Rahaf M Alsoufi Lab18

Clinical Chemistry
Electrolytes

Sodium (Na+)
Function -Major extracellular cation in the body
-Most important in determining the osmolality of the ECF
-Contributes to 90% of normal plasma osmolality
-Effects water distribution between body compartments
-Effects CSF volume
-Effects the size of cells
-Involved significantly in the:
~Co-transport
~Active transport
~Counter transport
of various molecules in and out of cells.
-Major contributor to electrochemical balance in the body
-Major role in transmitting nerve and muscles impulses, by the cycle of
depolarization (influx of sodium) and repolarization (efflux on potassium)
Regulation at -By Na+ K+ ATPase pump, pumps 3 Na+ out and 2 K+ in to the cell.
-Active transport process that requires ATP and Mg2+ as a cofactor enzyme.
cellular level -Low intercellular Na+
-High intercellular K+
→This creates an electro-chemical gradient that allows other substances to be
transported across the membrane such as: glucose, amino acids and fatty acids inside
the cell with the sodium (co-transport or counter-transport)
-Water also follows the movement of sodium in to the cell, regulating the size and
shape of cell and preventing osmotic rupture.
Regulation at -Kidney is the major site for Na+ regulation.
-Under normal conditions:
body level ~47% of filtered Na+ is reabsorbed in the proximal tubule
~25% in the loop of Henle
~25% in the distal tubule
~3% in the collecting duct

-All reabsorption is accompanied by the reabsorption of water

-Two hormones are involved in the active reabsorption of sodium:
a) ADH (Antidiuretic hormone)+Renin:-
-ADH is released from the posterior pituitary gland (produced in the hypothalamus)
due to the renin secretion from the kidneys, in response to low glomerulus filtration
ratio (due to decrease in volume)

b)Aldosterone (Renin-Angiotensin-Aldosterone system):-

-Stimulated by decrease in blood pressure (BP), decreased Na+, increased K+, stress
or secreted renin.
-Aldosterone causes:
~Increased Na+ reabsorption by tubules
~Increase K+ excretion in urine

1
 Rahaf M Alsoufi Lab18

~Increased BP with increased blood volume

~Increased H2O retention (with sodium)

-Renal threshold for Na+ (110-130 mmol/L)

Hyponatremia A)Increased Na+ loss:
1-Renal route loss:-
~Renal tubular disease (high Na+ in urine, low Na+ in blood)
~Adrenal insufficiency (Addison’s disease):- Hypoaldosteronism
~Diuretic therapy (prevent Na+ reabsorption)
~Metabolic alkalosis
-Urinary Na+ concentration will be >20 mmol/L

2)Loss of Na+ by non-renal route:-

~Vomiting ~Burns
~Diarrhoea ~Cystic fibrosis
~Excessive sweating ~Drainage from fistulas (surgery or any open wound)
-Urinary Na+ concentration <10 mmol/L

3)Isolation of Na+ in certain site (trapped):-

~Peritonitis (inflammation of peritoneum-abdominal)
~Small bowel obstruction
-In this condition, Na+ is not available for its physiological function
-Urinary Na+ concentration <10 mmol/L

B)Increased water retention (Increased blood fluid volume):

~Liver cirrhosis (No proteins produced →water cannot be absorbed from cells into
vessels)
~Cardiac failure (low blood pressure → low glomerular filtration → renin-
angiotensin-aldosterone system activates → reabsorbs water although originally there
is no problem with the water level, only the pressure.)
-Urinary Na+ concentration <10 mmol/L
Symptoms -Depends on degree and rate of loss:
~Weakness, fatigue, headache, hypotension,
~Muscle cramps (problem in repolarization phase due to low Na+)
~Shock, vomiting, anorexia, brain damage, death.
Treatments A) Na+ and fluid replacement
B)Water restriction
Hypernatremia A)Excessive loss of water in excess presence of salts:
~Profuse sweating. ~Diarrhoea
~Diabetes insipidus (loss of free water due to ADH problem)
~Increased urinary osmolarity (body’s trying to get rid of the excess salt in urine.)
~Renal disease (kidney can’t filtrate Na+)
~Irregular thirst mechanism (due to damage to hypothalamus)
-Low to normal urine osmolarity and Na+ concentration >20 mmol/L

B)Excessive treatment with Na+ salts:

~Excessive intake of Na+ containing compounds, e.g. NaCl

C)Hyperaldosteronism

2
 Rahaf M Alsoufi Lab18

Symptoms -Excessive thirst. ~Increased blood pressure.

