CLINICAL CHEMISTRY 2 (MT 6324) LABORATORY

nd
MS. Canellie Canlas & Asst. Prof Vivian Asuncion | 2 Shifting
d
CALCIUM & MAGNESIUM

CALCIUM
● 5th most common element
● Most prevalent cation in the body

FUNCTIONS
● It is an essential element because it is involved
in several processes such as:
○ Bone mineralization
○ Blood coagulation
○ Neutral transmission
○ Muscle contraction
○ Cardiac contractility & conduction
○ Hormonal secretion

DISTRIBUTION ○ Vitamin D is obtained from sunlight in

● Calcium is distributed in the following:
○ 99% in the bone and the remaining 1%
is found in the blood and ECF.
○ Of this 1%, calcium has three forms:
■ 45% - Ionized form of calcium
or free form of calcium- most
important; physiologically active
form
■ 40% bound to protein primarily
albumin
■ 15% bound to anions such as
citrate, bicarbonate and lactate.
the form of 7-dehydrocholesterol it
would be converted to Pre Vitamin D3
and then into Vitamin D. This Vitamin D
will be hydroxylated by the
25-hydroxylase enzyme from the liver
into 25-hydroxycholecalciferol. This
25-hydroxycholecalciferol is
hydroxylated by 1-α hydroxylase into
1,25-dihydroxycholecalciferol which is
the active form of vitamin D.
○ 1-α hydroxylase is being influenced by
the release of parathyroid hormone
(PTH); when PTH is released, this will
activate the kidneys to secrete 1-α
hydroxylase to convert the inactive of
vitamin D into its active form
1,25-dihydroxycholecalciferol, and this is
the one responsible for the absorption of
calcium ions in the kidneys and intestine

REGULATION
● Calcium is regulated by the following hormones:
● Promote Ca++ absorption
○ PTH (Parathyroid Hormone)
▪ Kidneys
▪ Bone
○ Vitamin D ○ As you can see in this image here, when
▪ Kidneys there is a low calcium concentration in
▪ Intestines the blood, this would trigger the release
of PTH and this hormone will act on the
bone and kidneys to increase Ca
absorption.

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 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING
○ Take note that the form of Ca that is
detected by the glands is the ionized
form.
○ In the bone it promotes bone resorption
while in the kidneys it promotes tubular
reabsorption of Ca. Therefore, leading
to an increased Ca levels. This is where
the Vitamin D comes in.
● Decrease Ca++ levels
○ Calcitonin
▪ total effect of calcitonin is to
have a decrease in calcium
levels
▪ inhibitory to PTH and Vitamin D
▪ when there is an increase in the
ionized calcium levels, this will
trigger the release of calcitonin
from the thyroid gland, and fill
have the following actions:
1. decrease in intestinal
absorption of calcium
ions
2. decrease in bone
resorption
3. a decrease in calcium
reabsorption in the
kidneys
METHODS OF DETERMINATION
Total Ca++ determination
● Colorimetric Method
○ Calcium + R → AR complex (calcium
binds with the reagent/dye to form a
colored complex, this will be the one
measured spectrophotometrically; an
increase in absorbance is directly
proportional with the ion concentration)
■ Ortho-cresolphthalein
complexone (CPC) → in the
experiment, this is the dye being
used
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●
Calcium binds to the
dye to form a colored
complex
(Calcium-oCPC
complex) and this will
be the one that is
measured
spectrophotometrically
● 8-hydroxyquinoline is
used to prevent or
removed any
interference from
magnesium ions
■ Arsenazo III dye
● The same principle
applies when the
Arsenazo III dye is
used. The calcium will
bind to this dye to form
a colored complex
● Atomic absorption spectrophotometer
○ reference method
Ionized Ca++ determination
● Ion Selective Electrode
○ use membranes impregnated with
special molecules that selectively, but
reversibly, bind Ca++ ions
○ an electric potential develops that is
proportional to the ionized Ca++
concentration
● As you can see in this image here, this electrode
(right portion) is the reference electrode while
this one (left portion) is selective for calcium ions
and within the electrodes, there are membranes
inside which are incorporated in the silver
chloride and this one will selectively bind to
calcium ions
● And then an electro-potential will develop
inside which is directly proportional to the
calcium ions
 MT 6324: CLINICAL CHEMISTRY 2 | LABORATORY | 2 ND SHIFTING

