CLINICAL CHEMISTRY 2 (MT 6324) LABORATORY

nd
Ms. Canellie Canlas & Asst Prof. Vivian Asuncion | 2 Shifting

ELECTROLYTES - SODIUM AND POTASSIUM

● The distribution of water in various fluid
ELECTROLYTES compartments is usually controlled when we
● Ions capable of carrying an electrical charge maintain the concentration of electrolytes as well
● Can be classified as: Based on the charge they as proteins in the individual compartments.
carry and how they migrate in an electrical field. ● Since most biological membranes are freely
○ Cations – positive charge (migrate permeable by water pero and ions and proteins
toward the negatively charged cathode) are not, yung concentration ngayon ng ions and
○ Anions – negative charge (migrate proteins on either side of the membrane will
toward the positively charged anode) influence the flow of water across the
● Body fluids should always contain equal number membrane. That is why yung mga electrolytes
of cations and anions are considered as osmoregulatory.
○ Balance of charges is called as ● We have 2 major compartments in the body, we
electroneutrality have the intracellular fluid compartment and
● Electrolytes have various functions in our body; extracellular fluid compartment.
they regulate many processes that is why it is ● The INC fluid is the fluid found inside the cells
very critical for the patient to have regulated and it usually accounts for ⅔ of the body's total
levels of these substances. water.
● In the laboratory, when we see a very high or ● The EC fluid accounts for the remaining ⅓ of the
very low level of these electrolytes, we total body water and can be subdivided into two:
immediately inform the attending physician so ○ Interstitial cell fluid
that they can perform necessary interventions ■ Surround the cell in the tissue
for the patient. and is usually separated from
the INC fluid by the cell
FUNCTIONS OF ELECTROLYTES membrane
1. Maintain volume and osmotic regulation (Na+, ○ Intravascular extracellular fluid or
K+, Cl-) plasma
○ Na and K are the major extracellular and ■ plasma is separated from the
intracellular electrolyte, respectively. interstitial fluid by your capillary
2. Myocardial rhythm and contractility (K+, Mg2+, walls of your blood vessels.
Ca2+)
3. Cofactors in enzyme activation (Mg2+, Ca2+, Zn2+)
4. Regulation of ATPase ion pumps (Mg 2+)
5. Acid-base balance (HCO3-, K+, Cl-)
6. Blood coagulation (Ca2+, Mg2+)
7. Neuromuscular excitability (K+, Ca2+, Mg2+)
8. Production and use of ATP from glucose (Mg 2+,
PO4-)

MAJOR EXTRACELLULAR ELECTROLYTES

1. Sodium
2. Chloride
3. Calcium

● Average water content of the body varies from

40% - 75% of the total body weight of the
human.
● Water serves as a solvent for many processes in MINOR INTRACELLULAR ELECTROLYTES
the body. It is particularly responsible for: 1. Potassium
○ Transporting nutrients to cell 2. Magnesium
○ Determines cell volume 3. Phosphate
○ Removal of waste products via the urine
○ Body Coolant - sweating
● Water is located in both the intracellular and
extracellular compartments within the body.

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

SODIUM
● Major extracellular cation (90%)
● Determines osmolality of plasma & maintenance
of acid-base balance
○ Normal plasma osmolality is
approximately 295 mmol/L
○ Sodium and associated anions accounts
for 270 mmol/L
● Na-K ATPase pump: makes sure that Na+ METHODS OF DETERMINATION
concentration in ECF is higher → preventing
osmotic rupture of cells Ion Selective Electrode (ISE)
○ NA-K ATPase pump moves 3 sodium
● Uses a semipermeable membrane to develop a
ions out of the cell, in exchange for 2
potential produced by having different io
potassium ions moving into the cell
concentration of either side of the membrane
○ During the movement, ATP is converted
● Method of choice
to ADP
● 2 electrodes used:
○ Because the water follow the
○ Reference electrode (constant potential)
electrolytes across cell membrane, the
○ Measuring electrode
continual removal of sodium from the
● Difference in potential between reference and
cell prevent osmotic rupture of the cell
measuring electrode is used to calculate
● Plasma concentration depends on intake and
“concentration” of ion in the solution
excretion of water & renal regulation
● Activity of the ion is the one being measured
● Regulation:
(not the concentration of ions)
○ Adrenal gland
● Glass aluminum silicate membrane
■ Releases aldosterone (direct
● 2 Types - based on sample preparation
reabsorption of sodium)
○ Direct ISE – undiluted samples
■ When blood sodium levels are
interact with the ISE membrane
low, the adrenal glands release
(preferred in hyperlipidemic and
aldosterone
hyperproteinemic samples)
○ Kidney
○ Indirect ISE – diluted samples are
■ Targeted by aldosterone
used
hormone
○ Generally, between the 2 types, there is
■ Reabsorbed 80% (proximal
no significant difference in the sample
tubules)
found except in cases of hyperlipidemic
■ 20-25% (loop of Henle)
or where there is increase lipids in the
○ Failure to properly regulate sodium
blood or proteinemic (increased protein
levels may results to hypernatremia or
level)
hyponatremia
○ For these samples, mas preferred natin
gumamit ng direct ISE measurement for
more accurate reading (this is to prevent
false decreased measurement)
○ In cases where in may hyperlipidemia or
hyperproteinemia si patient, we use the