Sample -Serum (Coagulated blood)
-Plasma (Anti-coagulant):
~Li-heparin
~Li-oxalate
~Li-ammonium
-Urine (24 hour collection)
Detection -Flame Emission spectrophotometer (FES)
~Reference method
-Ion Selective Electrode (ISE)
~Direct
-Ion Selective Conductometry
~Or indirect ISE

3
Rahaf M Alsoufi Lab18

Detection Methods
Method FES ISE
Principle -When aspirated into a flam, Na -A glass membrane separates the
emits light at a specific sample from the electrode. It allows
wavelength (589nm). the selective diffusion of Na from the
-This light can be measure by sample into the electrode. Originally a
comparing with the standard, constant potential is maintained. The
after separating it from the difference in the potential of the
interfering light. electrode in the buffer chamber
-Lithium is used as an intern (standard/reference electrode) and the
standard. electrode in the sample is directly
proportional to the activity of Na.
-The activity of Na in the sample is
compared to the activity of the
standard in order to determine the
concentration of the Na in the unknow
sample.
Key points -Dilution is done in order to
decrease the concentration of
proteins and prevent the
plugging of the atomizer with
the large particle.
-Hyperproteinaemia,
hyperglycaemia and
hyperlipidaemia all causes
falsely low Na because the size
of these substances are similar to
water so they will displace a
certain amount of the water
volume.
-Disadvantage:
~Require flame (fire hazard)
~Specimen requires dilution
(dilution error could occur)
~Interference substances may be
present in sample.
~System set up and optimization
are important but time
consuming.
Notes -No dilution is required for direct ISE but must be done for indirect ISE
and FES
-Hyperproteinaemia specimens can only be used in direct ISE
-Hyperlipidaemia specimens must be centrifuged and excess lipids are
removed before dilution in indirect ISE or straight away used in direct
ISE
-Hyperglycaemia must be corrected as it falsely decrease Na+ value, it
draws water from intercellular spaces and into ECF and vascular spaces.

4
 Rahaf M Alsoufi Lab18

Potassium (K+)
Function -Major Intercellular cation in the body (20x greater inside)
-Important in many regulatory mechanisms:
~Regulation of neuromuscular excitability
~Regulation of co-transport of substances into the cell indirectly (Na+ K+ ATPase
pump)
~Regulation of heart contractions (Repolarization phase)
~Regulation of intracellular fluid volumes with NA+
~Regulation of H+ concentration (pH balance)
~Maintenance of electrochemical balance inside the cell
Regulation at -By Na+ K+ ATPase pump, pumps 3 Na+ out and 2 K+ in to the cell.
-Active transport process that requires ATP and Mg2+ as a cofactor enzyme.
cellular level -Low intercellular Na+
-High intercellular K+
~This maintains a negative intracellular potential (resting potential)
~Also, the conduction of impulses and maintenance of electrochemical gradient are
dependent on this type of mechanism.
Regulation at -Kidney is the major site for K+ regulation.
-Has no renal threshold, so even low levels of K is found in the urine.
body level -K filtered by the glomerular is nearly all reabsorbed by the proximal tubule and loop
of Henle
-At distal tubule and collecting duct, 10-20% of the filtered K will be secreted and
excreted due to the action of aldosterone, in exchange for Na+
-Increase in K in the body → Increase in aldosterone secretion→Increase secretion of
K in urine.
-Decrease K in the body →No aldosterone secreted →Increase in K reabsorption and
decrease in K excretion.

-H+ concentration influences K+ in the body:

~Acidosis (high [H+]):-
(At a body level)
As [H+] is high → the kidney increases in H+ excretion in exchange for Na+
reabsorption and K+ is excreted → this increases the K+ in the body.
(At a cellular level)
As [H+] is high → H+ enters the cell in exchange for K+ leaving the cell (H+ K+
pump)→this increases the circulating K+.

~Alkalosis:-
(At a body level)
As the [H+] is low → the kidney excrete K+ in exchange for H+ → K+ decrease in
the body.
(At a cellular level)
As the [H+] is low → the K+ enters the cell in exchange for H+ to enter the
circulation → K+ decreases in circulation.

~Hypokalaemia:-
Low K+ → K leaves the cell and enters the circulation in exchange for H+ and Na to
enter the cell → decreases [H+] in the circulation → Alkalosis

5
 Rahaf M Alsoufi Lab18

Notes -Alkalosis leads to hypokalaemia and hypokalaemia leads to alkalosis.

-Acidosis leads to hyperkalaemia and Hyperkalaemia leads to acidosis.

