- LD1 and LD2 flipped pattern: MI, hemolytic -chronic acute pancreatitis: acinar cell
anemia degradation occurs resulting in loss of amylase
- LD5: moderately increased in acute viral and lipase production
hepatitis, and cirrhosis; markedly increased in -LPS is also elevated in pancreatic duct
hepatic carcinoma and toxic hepatitis obstruction and tumors of the pancreas
 Clinical Significance:
- Monitor those exposed to cholinesterase
inhibitors
- Pseudocholinesterase production reflects
synthetic function of the liver

ANGIOTENSIN-CONVERTING ENZYME (EC 3.5.15.1)

- also known as kininase 11 or peptidyl
dipeptidase A
- responsible for conversion of angiotensin I to
GLUCOSE-6-PHOSPHATE DEHYDROGENASE angiotensin H, and inactivation of bradykinin,
-oxidoreductase; catalyzes the oxidation of G6P to enkephalin, tachykinin
6-phosphogluconate - enzyme structure is described as a single
-important as the 1st step in the pentose phosphate polypeptide chain with zinc at the active center
shunt - most activity is present in the lungs but it is found
in endothelial cells throughout the body
LEUCINE AMINOPEPTIDASE Measurement:
-exhibits naphthyl amidase activity: enzyme attacks the - activity is measured by ACE’s ability to cleave
free amino end of the peptide chain synthetic peptides, releasing hippuric acid, of
-rich in pancreas other indicator molecules
Substrate: Acyl β naphthylamide Disease Correlation:
Increase LAP - most common reason for ordering ACE levels is
- Hepatobiliary disease for diagnosing and monitoring sarcoidosis
- Pancreatic cancer - as the disease progresses to fibrosis, ACE levels
- Last trimester of pregnancy decline
- Obstructive biliary disease - ACE elevations are also seen pulmonary
involvement
MYELOPEROXIDASE
- Stored in azurophilic granules of PMNs and
monocytes
- Catalyzes the conversion of chloride anion and
hydrogen peroxide to hypochlorite
Clinical Significance:
- MPO is released into the extracellular fluid and
5’ NUCLEOTIDASE
general circulation during inflammatory
-used to differentiate obstructive from osseous disease
conditions
Increased: post-hepatic jaundice, intrahepatic
- Active mediator in the atherosclerotic CV
cholestasis, and infiltrative lesions of liver
disease
Slightly Increased: hepatocellular jaundice
Normal: bone disease ALDOLASE
ORNITHINE CARBAMOYLTRANSFERASE - Enzyme involved in the conversion of fructose
-found most exclusively in the liver 1,6-biphosphate into dihydroxyacetone
-excellent marker for liver disease but it is rarely used phosphate and glyceraldehyde-3-phosphate
- Elevated aldolase level can be seen in skeletal
CHOLINESTERASE (EC 3.1.1.7) muscle damage, IM, muscular dystrophy
PSEUDOCHOLLNESTERASE (EC 3.1.1.8)
-enzymes cleave one of the body’s major neuro-
transmitter known as acetylcholine
True Cholinesterase – has a high activity in CNS, RBC,
lung, and spleen
Pseudocholinesterase – it is important in the cleavage
of succinylcholine (muscle relaxant used during surgery)
- It is primarily produced in the liver, but is also
synthesized by myocardium and pancreas
Measurement:
- True cholinesterase uses acetylcholine while
pseudocholinesterase uses butyryl-thiocholine
as a substrate
- The released thiocholine reacts with Ellman’s
reagent
 ELECTROLYTES -Atrial natriuretic factor: produced by the atrial
-ions that carry an electrical charge myocardium (promotes natriuresis and relaxation of the
-dissociation of solutes into charged particles depends vascular smooth muscle)
on the chemical composition of the compound and on the HYPONATREMIA
concentration of other charged particles in the medium -an electrolyte disturbance in which the sodium
Functions: concentration in the serum is lower than normal
- Volume and osmotic regulation -defined as a serum level of less than 135mEq/L and is
- Myocardial rhythm and contractility considered severe serum level is below 125mEq/L
- Cofactors in enzyme regulation Causes of Hyponatremia:
- Regulation od ATPase ion pumps - Use of diuretics
- Acid base balance - Syndrome of Inappropriate ADH secretion
- Blood coagulation - Aldosterone deficit secondary to Addison’s disease
- Neuromuscular excitability - Bartter’s syndrome: rare condition wherein sodium
- Production and use of ATP from glucose chloride gradients cannot form in the loop of henle
REGULATION OF ELECTROLYTES causing the retention of chloride ion that is not
Active Transport- mechanism that requires energy to available for the countercurrent mechanism
move ions across cellular membranes - Diabetic hyperosmolar state: causes efflux of
Diffusion- passive movement of ions across a cellular water with consequent osmotic dilution of
membrane serum sodium
- Congestive heart failure
ANION GAP - Azotemia
-refers to the difference between the sums of the - Burns
concentration of the principal cations (Na+ and K+) and - Vomiting
of the principal anions