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

Indirect ISE measurement in order to TRINDER METHOD

avoid false decreased levels
● Source of error: MATERIALS/INSTRUMENTATION
○ Protein buildup on membrane (because ● Automatic pipettor (1.0 mL and 0.2 mL capacity)
of its continuous use, since continuous ● Pipette tips
ang use ng ISE for the determination of ● Tissue
electrolytes, it causes protein build up in ● Parafilm
the membrane) ● Specimen (controls and unknowns – serum or
■ poor selectivity → poor plasma)
reproducibility of results ● Spectrophotometer set at 405 nm and 37C
■ That is why it is very essential ● Test tubes
for ISE to have a regular ● Cuvettes with 1 cm path length
maintenance in order to remove ● Centrifuge machine
or avoid protein buildup and to
ensure the quality of results REAGENTS
■ In the laboratory, usually we ● Sodium reagent kit:
perform the ISE maintenance ○ Precipitant of sodium: uranyl acetate
during the night duty (everyday) and magnesium acetate
● Other methods of Sodium Determination ○ Main Reagent: ammonium thioglycolate
○ FEP = Flame Emission and ammonia to which the remaining
Spectrophotometry uranyl ions upon sodium precipitation
■ it is used for the measurement will react to
of your excited ions such as ○ Standard: sodium (150.0 mmol/L); may
your sodium and potassium, vary
wherein your Na+ would ○ TRINDER METHOD
produce an intense yellow in the
flame PROCEDURES
○ AAS = Atomic Absorption ● Prepare and label cuvettes properly
Spectrophotometry ○ 2 sets of tubes
○ Chemical Methods ■ 1st set = used for precipitation
■ However, yung chemical procedure (1 tube for each
methods ngayon for Na controls and sample)
determination is no longer used ■ 2nd set = actual measurement
because it requires large ● prepare 1 tube for each
sample volume and lack of reagent blank, standard,
precision CN, CP and Unknown)
● Working reagent is ready for use
Colorimetric Method ● Prepare two blank solutions (water blank and
● Suderman and Delory – zinc uranyl acetate as reagent blank)
the precipitating agent ● Follow the semi-micropipetting scheme
● Trinder – magnesium uranyl acetate as the ● Prior to actual measurement, samples (including
precipitating agent controls) must be precipitated using the
precipitant
GENERAL PRINCIPLE ○ Standard is ready for measurement and
1. Sodium is precipitated with magnesium-uranyl does not require precipitation
acetate
2. Remaining Uranyl ions in suspension PRECIPITATION
(supernatant) + thioglycolic acid → yellow brown
complex Pipette into CN CP U
3. Reagent blank with precipitant is prepared cuvettes
4. Absorbance difference between reagent blank Control normal 0.02 ml
and samples is observed – proportional to serum
sodium concentration
Control pathologic 0.02 ml
● The absorbance difference between the reagent
serum
blank and the samples is being observed here,
Patient’s serum 0.02 ml
which is proportional to the sodium
concentration Precipitant 1.0 ml 1.0 ml 1.0 ml