-K levels and excretion is influenced by:

~Aldosterone
~Na level
~[H+]
~K blood level

-Circulation k is only 2% of total K

-70% of K is in the muscles
-20% of K is in the brain and large viscera
-10% of K is in the skin
Hypokalaemia A)Increased K+ loss:
1)Gastrointestinal route loss:-
~Vomiting. ~Diarrhoea ~Gastric suction
~Discharge from fistula

2)Renal route loss:-

~Renal tubular acidosis. ~K-losing nephritis
~Hyperaldosterism
~Diuretic drugs (Prevents the reabsorption of K+)

B)Reduced dietary intake of K

~Starvation ~Anorexia nervosa

C)K redistribution:
~Metabolic alkalosis
~Insulin (stimulates Na+ K+ ATPases → pumps K in the cell)
~Decrease Mg (decreases activity of Na+ K+ pump, therefore high K in the
body→the kidney will get rid of all that K → causes a decrease in K in the body)
Symptoms -Variable and not specific:
~Weakness in muscles ~Cramps
~Convulsions (increase repolarisation time period)
~Anorexia (due to low neuromuscular impulses → weakness of smooth GI muscle.
~Irregular heart beat (cardiac arrhythmias)
~Irregular ECG pattern
~Hypotension ~Circulatory failure
Treatments -Replacing lost K:
~Oral or intravenous injection
~Increase intake of K rich food (bananas, orange juice, dates…)
Hyperkalaemia A)Excessive or rapid administration of K

B)Reduced renal excretion of K

~Renal failure with reduced urine output (Anuria→no urine produced)
(Oliguria→little urine produced)
~Addison’s disease (low aldosterone→increase in K reabsorption→ no excretion of
K in urine)
~Severe exercise (breaking of muscle cells releases K)

6
 Rahaf M Alsoufi Lab18

~Na depletion (Na+ K+ ATPase pump can’t function, causes K to remain outside the
cell.)
~Severe haemolysis
~Tissue ischemia (No blood supply to dying tissues→No O2→No ATP
formed→Inactive Na+ K+ ATPase →K remains outside the cell)

C)K Redistribution:
~Massive haemolysis (transfusion problems)
~Metabolic acidosis
~Severe tissue damage
~Epilepsy (hyper kinetic energy – fast repolarisation)
~Dehydration (low blood volume→ low pressure→ low K filtered in the kidney)
~Massive transfusion of store blood (K leaks from RBC while it is stored, effects
infants and children as they have a small blood volume.
Symptoms -Irregular heartbeat and ECG patterns
-Muscle weakness
-Cardiac arrest (constant state of depolarization due to high K inside the membrane
and outside, making repolarization hard, muscle can’t instead of returning to resting
membrane potential)
Treatments -Cause must be known:
~Ca infusion (antagonist to K in the heart- allows normal pattern of depolarization
and repolarization) e.g. NaHCO3, Ca gluconate, etc…
~Insulin (moves K back into the cell by pump)
~Glucose (to stimulate insulin production)
-If all fails, peritoneal dialysis or haemodialysis is done.
Sample -Heparinized plasma is preferred to serum (Clotting process: K leaks from platelets
and RBC, causing falsely elevated K.
-Store at room temperature (cold causes K to leak)
-Avoid haemolysis (ruptured RBC release K)
-Prolonged tourniquet and clenched fists causes falsely decrease K due to the
movement of K back into the cell due to high pressure.
-Urine (24 hour urine sample)
Detection -Flame Emission spectrophotometer (FES)
~Reference method
-Ion Selective Electrode (ISE)
~Direct
~Valinomycin membrane is selective to K
-Ion Selective Conductometry
~Or indirect ISE

7
 Rahaf M Alsoufi Lab18

Chloride (Cl-)
Function -Major extracellular anion
-Involved in many vital (important) body functions:
~Maintaining osmotic pressure
~Maintaining proper body hydration
~Maintaining electric neutrality
~Maintaining electrolyte balance
-Due to its passive roles, it acts as a rate limiting factor for:
~Reabsorption of Na and K (could bind to them forming NaCl or KCl)
~Exchange in bicarbonate
Key Points -Is abundant in many dietary sources, especially in salty food (NaCl)
-Chloride is filtered by the glomerulus, reabsorbed passively by the proximal
tubules bound to Na.
-Excess Cl is excreted in urine and sweat.
-Aldosterone binds Na and Cl in order to reserve it in the body if there is a decrease
in Na or Cl or there is excess sweating
Hypochloraemia A)Excessive loss of chloride:
~Prolonged vomiting ~ Diarrhoea →GI route
~Diabetic ketoacidosis
~Mineralocorticoid deficiency (Hypoaldosteronism)
~Pyelonephritis (inflammation of the kidney)
~Increased bicarbonate (due to metabolic alkalosis)
Hyperchloremia -Occurs when there is excess loss of bicarbonate;
~Metabolic acidosis (increase in H+→causes a decrease a HCO3-→causes a
decrease in Cl excretion)
~Renal tubular acidosis
~Increased Mineralocorticoid (Hyperaldosteronism)
Sample -Fasting serum
-Fasting Plasma (Li heparin)
- Urine (24 hour collection)
-Sweat (for cystic fibrosis)
Detection -Amperometric-coulometric titration
(see table below for ~Reference method
explanation) -Mercurimetric titration
-Colorimetric procedure
-ISE