Increase AG:
- Uremia
- Ketoacidosis
- Methanol, aspirin, or ethylene glycol poisoning
- Severe hydration
- Lactic acidosis Symptoms of Hyponatremia:
Decreased AG: - Gastrointestinal: 125-130mmol/L
- Multiple myeloma - Neuropsychiatric: below 125mmol/L
- Protein and instrument error - Nausea and vomiting
SODIUM - Muscular weakness
-major cation in the extracellular fluid; renal regulation - Headache, lethargy, and ataxia
-plays a central role in maintaining the normal (Severe symptoms)
distribution of water and osmotic pressure in the ECF - Seizures, coma, and respiratory depression
compartments - Acute hyponatremia: below 120mmol/L for 48
-principal osmotic particle outside the cell hours (considered as medical emergency)
-for every 100mg/dl increase in blood glucose, serum PSEUDOHYPONATREMIA
sodium decreases by 1.6mmol/L -occurs when sodium is measured using indirect ion
REGULATION OF SODIUM selective electrodes in a patient who is hyperproteinemia
-diet or hyperlipidemic
-kidney (renal threshold for sodium (110-130mmol/L)) HYPERNATREMIA
70-80% are reabsorbed at the proximal tubule -a serum sodium concentration above the upper limit of
-RAA: release of aldosterone the reference interval
Causes of Hypernatremia:
- Diabetes insipidus
- Hyperaldosteronism HYPERKALEMIA
- Hyperadrenocorticism
-serum potassium concentration above the upper limit of
the reference interval
Seen in the following conditions:
- Dehydration
- Diabetes insipidus
- Hypoadrenalism
- Acidosis
- Hemolysis
Causes of Hyperkalemia:

Symptoms of Hypernatremia:
- CNS: altered mental status, lethargy, irritability,
restlessness, seizure, muscle twitching, hyper
reflexes, fever, nausea or vomiting, difficult
respiration, and increased thirst
- 160mmol/L is associated with a mortality rate of 60-
75%
Methods of Determination:
- Ion specific electrodes
- Atomic absorption spectrophotometry
- Flame emission spectrophotometry/ emission Symptoms of Hyperkalemia:
flame photometry - Muscle weakness, tingling, numbness, or mental
- Colorimetric method: Albanese Lein; combining confusion by altering neuromuscular conduction
sodium with zinc uranyl acetate (addition of water - Hyperkalemia disturbs cardiac conduction:
produces yellow solution) cardiac arrhythmias and possible cardiac arrest
- 6-7mmol/L may alter the electrocardiogram
- >10mmol/L may cause fatal cardiac arrest
HYPOKALEMIA
-serum potassium concentration below the lower limit of
the reference interval
Seen in the following conditions:
- Infusion of insulin to diabetes
- Alkalosis
POTASSIUM - Vomiting
-major intracellular cation - Use of loop diuretics
Function: - Syndrome of Inappropriate ADH secretion
- Neuromuscular excitability - Bartter’s syndrome
- Contraction of the heart Causes of Hyperkalemia:
- Intracellular fluid volume - Acute myelogenous leukemia
- Hydrogen ion concentration - Acute myelomonocytic leukemia
- Acute lymphocyte leukemia
REGULATION OF POTASSIUM
 - Increased gastric juice secretion
- Salt losing nephritis
- Addison’s disease
Analytical Methods:
- Ion selective electrode
- Mercurimetric titration (Shales-shales method)
- Colorimetric method uses mercuric thiocyanate
and ferric nitrate to form a reddish colored
complex with a peak at 480nm
- Coulometric-amperometric titration
Reference Interval:

CALCIUM
Symptoms of Hyperkalemia: -fifth most common element and most prevalent cation in
the human body
- Weakness
Functions:
- Fatigue
- Important in skeletal mineralization
- Constipation
- Plays a vital role in: blood coagulation, neural
- Muscle weakness or paralysis
transmission, enzyme activity, maintenance of
- Sudden death would be caused by arrhythmia
normal tone, excitability of skeletal and cardiac
Analytical Methods:
muscle
- Ion selective: method of choice
- Involved in glandular synthesis and regulation of
- Atomic absorption spectrophotometry
exocrine and endocrine glands
- Flame emission spectrophotometry
- Preserves the cell membranes integrity and
Reference Interval:
permeability particularly in terms of sodium and
potassium exchange
REGULATION
- PTH, vit. D and calcitonin are known to regulate
serum Ca2 by altering their secretion rate in
response to changes in ionized Ca2
(PTH)
- PTH secretion in blood is stimulated by a decrease
in ionized Ca2 and conversely, PTH secretion is
stopped by an increase in ionized Ca2
CHLORIDE - In the bone, PTH activates a process known as
-major extracellular anion bone resorption, in which activated osteoclast break
-together with sodium, they represent the majority of the down bone and subsequently release Ca2 into the
osmotically active constituent of the plasma ECF
-maintaining electrical neutrality - In the kidneys, PTH conserves Ca2 by increasing
-regulate fluid content on the body and its influence in tubular reabsorption of Ca2 ions
the kidney - PTH also stimulates renal production of active vit. D
HYPERCHOLOREMIA (Vitamin D)
- Obtained from the diet or exposure of skin to
sunlight
Seen in the following conditions: - 1,25-dihydroxycholecalciferol: biological active
- Dehydration form
- Renal tubular acidosis - This active form of vit. D increases Ca2
- Acute renal failure absorption in the intestine and enhances the
- Metabolic acidosis associated with prolonged effect of PTH on bone resorption
diarrhea (Calcitonin)
- Prolonged vomiting - Medullary cells of the thyroid gland
- Profuse sweating - Secreted when the concentration of Ca2 in blood
increase
 - Calcitonin exerts its Ca2: lowering effect by - Hypomagnesemia
inhibiting the actions of both PTH and vit. D - Acute pancreatitis
- Not secreted during normal regulation of the Analytical Methods:
ionized Ca2 concentration in blood, it is secreted  Total Calcium
in response to a hypercalcemic stimulus - Spectrophotometric analysis with the
metallochromic indicators
Distribution: - Titration of fluorescent calcium complex with
- Free or ionized form (50%) EDTA or EGTA
- Bound to plasma protein (40%) - Atomic absorption spectrophotometry
- Complex form (10%) - Redox titration method
Clinical Significance:  Ionized Calcium
- Increased calcium levels are seen in: - Ion selective electrode
o periods of growth in children Reference Interval:
o pregnancy
o lactation

- Decrease calcium levels are seen in:

o Old age
Factors Influencing Calcium Levels:
- Increase calcium absorption
o Vit. D (major stimulus of calcium
absorption)
o Growth hormone
o Increased dietary protein
- Decreased calcium absorption
o Formation of insoluble salts with MAGNESIUM
phosphorus -essential for the function of cellular enzymes and energy
o Phytic acid metabolism
o Dietary oxalate -has an important role in membrane stabilization, nerve
o Fatty acids conduction, and ion transport and calcium channel
o Cortisol activity
- Increased urinary calcium secretion -plays an important role in maintenance of intracellular K
o Hypercalcemia concentration
o Phosphate deprivation REGULATION
o Acidosis - Loop of Henle is the major renal regulatory site:
o Glucocorticoid
50-60% of filtered magnesium is reabsorbed in
- Diminished urinary calcium excretion the ascending limb
o PTH - Renal threshold for magnesium is approximately
o Certain diuretics 0.60-0.85mmol/L
o Vit. D - Parathyroid hormone (PTH) increases the renal
HYPERCALCEMIA reabsorption of magnesium and enhances the
-a condition characterized by an increased serum absorption of magnesium in the intestine
calcium level - Aldosterone and thyroxine: opposite effect of
-associated with anorexia, nausea, vomiting, PTH in the kidney ---increasing the renal
constipation, hypotonia, depression, and coma excretion of magnesium
Causes:
- Primary hyperthyroidism HYPERMAGNASEMIA
- Multiple endocrine neoplasia -condition with high level of serum magnesium
- Familial hypercalciuria hypercalcemia -increased magnesium level in the blood is rare and
- Vit. D intoxication usually iatrogenic
- Thyrotoxicosis -elderly and patients with bowel disorder and renal
- Hypoadrenalism insufficiently are the most at risk
Clinical manifestation: hypotension, bradycardia,
- Multiple myeloma
respiratory depression, depressed mental status and
HYPOCALCEMIA electrocardiography abnormalities
-condition characterized by a low serum calcium level
-severe hypocalcemia will eventually lead to tetany
Causes:
- Hypoparathyroidism
- Pseudohypoparathyroidism
- Deficiency in vit. D or its metabolite
- Chronic renal failure
Causes: - Photometric methods on automated analyzers:
these methods employ metallochromic
indicators or dyes such as calmagite, formazan
dye, magon, and titan yellow dye
 Ionized (Free) Magnesium
 Ion selective electrode for magnesium
 Fluorescence measurement using furapta
(magnesium binder)
 Nuclear magnetic resonance spectroscopy
 Ion selective microelectrode
 Electroprobe microanalysis
Reference Interval:

Symptoms:

PHOSPHORUS
-important constituent in nucleic acid, phospholipid, and
phosphoproteins
-forms high energy compounds such as ATP and
cofactor (NADP) and is involved in intermediary
metabolism and various systems
-essential for muscle contractility, neurologic function,
and electrolyte transport and oxygen carrying by
hemoglobin
HYPERPHOSPHATEMIA
-condition characterized by a serum phosphorus
concentration above the upper limit of the reference
HYPOMAGNASEMIA interval
-condition with low serum magnesium level Causes:
Causes: - Decrease renal excretion in acute and chronic
- Loss of magnesium in the GI tract as in chronic renal failure
diarrhea and malabsorption steatorrhea - Increase intake with excessive oral, rectal,
- Diabetes mellitus secondary to glycosuria and intravenous administration
osmotic diuresis - Increase extracellular load due to transcellular
- Alcohol shift in acidosis
- Stress - Secondary to over medication with vit. D and
Symptoms: production of vitamin by granulomatous tissue
HYPOPHOPHATEMIA
-condition characterized by a serum phosphorus
concentration below the lower limit of the reference
interval
Can be seen in:
- Alcohol abuse
- Intestinal loss due to vomiting, diarrhea, and use
of phosphate binding antacids
- Induced by a shift of phosphorus from
extracellular fluid into cells
- Increased urinary excretion, secondary to
hyperparathyroidism, renal tubular defects and
diuretic therapy
Analytical Methods: - Decreased intestinal absorption is observed in
 Total Magnesium malabsorption
- Atomic absorption spectrophotometry is the - Vit. D deficiency and steatorrhea
reference method but it is not routinely done in
the clinical laboratory
Analytical Methods:
- Reaction of phosphate with ammonium
molybdate
- Reduction of phosphomolybdate to molybdenum
blue which can be measured at 600-700nm
spectrophotometrically
- Enzymatic method
Reference Interval:

ELECTROLYTE AND RENAL FUNCTION

RENAL TUBULES
-phosphate reabsorption is inhibited by PTH and
increased by 1,25-dihydroxycholecalciferol
-excretion of PO4 is stimulated by calcitonin
-Ca2 is reabsorbed under the influence of PTH and
1,25-dihydroxycholecalciferol
-calcitonin stimulates excretion of Ca2
-Mg2 reabsorption occurs largely in the thick ascending
limb of Henle’s loop
-Cl is reabsorbed, in part, by passive transport in the
proximal tubule along the concentration gradient created
by Na
 ELECTROLYTES - It is associated with hyperlipidemia or
-ions capable of carrying an electric charge hyperproteinemia
-essential component in numerous processes, including Methods:
volume and osmotic regulation - Emission Flame Photometry: YELLOW
-measured or quantified using Ion selective electrodes - ISE (Glass Aluminum Silicate): common
(ISE) - AAS (Atomic Absorption Spectrophotometry)
Cations: electrolytes with a positive charge, they move - Colorimetry (Albanese-Lein Method)
toward the cathode POTASSIUM
Anions: electrolytes with a negative charge, they move -known as “Kalium”
toward the anode -Primary intracellular cation, permits neural signal to
Electrolytes: Na, K, Cl move down the nerve fiber
Functions: -single most important analyte in terms of an abnormality
- Volume and osmotic regulation being immediately life threatening
- Myocardial rhythm and contractility -it is a cardiac ion
- Cofactors in enzyme activation
- Regulation of ATPase ion pumps
- Neuromuscular excitability
- Production and use of ATP
- Acid base balance
- Replication of DNA
SODIUM Hyperkalemia:
-known as “Natrium” - >5.0mmol/L
-most abundant cation in ECF; largest constituent of - Impaired renal excretion/ renal failure
plasma osmolality - Reduced aldosterone
-major extracellular cation, major contributor of Hypokalemia:
osmolality/ osmotic pressure - <3.0mmol/L
-changes in sodium result in changes in plasma volume - Impaired renal function/ renal loss
-excreted in urine when the human threshold for serum - Metabolic alkalosis
sodium exceeds 110-130mmol/L Artificial Hyperkalemia:
Reference value: 135-145mmol/L - Sample hemolysis
- Prolonged tourniquet application/ fist clenching
Clinical Significance: Pseudo-hypokalemia:
HYPONATREMIA - Leukocytosis: K is taken up by wbc
-occurs when the level is <135mmol/L Methods:
Depletional Hyponatremia: can be due to diuretics, - Emission Flame Photometry: VIOLET
hypoaldosteronism (Addison disease), diarrhea or - ISE with Valinomycin gel
vomiting, and severe burns or trauma - AAS
Dilutional Hyponatremia: can be due to overhydration, - Colorimetry (Lockhead and Purcell)
syndrome of inappropriate anti-diuretic hormone
CHLORIDE
(SIADH), congestive heart failure, cirrhosis, and
-major extracellular anion
nephrotic syndrome
-chief counter ion and sodium
HYPERNATREMIA -excreted in urine and sweat
-occurs when the level is >150mmol/L
-usually occurs when water is lost through diarrhea,
excessive sweating, or diabetes insipidus and when Methods:
sodium is retained through acute ingestion, - Mercuric Titration: (schales and schales) based
hypoaldosteronism, or infusion of hypertonic solution on the reaction of chloride ions to mercuric ions to
during dialysis form mercuric chloride. Excess mercuric ions are
Hypernatremia: then made to react with diphenylcarbazone in
- >150mmol/L order to form violet blue color
- Usually results from excessive water loss - Spectrophotometric: uses mercuric thiocyanate
- THIRST is the major defense against and ferric nitrate to form ferric thiocyanate, which
hyperosmolality and hypernatremia is reddish colored complex with a peak
Hyponatremia: absorbance at 480nm
- <135mmol/L - Coulometric Amperometric Titration: (Cotlove
- Most common electrolyte disorder Chloridometer) uses silver ions which combine
- Excessive water intake with chloride is used to indicate endpoint
Artificial Hyponatremia: -can be used in Cystic Fibrosis
- In vitro hemolysis
 - ISE: most common method for Na, K, Cl; Methods:
membrane used is a combination of silver wire - Precipitation and redox titration
coated with AgCl - Colorimetric methods/ spectrophotometric (orto-
Hypochloremia: cresolpthalein complexone, arseno III dye)
- Occurs when level is <98mmol/L - ISE (liquid membrane): measure the free form.
- Results from excessive vomiting, use of Temperature sensitive and performed at 37C
diuretics, burns, aldosterone deficiency - AAS: reference method
Hyperchloremia: - Emission Flame Photometry: ORANGE
- Occurs when level is >107mmol/L Sources of Error:
- Results from prolonged diarrhea, renal tubular - Cannot use oxalate, EDTA anticoagulants for
disease, dehydration, excess loss of spectro
bicarbonate - Hemolysis, icterus, lipemia
CALCIUM - Protein buildup in electrode and change of blood
-essential for myocardial contraction pH in vitro before analysis for ISE
-blood coagulation
-enzyme activation
-excitability of cardiac and skeletal muscle
-decrease free calcium levels cause muscle spasms and
uncontrolled muscle contractions called “Tetany”
3 Forms of Calcium:
- Ionized calcium (free) -50%
- Protein bound calcium - 40%
- Complexed with anions – 10%
Factors Affecting Serum Calcium Levels:
 1,25 Dihydroxycholecalciferol
- Activated vit. D3
- Increased intestinal absorption of calcium
- Obtained by diet or exposure to sunlight
- Initially, vit. D is transported to liver, where it is
hydroxylated but still inactive. Then, the POTENTIOMETRY
hydroxylated form is transported to kidneys -technique used to determine the concentration of a
where it is converted to the active form of vit. D substance in solution employing an electrochemical cell
 PTH (Parathyroid hormone) consisting of two half-cells, where the potential difference
- Main hypercalcemic hormone between the indicator electrode and reference electrode
- For calcium hemostasis is measured
- A decreased in ionized calcium will stimulate the ISE/ION Selective Electrode
release of PTH by the parathyroid gland - Consist of liquid ion exchange membrane made
- Rise in ionized calcium terminate PTH release of inert solvent and ion selective carrier material
- In bone, PTH activated osteoclasts to - Collodion membrane may be used to separate
breakdown bone with the release of calcium membrane solution from sample solution
- In kidneys, PTH increases tubular reabsorption  Potassium: antibiotic VALINOMYCIN, because
of calcium and stimulates hydroxylation of vit. D of its ability to bind K+ selective membrane
to the active form  Sodium: utilizes GLASS MEMBRANE
 Calcitonin ELECTRODE; consist of silicon dioxide, sodium
- Inhibits PTH and activated vit. D3 oxide, and aluminum oxide
- Released by parafollicular cells of the thyroid
gland when serum calcium levels increase