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

Mix well. Stand for 5 minutes. Shake intensively for 30 ● Hemolysis does not cause significant change in
seconds. Stand for 30 minutes. Centrifuge at high values (since majority of the sodium is found
speed for 5-10 minutes. Obtain the supernatant. outside the cell)
● 0.02 ml = 20 ul ○ Marked hemolysis – falsely decreased
● 1.0 ml = 1000 ul levels (dilutional effect)
● Specimen of choice in urine Na+ analyses –
Pipette into RB S CN CP U 24-hour collection
cuvettes ● Sweat is also suitable for analysis
Standard 0.02
solution m CLINICAL SIGNIFICANCE
Control 0.02 ● As said earlier, failure of regulation of sodium
normal ml levels can lead to either hypernatremia or
serum hyponatremia.
Control 0.02
pathologic ml HYPERNATREMIA
serum ● Increased plasma or serum concentrations of
Unknown 0.02 sodium (>145 mmol/L)
supernatant ml ● Results from excess loss of water, decreased
Precipitant 0.02 water intake, or increased sodium intake or
ml retention
Working 1.0 ml 1.0 1.0 ml 1.0 ml 1.0 ml ● Symptoms involve CNS as a result of
reagent ml hyperosmolar state
Mix well. Measure absorbance of RB, standard, and ○ Hyperosmolarity - a condition wherein
samples against distilled water at 405 nm, after 5-30 the blood has very high
minutes. concentrations of the sodium, glucose
and other substances causing water
● RB = Reagent Blank
to draw out of the body’s organs
● Pipette 0.02 ml of the obtained supernatant from
including the brain which leads to
the precipitation step from the freshly prepared
CNS symptoms
tubes for CN, CP and U.
● >160 mmol/L – associated with 60-75%
● Take note: Add the samples first directly to the
mortality rate
bottom of the tube prior to adding the reagent to
○ Hypernatremia is less commonly seen
the side of the tube.
in hospitalized patients than
hyponatremia
CALCULATIONS
Causes of Hypernatremia
𝐴 𝑅𝐵 − 𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 Excess water loss - may occur either through kidney
𝐶 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐴 𝑅𝐵 − 𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 or through extrarenal conditions, such as diarrhea,
severe burns and sweating
● Unit of measurement: mmol/L or mEq/L 1. Diabetes Insipidus (DI)
● Conversion factor: 1.0 ● Kidney cannot respond to AVP/ADH or
vasopressin secretion is usually impaired
REFERENCE RANGES ● AVP is an antidiuretic hormone which
promotes reabsorption of water
2. Renal tubular disorder
Reference Ranges for Sodium ● Acute tubular necrosis - tubules can
Serum, plasma 135-145 mmol/L concentrate urine
Urine (24 h) 40-220 mmol/day, varies 3. Prolonged diarrhea
with diet Decreased water intake
Cerebrospinal fluid 135-150 mmol/L (slightly ● Osmolality measurement for evaluation of
higher than serum and hypernatremia
plasma) ● Renal loss of water - low or normal osmolality
● Extrarenal such as burn - increased osmolality
SPECIMEN CONSIDERATIONS Increased sodium intake or retention
● Serum, plasma, urine 1. Hyperaldosteronism (Conn’s disease)
● To obtain plasma samples, we use lithium ● Continuous reabsorption of sodium
heparin, ammonium heparin, lithium oxalate – ● Retarded AVP release
suitable anticoagulants 2. NaCO3 infusion

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

● Neonates are prone (3) Under the influence of aldosterone,

3. Increased oral or IV intake of NaCl additional potassium is secreted in the
urine in exchange for sodium (this
HYPONATREMIA happens both in distal tubules and
● Na concentration = <135 mmol/L collecting ducts).
○ most common electrolyte disorder for ● In contrast to the effects on sodium, the
hospitalized and non-hospitalize aldosterone causes the decrease in
● Clinically significant = <130 mmol/L level of potassium in the blood.
● Results from increased sodium loss, ● ↑ Potassium, ↓ Sodium would trigger the
increased water retention, or water imbalance release of aldosterone from the adrenal
● 125-130 mmol/L – GI symptoms cortex. This aldosterone will target your
● <125 mmol/L – neuropsychiatric symptoms kidney to release potassium and to
○ nausea, seizure, coma, respiratory reabsorb sodium normalizing the levels
depression, vomiting, muscular of these electrolytes in your body.
weakness, headache ● Regulates neuromuscular activity, contraction of
● <120 mmol/L (48 hours) – medical emergency the heart, intracellular fluid volume, and H+
Causes of Hyponatremia concentration. Potassium also plays a role in the
Increased sodium loss acid-base balance.
1. Diuretic use ● Most individuals can consume far more
2. Hypoadrenalism potassium than needed. The excess is usually
● Decreased aldosterone excreted in the urine but may accumulate toxic
3. Potassium deficiency levels if renal failure occurs.
● When K is low, renal tubules conserve
potassium and excrete sodium
Increased water retention - dilution of plasma
sodium
1. Renal failure
2. Nephrotic syndrome
3. Hepatic cirrhosis
4. Aldosterone deficiency
Water imbalance
1. Excess water intake
● Polydipsia
● Must be chronic
2. SIADH
● Syndrome of Inappropriate Antidiuretic
Hormone - increased AVP, increased
water retention, decreased sodium levels
3. Pseudohyponatremia
● Sodium is measured using indirect ISE
especially when patients are
hyperproteinemic or hyperlipidemic