8
Rahaf M Alsoufi Lab18

Amperometric-coulometric titration
Principle -Just like electrolysis
-Ag+ are generated at a constant voltage from a silver electrode
coulometrically (producing colours)
-Ag combines with the Cl- present in the sample, forming a insoluble
compound.
-Once all the Cl has been consumed and free Ag+ appears, that is the end-
point.
-The time taken to reach the end-point is proportional to the concentration of
Cl
Notes -Highly accurate
-Gelatine (original method) or polyvinyl pyrrolidone (currently used)
prevents the dissociations of AgCl at the indicator electrode →releasing the
Cl and Ag→ increases the time for Cl to all be consumed→falsely elevates
[Cl-]
-pH must remain acidic to prevent the formation of poorly insoluble basic
silver salts.
-Nitric acid is used to provide with the acidic medium and helps in the ionic
conductivity (since it is charged)
-Acetic acid could also be used to provide with the acidic medium and
reduced the solubility of AgCl (makes it more insoluble)
-Suitable for measuring Cl in serum, plasma, sweat and CSF.
-Br-, I-, CN-,SCN-, -SH →interfere →falsely elevates that values.
-Haemolysis, lipideamia and jaundice do not interfere with the
measurements
-Commercial analysers used:
~ Astra 8 (Beckman Instruments)
~Coltove Chloridometer (Buchler Instruments Inc.)

9
 Rahaf M Alsoufi Lab18

Calcium (Ca2+)
Function -Extracellular cation (except a few exceptions where some compartments have
higher Ca2+ inside the cell-such as muscles, heart…)
-In the bone, Ca and Pi form a insoluble crystal (hydroxyapatite crystals) which
provides the bone with strength, and is a major storage area for Ca
-Blood coagulation requires Ca as co-factor IV
-Involved in muscular and heart contractions
-Involved in regulation of membrane ion transport and membrane permeability
-Involved in milk production
-Involved in activation of cAMP (secondary messenger mechanism of hormone
action)
-Involved in cellular secretions
Key Points -98% of Ca is in the bone and teeth
-1% is in the body fluids
-1% is in soft tissues
-Most circulating Ca is in the plasma
-Little or none in RBCs
-Milk and meat are rich in Ca
-Total Ca (tCa):
~60% free calcium (ionized-Ca2+) →physiologically active form
~35% bound to proteins (mainly albumin)
~5% forms a complex with: citrate, oxalate, phosphate…. (minerals)
Factors A)Non hormonal factors:
-pH
Involved in -Total protein
regulation of -Serum Pi (inorganic phosphate) levels
Ca -Oxalate and citrate levels
(See table below for
explanation) B)Hormonal control:
-Parathyroid hormone (PTH)
-1,25-dihydroxycholecalciferol (Vitamin D3)
-Calcitonin
-Thyroid hormone (T3 and T4)
-Glucocorticoids
-Sex hormones
Hypocalcaemia -Hypoparathyroidism
-Mg deficiency
-Vit D deficiency
-Steatorrhea (fat in stool)
-Nephrosis (loss of proteins in urine)
-Nephritis (inflammation-decreases reabsorption of Ca)
-Acute pancreatitis (formation of Ca soap)
-Hyperphosphatemia
-Hypoalbuminemia
-Increased bone uptake (post-parathyroidectomy or post-thyroidectomy)
-Drug therapy (EDTA- chelates Ca , mithramycin- antibiotic , calcitonin)
-Physiological (non-pathogenic):
~Pregnancy
~Lactation (breast-feeding)

10
 Rahaf M Alsoufi Lab18

Symptoms -Increased neuromuscular irritability

-Tetany (muscular spasm due to hypoparathyroidism)
-Cataract (clouding of the lens in the eye which leads to a decrease in vision)
-Depression
-Thyrotoxicosis (hyperthyroidism)

-Lab findings in hypoparathyroidism:

~Decreased PTH, tCa, Ca2+, urinary Ca and Pi
~Increased or normal Pi

-Lab findings in other conditions:

~Decreased bound and total Ca
~Increased PTH (to compensate for the lost Ca)
~ Normal Ca2+
Treatment -Calcium and Vitamin D supplements
Hypercalcemia 1)Hyperparathyroidism:
-Primary hyperparathyroidism (disorder of one or more of the parathyroid glands)
-Secondary hyperparathyroidism (disease outside of the parathyroid glands causing
all of the parathyroid glands to become enlarged and hyperactive)
-Multiple endocrine neoplasia (endocrine glands cancer)

2)Malignancy:
A) Solid tumours:
~Testicles, thyroid, kidney, stomach, liver, bone, thyroid, prostate, bone..
~Ectopic production of PTH by various tumours (other organs produces hormones
are not originally secreted from them)

B) Haematological:
-Multiple myeloma
-Waldenstrom’s macroglobulinemia (slow-growing blood cancer)
-Hodgkin’s disease (lymphoma- cancer of immune system)
-Non-Hodgkin’s disease (cancer that originates in your lymphatic system)
-leukaemia (blood-forming tissue cancer)
-Polycythaemia Vera (slow-growing blood cancer)

3)Non-malignant, non-parathyroid:
A)Increased Ca intake or absorption:
-Hypervitaminosis D
-Milk-alkali syndrome (used in treatment of peptic ulcers)
-Sarcoidosis (inflammatory disease that affects multiple organs in the body)

B)Increased bone resorption:

-Immobilization from the bone (by different factors or hormones mentioned in table)

C)Hyperalbuminemia:
-Dehydration
-Prolonged tourniquet application (falsely elevated/positive Ca)

D)Renal failure:
-Post-dialysis

11
 Rahaf M Alsoufi Lab18

-Post-acute

E)Increased renal absorption:

-Thiazide diuretics (all diuretic drugs prevent the secretion of electrolytes)
-Lithium therapy
-Familial hypocalciuric hypercalcemia (calcium-sensing receptor genetic disorder)

F)Other causes:
-Addison’s disease
-Myxedema (disease caused by severe hypothyroidism)
-Acromegaly (pituitary gland produces too much growth hormone)
-Vitamin A toxicity
-Pheochromocytoma (rare tumour of adrenal gland tissue)
-Idiopathic hypercalcemia of infancy (disorder of calcium metabolism, cause is
Unknown)
-Familial benign hypercalcemia (calcium-sensing receptor genetic disorder)
Symptoms -Symptoms associated with hypercalcemia:-
~Nausea. ~Vomiting. ~Abdominal pain. ~Polyurea
~CaPi kidney stone formation. ~Abnormal calcification of soft tissues.

-Lab findings in primary hyperparathyroidism:

~Increased Ca2+ , tCa , urinary Ca and Pi , PTH
~Decreased serum Pi

-Lab findings in hypervitaminosis D and multiple myeloma

~Increased tCa and Pi
~Normal or slighly decreased PTH
~No change in Ca2+
Sample -Fasting serum
-Fasting heparinized plasma
-Urine (24 hour collection – then neutralized with 10 mL 6M HCL)
-Never use oxalate or citrate (forms insoluble complex)
-Never use EDTA (Ca chelating)
-Avoid prolonged tourniquet application (increases tCa and bound Ca)
-Do not expose whole blood to open air → change in pH affects Ca2+
-Pasture must be lower than standing up.
Detection -Atomic Absorption Spectrophotometry (ASS)
(See table below for ~Reference method
explanation) -Colorimetric procedures
~ O-Cresolphthalein complexone
~Most commonly used in routine clinical labs
-Titration
~EGTA titration
-Precipitation methods
~Oxalate precipitation followed by redox titration
~Precipitation with chloranilic acid
-FES
~Same as Na and K but lacks sensitivity

12
Rahaf M Alsoufi Lab18

Non Hormonal Factors

pH -Increase in pH→decreases Ca2+ and increases bound Ca
-Decrease in pH→increases Ca2+ and decreases bound Ca
~No change in tCa
Total Protein -Hyperproteinaemia→increases bound Ca and increases tCa
Hypoproteinaemia → decreases bound ca and decreases tCa
~No change in Ca2+
Serum Pi level -An inversely proportional relationship between Ca and Pi
-Pi increases → Ca decreases
-Pi decreases → Ca increases
-Uraemia:-
~Pi increases due to renal retention→ Decreases Ca (increases Ca
excretion)
-Increased level of Pi may result in kidney stones (Ca phosphate
precipitation)
-In alkaline urine, phosphate crystals (e.g. amorphous phosphate
crystals)
Oxalate and - An inversely proportional relationship between Ca and citrate and
oxalate.
citrate levels -Increased levels of oxalate and citrate→decreases Ca (due to the
binding and formation of Ca-oxalate or Ca-citrate crystals)
-Increased levels of oxalate and citrate may result in kidney stones
(crystals precipitation)
--In alkaline urine, oxalate crystals (e.g. amorphous oxalate crystals)

13
Rahaf M Alsoufi Lab18

Hormonal Control
Parathyroid hormone -Polypeptide hormone produced by the parathyroid
glands
(PTH) -Released in response to decreased level of circulation
Ca2+

-This results in:

~Increases bone resorption (extraction of Ca from bone
into the circulation)
~Increases Ca reabsorption by renal tubules
~Decreases tubular reabsorption of Pi
~Stimulation of synthesis of (1,25-(OH)2 VitD3)
1,25- -Physiologically active form of vitamin D3
-Stimulated by the action of PTH
dihydroxycholecalciferol -Results in:
(1,25-(OH)2 VitD3) ~Increases Ca2+ absorption from intestine
~Increases bone resorption
~Increases Ca reabsorption by renal tubules