POTASSIUM
● Major intracellular cation
● Concentration in RBC – 20x greater (inside the
cells than outside) METHODS OF DETERMINATION
● Many cellular functions would require that the
body will maintain a low extracellular fluid Ion Selective Electrode (ISE)
concentration potassium ions ● Method of choice
● As a result, only 2% is found in plasma ● Valinomycin – potassium carrier in the
● Filtered in the glomeruli, reabsorbed in the membrane
proximal tubules, and secreted in the distal ● KCl – inner electrolyte solution
tubules (under influence of aldosterone)
● (1) Filtered in the glomeruli Other methods:
(2) Initially, reabsorbed in proximal ● Flame emission spectrophotometer (FES)
tubules

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

○ Potassium produces a violet color in the ■Sodium hydroxide is used to

flame. maintain the alkalinity of the
○ Sodium produces intense yellow in the medium
flame. ○ Standard: prediluted potassium (5.0
● Atomic absorption spectrophotometer (AAS) mmol/L); may vary

Colorimetric Method PROCEDURES

● Lockhead and Purcell – sodium cobaltrinitrite ● Prepare and label cuvettes
● Hoffman – sodium tetraphenylboron ○ Prepare 2 sets of tubes:
1. Precipitation method
PRINCIPLE 2. Actual measurement
PPF (K+) + sodium tetraphenylboron → potassium ● Prepare working reagent: sodium
tetraphenylboron tetraphenylboron + sodium hydroxide (1:1)
● The reaction of potassium ion in a protein free ● Stand mixture for 15-30 minutes prior to use
alkaline medium and sodium tetraphenylboron ● Follow the semi-micropipetting scheme
will produce a turbid suspension which is the ● Prior to actual measurement, samples (including
potassium tetraphenylboron controls) must be precipitated using the
precipitant.
● Reaction and turbidity is read at 578nm, which is ○ The standard is ready for measurement
proportional to potassium concentration and does not require the precipitation
step.
HOFFMAN METHOD
PRECIPITATION
MATERIALS/INSTRUMENTATION
● Automatic pipettor (1.0 mL, 0.1 mL, and 0.05 mL Pipette into CN CP U
capacity) cuvettes
● Pipette tips Control normal 0.05 ml
● Tissue serum
● Parafilm Control pathologic 0.05 ml
● Specimen (controls and unknowns – serum or serum
plasma) Patient’s serum 0.05 ml
● Spectrophotometer set at 578 nm and 37C Precipitant 0.5 ml 0.5 ml 0.5 ml
● Test tubes NOTE: Standard is ready for use. No need to precipitate
● Cuvettes with 1 cm path length 9. Centrifuge the standard solution.
machine
Pipette into B S CN CP U
REAGENTS
● Potassium reagent kit:
cuvettes
○ Precipitant: trichloroacetic acid Working 1.0 ml 1.0 1.0 ml 1.0 ml 1.0 ml
■ Precipitates protein for accurate reagent ml
measurement of turbidity that Standard 0.1 m
will be produced during the solution
reaction. Control 0.1 ml
■ For colorimetric measurement of normal
potassium, we have to serum
precipitate the protein because Control 0.1 ml
the final product in the reaction, pathologic
we are monitoring the turbidity serum
of the solution that is produced Unknown 0.1 ml
upon the addition of reagent to supernatant
the sample. As much as
possible, we have to eliminate Reminders:
interferences coming from the ● Pipette working reagents first before adding
proteins. the standard or clear supernatant
○ Reagent: sodium tetraphenylboron and ● Add the standard or clear supernatant to the
sodium hydroxide center of the surface of the working reagent
in the cuvette – homogenous turbidity