-In chronic disease, 1,25-(OH)2 VitD3 synthesis is

decreased→ decreases absorption of Ca2+
Calcitonin -Polypeptide hormone, produced by parafollicular C
cells in the thyroid, parathyroid and thymus glands.
-Stimulated by increased level of Ca
-Results in:
~Inhibition of bone resorption
~Stimulation of bone formation
~Mobilization of Ca and Pi from ECF into the bone
and bone fluid
~Increases secretion of Ca, Pi and Na in the urine
Thyroid hormone (T3 -Produced by the thyroid gland in response to TSH
excretion
and T4) -Increases mobilization of Ca2+ into the bone (bone
formation)
Glucocorticoids -Produced by adrenal cortex gland
-Main glucocorticoid id cortisol
-Increases bone resorption
-Decreases bone formation
-Decreases levels of 1,25-(OH)2 VitD3
Sex hormones -Androgens (testosterone) in males
-Oestrogen (oestradiol) in female
~> Stimulates Ca and Pi mobilization into the bone
-Stimulates bone formation and maintenance
-At menopause:
~oestrogen levels decreases causing increased bone
resorption
At old age in male:
~Testosterone levels decreases causing increased bone
resorption.

14
 Rahaf M Alsoufi Lab18

Detection Methods For tCa

Principle Interference
AAS -When introduced into a flame, Ca- -The presence of phosphates will bind
containing compounds will to Ca forming an insoluble crystal
dissociate and give free Ca atoms ~Add Lanthanum chloride (LaCl3): it
(Ca2+). prefers Pi and will form a tight complex
-Ca will absorb light (produced with it.
from a hallow cathode Ca lamp) at
a specific wavelength (422.7 nm). -The presence of proteins causes a clog
-The amount of light absorbed is in the tube leading falsely low tCa
directly proportional to the ~Precipitate proteins with trichoroacetic
concentration. acid
~This will improve the precision and
accuracy of the procedure.
O-cresolphthalein -In alkaline pH (10-12) and -Mg reacts with O-cresolphthalein
complexone method diethylamine or 20-amino-2- complexon instead of Ca (leads to
methyl-1-propanol or lysine falsely low Ca reading);
hydroxide buffer, Ca in serum ~Add 8-hydroxyquinoline, binds to Mg
forms a coloured complex with O- and prevent it from reacting with the
cresolphthalein complexon. dye.
-Ca will absorb light at 570nm
-The absorbance is directly
proportional to Ca concentration
EGTA titration -In alkaline medium, the dye
‘calcein’ is fluorescent.
-If a sample contains Ca, calcein
will bind to it and emits the
fluorescent light.
-EGTA has a higher affinity to Ca
than calcein.
-EGTA is added to the cuvette until
all the Ca has become bound to it
and initial fluorescence is restored
(reached end-point).
-The amount of EGTA add until
end-point is reached is directly
proportional to the Ca
concentration.

15
Rahaf M Alsoufi Lab18

Detection Methods For Ca2+

McLean-Hasting Formula ISE
-Used when tCa (mg/dL) and tProtein -Fast blood specimen is collected
(g/dL) are known. anaerobically (to prevent changes to pH) in
-pH of the blood 7.35 – 7.45 a plain tube.
-Measurements of tCa and tProtein are -Sample allowed to collect in room
carried out at 25 °C temperature.
-Protein concentration is within the normal -Immediately after clot, the serum is
range separated (into 1mL tuberculin syringe)
-No protein disorder -Analyse immediately using ISE
-Lipid concentration is normal -Serum can be stored in fridge (for 4 hours)
-NORMAL NORMAL NORMAL or -20°C for 4 days
-Equation:- -Activity of Ca2+ is measured and
1
(6×𝑡𝐶𝑎)−( ×𝑡𝑃𝑟𝑜𝑡𝑒𝑖𝑛) concerted to concentration by comparing it
Ca2+(mg/dL) = 3
to the activity of a standard solution.
6+𝑡𝑃𝑟𝑜𝑡𝑒𝑖𝑛
-Standard solution is isotonic (contains Na+
-Ca concentration conversion: and Mg2+ which interferes with the
~ mmol/L → mg/dL (x4) electrode)

-Protein concentration conversion:

~ g/L →g/dL (÷10)

-Ca2+ conversion:
~mg/dL →mmol/L (÷ 4)

-Important note:
~Before calculating Ca2+, correct tCa using
the albumin equation first.

16
Rahaf M Alsoufi Lab18

Correction of Calcium for Albumin

Why do we need to correct it? -Calcium in serum is bound to proteins
(mainly to albumin)
-The serum test done measures not only
pure Ca but also the ones bound to the
proteins (so protein-albumin- level affects
Ca levels)
-Patients with high or low serum albumin
level will affect the total serum Ca
concentration, leading to false results (not
accurately reflect the physiologically
important ionized Ca concentration)
Equation -If albumin <40 g/L
𝑚𝑚𝑜𝑙
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝐶𝑎 ( ) = (𝑡𝐶𝑎) + (0.02 × [40 − {𝐴𝑙𝑏}])
𝐿

-If albumin >45 g/L

𝑚𝑚𝑜𝑙
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝐶𝑎 ( ) = (𝑡𝐶𝑎) + (0.02 × [45 − {𝐴𝑙𝑏}])
𝐿

Notes -Higher tCa values in children and elderly

-Lower tCa in pregnant and lactating
mothers
-Compare and analyse tCa before and after
correction in order to obtain proper
diagnosis.
Effects of acidosis and alkalosis -Metabolic acidosis:
~Decreases protein binding of calcium
on plasma calcium levels →Increases Ca2+
~H+ competes with calcium for protein
binding

-Metabolic alkalosis:
~Increases protein binding of calcium
→decreases Ca2+

To obtain a good diagnostic picture of Ca:

-Determine tCa, Ca2+, acid-base status, tProtein, albumin level, Pi, Mg, VitD3, PTH
-Check if patient is taking any drugs that affects Ca concentration:
~ phenobarbital and phenotoyn inhibits synthesis of vitamin D3→decreases absorption of
Ca→hypocalcemia
-Check if the patient is under any hormonal therapy

17
 Rahaf M Alsoufi Lab18

Magnesium (Mg2+)
Function -Second most abundant intracellular cation after K
-Important co-factor that requires ATP
-Essential for many biological systems:
~Carbohydrate metabolism
~Nerve conduction
~Neuromuscular contraction
~Blood coagulation
-Co-factor for over 300 enzymes in the body, such as; Na+ K+ ATPase
-Essential for maintaining the macromolecular structure of RNA, DNA and
ribosomes
-Concentration in RBC is greater than in plasma
-Dietary source of Mg:
~Whole grain cereals, legumes, milk, nuts, bananas, leafy vegetables.
Key Points -65% in the bone and teeth (structural role)
-34% intracellular fluids (co-factor for enzymes and is essential for cell
function)
-1% extracellular fluid:
~65% free form (Mg2+)
~22-27% bound to albumin
~8% bound to other globulins
~1-5% complexed with citrate, phosphate, oxalate, carbonate
Factors Involved in 1)pH:
-Decrease in pH→ increases Mg2+ and decreases bound Mg
regulation of Mg -Increase in pH→ decreases Mg2+ and increases bound Mg
~Total Mg is not affected

2)Total protein:
-Hyperproteinemia→ increases bound Mg and increases tMg
-Hypoproteinemia→ decreases bound Mg and decreases tMg
~Mg2+ is not affected

3)PTH, GH
-Increases tubular reabsorption of Mg

4)Thyroid hormones, calcitonin, hyperaldosteronism

-Decrease renal reabsorption of Mg

5)Increase Na+, Increase Ca, Alcohol ingestion

-Decrease renal absorption of Mg
Hypomagnesaemia -Chronic diarrhoea. -Malabsorption. -Hepatic cirrhosis
-Pancreatitis -Ulcerative colitis -Haemodialysis
-Alcoholism. -Diuretic drugs -Insulin administration
-Osmotic diuresis -Infants on high Pi drink
-Hypercalcemia -Hyperaldosteronism. -Hypoparathyroidism
-Hyperthyroidism

Symptoms -Similar to hypocalcaemia;

-Weakness. -Sever neuromuscular irritability (tetany)

18
 Rahaf M Alsoufi Lab18

-Hyperreflexia (overresponsive reflexes)

-Convulsions
-Disorientation
-Cardiac arrythmias
Hypermagnesemia -Oliguria -Dehydration. -Addison’s disease
-Diabetic acidosis. -Chronic renal failure
-Hypothyroidism
-Ingestion of Mg containing gastroenteric medication (Milk of magnesia)
Symptoms -Depressed neuromuscular activity (sedation)
-Depressed cardiac activity
-Respiratory depression (That’s a lot of depression…)
-Nausea. -Drowsiness
-Hypotension -Cardiac arrest. -Death
Sample -Fasting serum
-Fasting Li-heparin plasma
-Separate immediately after collection (to avoid hemolysis and leakage of Mg
from RBCs)
-Urine (24 hour collection-acidify with 6M HCl)
-Do not use EDTA, citrate or oxalate tubes
-Avoid hemolysis (increases Mg)
-Avoid prolonged tourniquet application
-Avoid metal containers (will react with Mg)
Detection -AAS
(See table below for ~Reference method
explanation)
-Colorimetric method (same method as Colorimetric in Ca)
~Mg forms coloured complexes with certain dyes:
~Titan yellow dye, Methylthymol blue (MTB), Calmagite, Magon, Erichrome,
Black T

-Complexometric method (same method as Colorimetric in Ca)

~8-hydroxyquinoline has a high affinity to Mg

-EDTA titration

19
 Rahaf M Alsoufi Lab18

Detection Methods For Mg

Principle Interference
AAS -Same as principle Ca -Presence of phosphate:
-Wave length used, 28.5 nm ~Add Lanthanum chloride
-Sensitive ~Dilute solution
-Specific -Presence of proteins:
-Easy to apply ~Add trichloroacetic acid, centrifuge
and use supernatant
-Urine samples:
~Add lanthanum chloride and dilute

Major Precautions
Colorimetric method Complexometric method
-Ca causes a major interference since it reacts with the Mg agents
-Ba-EGTA is added to chelate (remove) Ca
-The linearity of the measurements obtained has to be checked with a range of Mg standards:
~Beers law only applies to a short range of Mg concentration, not all.