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

● Mix carefully before proceeding to the next CLINICAL SIGNIFICANCE

sample
● Allow the mixture to stand for at least 5 minutes HYPERKALEMIA
prior to reading ● Plasma K level = 5.5 mmol/L
● Measure absorbance against the reagent blank ● Almost always due to impaired renal excretion
between 5-30 minutes because of reduce aldosterone or renal failure
(Sodium determination: dH2O Potassium ● Can cause muscle weakness, tingling,
determination:Reagent blank) numbness, or mental confusion by altering
neuromuscular conduction
CALCULATIONS ○ >8 mmol/L – Muscle weakness
develops
𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 ● Disturb of cardiac conduction which can lead
𝐶 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 to cardiac arrhythmia and possible cardiac
arrest or cessation of the heart
● Unit of measurement: mmol/L or mEq/L ○ 6-7 mmol/L – alter ECG
● Conversion factor: 1.0 ○ >10 mmol/L – fatal cardiac arrest
Causes of Hyperkalemia
REFERENCE RANGES Decreased renal excretion
1. Renal failure
Reference Ranges for Potassium 2. Addison’s disease (Hypoaldosteronism) -
Serum 3.5-5.1 mmol/L decrease in aldosterone level/responsiveness
Plasma Males: 3.5-4.5 mmol/L Extracellular shift
Females: 3.4-4.4 mmol/L 1. Metabolic Acidosis - excess hydrogen ions
Urine (24 h) 25-125 mmol/day moves intracellularly to be buffered results to
potassium leaving the cell in order to maintain
SPECIMEN CONSIDERATIONS electroneutrality (this is because hydrogen
● Serum, plasma, urine ions and potassium are both positively
● Heparin – anticoagulant of choice charged)
● Urine samples – collected over a 24-hour period 2. Muscle/cellular injury
(eliminate diurnal variation) ● Potassium is released into the
● Avoid hemolysis (most common cause of extracellular fluid during tissue
artifactual hyperkalemia) breakdown or catabolism.
○ Slight hemolysis (50 mg/dL hemoglobin) ● This is specially true when it is
– 3% increase accompanied by renal insufficiency
○ Gross hemolysis (>500 mg/dL 3. Hemolysis
hemoglobin) – up to 30% increase ● increased cellular breakdown can be
● Coagulation process releases K+ from platelets caused by trauma, various cytotoxic
– serum levels → 0.1-0.7 mmol/L higher than in agents. massive hemolysis
plasma Increased intake
○ In cases of thrombocytosis(increase 1. Oral or IV infusion
platelet count)plasma sample is ● in healthy individuals, an acute oral
preferred (avoid false increased) load of potassium will just briefly
● Avoid prolonged tourniquet application and increase the plasma concentration.
excessive fist clenching during venipuncture This is because most of the absorbed
may cause release of potassium in plasma potassium rapidly moves
causing false increase intracellularly
● Do not store whole blood on ice (promotes
release of K+ from cells) PSEUDOHYPERKALEMIA
○ Store at RT and analyze immediately ● Causes: sample hemolysis, thrombocytosis,
○ Centrifuge to remove cells from prolonged tourniquet application, excessive
plasma/serum ● fist clenching, blood stored in ice, IV fluid, high
blast counts
● Usually due to potassium in cells leaking out
into plasma → falsely elevated levels

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

HYPOKALEMIA
● Plasma K level = below 3.0 mmol/L
● Symptoms: muscular weakness, fatigue,
muscle cramps, hypotension, decreased
reflexes, palpitation, cardiac arrhythmias,
cardiac arrest
Causes of Hypokalemia
1. Gastrointestinal loss
● Occur through vomiting, diarrhea or
gastric suction
2. Renal loss
● increased renal wasting of potassium
a. Cushing’s syndrome
b. Hyperaldosteronism
- increased activity of
aldosterone or other
mineralocorticoids
- Aldosterone promotes sodium
retention and potassium loss
- can lead to hypokalemia and
metabolic alkalosis
3. Intracellular shift
- increase intracellular uptake of
potassium
a. Alkalosis: promotes intracellular loss
of potassium to minimize the elevation
of intracellular pH.
- If na withdraw ng hydrogen
ions intracellularly, both the
potassium and sodium ions
would enter the cells to
preserve the electroneutrality
b. Insulin overdose
- Insulin promotes the entry of
potassium into the skeletal
muscles and liver cells.
4. Decreased intake

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