-Therefor;
~AAS is the reference and most used method for Mg, since it is more accurate and precise.

20
 Rahaf M Alsoufi Lab18

Inorganic phosphate (Pi)

Function -Intracellular electrolyte
-Plays a role in:
~structural (bone)
~Energy (ATP, ADP, GTP,GDP, AMO, Creatine phosphate)
~Intermediate metabolism (G-6-P)
~Carbohydrates and lipid metabolism
~Regulation of Ca levels
~Regulation of acid-base balance
~In nucleic acid
~Cell membrane
Key Points -Total Pi = 650g in adult
-80-85% in teeth and bones
-14% in intracellular
-1% in blood and CSF
-Circulating Pi shows circadian (changes according to time) variations:
~Highest level in the late morning
-Lowest level in the evening
-Dietary source: milk, meat (especially fish)
-Maximal absorption in jejunum (favours acidic pH)
-At alkaline pH, Pi forms insoluble complex with Ca
-Types of phosphates in the blood:
~Organic phosphate (ATP, ADP, G-6-P)
~Inorganic phosphate (Pi- physiologically active form)
Hypophosphatemia -Prolonged vomiting
-Prolonged diarrhoea
-Rickets (vitamin D deficiency)
-Osteomalacia (bone softening)
-Fanconi syndrome (defect in reabsorption of Pi and other substances by the
renal tubules)
-Malnutrition
-Renal tubular acidosis
-Treatment of diabetic acidosis
-Extended I.V infusion of dextrose 5%
-Ingestion of phosphate-binding antacids
-Therapy with;
~Acetazolamide
~Insulin
~Adrenalin (epinephrine)
-Non-pathological:
~After meal, when Pi is mobilized into cell for ATP production)
Hyperphosphatemia -Hypervitaminosis D
-Hypoparathyroidism
-Renal failure. ~Diabetic acidosis
-Acromegaly ~Intestinal obstruction
-Non-pathological:
~Bone regrowth after fracture (bone healing)
Sample -Fasting serum
-Separate as soon as clot forms

21
 Rahaf M Alsoufi Lab18

-Analyse immediately:
~Freeze at -20 °C if not assayed soon.
-If freezing is delayed; Organic phosphate dissociates into Pi →falsely
increases Pi
-Avoid haemolysis (falsely increases Pi)
-Never collect after meal (decreased Pi)
-Check for any treatments or drugs.
-Urine (24 hour sample)
Detection -All methods depend on the specific reaction between Pi and ammonium
molybdate
-Acidic medium is used ( in order to prevent; organic phosphate dissociates
into Pi →falsely increases Pi)
-Complex formed (phosphomolybdate complex) will react with a reducing
agent to produce a colour (Molybdenum blue)
-Spectrophotometry is used to detect the absorbance (read at 660 nm)
-Colour (absorbance) is directly proportional to Pi concentration
-Reducing agents used (wave length and colour differs between each one):
~Ascorbic acid
~Stannous Chloride
~Fe2+
~Malachite green
-Presence of proteins acts as an interfering substance:
~Protein precipitation
-Adding the agent to the phosphomolybdate complex must rapid as
phosphomolybdate dissociates fast → falsely decreases results.

22
Rahaf M Alsoufi Lab18

Anion Gap
-Cation in plasma and the body = Anions present
-Not every ion constituent is measured and reported in the lab such as trace elements
-Only:
~[Na2+] [K+] [Cl-] [HCO3-]
-In a healthy individual:
~Na+ and K+ makes up about 95% of the total serum cations
~HCO3- makes up for about 85% of the total serum anions
-Normal ion gap is also due to various unmeasured anions;
~Plasma proteins
~Phosphate
~Sulphate
-An increase in anion gap:
~Presences of excessive amount of either the cations or anions
-Equation:
~If K is measured:
([𝑁𝑎] + [𝐾]) − ([𝐶𝑙] + [𝐻𝑐𝑜3])
~If K is not measure:
([𝑁𝑎]) − ([𝐶𝑙] + [𝐻𝑐𝑜3])
-Sometimes tCO2 (mmol/L) may be used instead of HCO3-
-Results can be normal, high or low, all leading to different clinical diagnosis
-Especially used in differential diagnosis of acid-base disorders.

23