MATERIALS/INSTRUMENTATION PROCEDURES
● Automatic pipettor 1. Prepare and label cuvettes properly
● Pipette tips 2. Prepare the working reagent by mixing equal
● Tissue volumes of color reagent and buffer
● Parafilm 3. Follow the micropipetting scheme in the table
● Specimen (controls & unknowns - serum or below:
plasma)
● Spectrophotometer set at 570 nm and 37C Pipette into RB S CN CP U
cuvettes
(body temperature)
Standard - 0.02 mL - - -
● Cuvettes solution
● Centrifuge machine Control - - 0.02 mL - -
● Reagents normal
○ Calcium reagent kit supernatant
Control - - - 0.02 mL -
■ 8-hydroxyquinoline pathologic
● to remove any supernatant
interference from Unknown - - - - 0.02 mL
magnesium ions supernatant
■ O-cresolphthalein complexone Working 1.0 1.0 mL 1.0 mL 1.0 mL 1.0 mL
reagent mL
● dye to be used
Mix well. Measure absorbance of sample and standard
■ Hydrochloric acid against reagent blank within 5 to 50 minutes
● used to release the
calcium ions from the
CALCULATION FOR CALCIUM DETERMINATION
protein carriers
● So before the reaction (𝐴 𝑠𝑎𝑚𝑝𝑙𝑒)
to proceed, the calcium 𝐶 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 (𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
𝑖𝑛 𝑚𝑔/𝑑𝐿 𝑜𝑟 𝑚𝑚𝑜𝑙/𝐿
ions must be released
from the protein carriers Conversion factor: multiply by 0.25 by mg/dL = mmol
by acidification of the
sample REFERENCE RANGES
○ Buffer & stabilizer
■ Lysine buffer (pH 11.1) Reference Range for Calcium
● alkaline pH of the buffer Serum Child 2.20-2.70 mmol/L
because the reaction or < 12 years (8.8-10.8 mg/dL)
will proceed in an Total plasma Adults 2.15 - 2.50 mmol/L
Calcium (8.6 - 10.0 mg/dL)
alkaline pH
Urine 2.50-7.50 mmol/day (100-300 mg/day)
■ Sodium azide (24h) varies with diet
○ Standard: Calcium (2mmol/L or 8 Child 1.20-1.38 mmol/L
mg/dL) (4.8-5.5 mg/dL)
Ionized Serum Adult 1.16 - 1.23 mmol/dL
calcium (4.6-5.3 mg/dL)
SPECIMEN
Plasma Adult 1.03 - 1.23 mmol/L
(4.1-4.9 mg/dL)
Total Ca++ determination Whole Adult 1.15 - 1.27 mmol/L
blood (4.6-5.1 mg/dL)
● Serum or lithium heparin plasma - preferred
● EDTA, oxalate
○ bind Ca++ CLINICAL SIGNIFICANCE
○ Anticoagulant such as EDTA and
oxalate will bind Ca++, therefore leading HYPERCALCEMIA
to a falsely decreased ion ● Primary hyperthyroidism
concentration ○ As mentioned earlier, parathyroid
hormone increases calcium absorption in
the kidneys and in the bone.
Ionized Ca++ determination ○ Increase in parathyroid hormone
● Samples must be collected anaerobically secretion → increase in calcium levels
● Heparinized whole blood - preferred ○ Adenoma of the gland
● Serum collected in sealed evacuated blood ○ Glandular hyperplasia
● Hyperthyroidism
tubes at room temperature ○ Because of the proximity or anatomical
○ may be used if clotting and location of the parathyroid gland to the
centrifugation is done quickly thyroid gland, an increase in the activity

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

of the thyroid gland will also lead to an
increase in the activity of the parathyroid
gland.
● Benign familial hypocalciuria
○ Calcium sensing receptor in the
parathyroid gland and in the kidneys,
normally this calcium sensing receptor
influence the secretion of PTH and the
renal calcium secretion, an increase in
the Ca ions, these calcium sensing
receptors inhibits PTH and the renal
absorption of Ca. Benign familial
hypocalciuria, defect in the calcium
sensing receptor therefore increases in
Ca levels and to hypercalcemia.
● Malignancy (PTH-rp)
○ tumor cells PTH related peptide, acts like
PTH, leading to hypercalcemia
● Multiple myeloma and prolonged immobilization
○ increase in bone resorption
● Increased Vitamin D
○ more absorption of Ca ions
● Thiazide diuretics
○ Inc tubular reabsorption of Ca
● Prolonged immobilization
○ No active form of vitamin D that will be
formed
● Rhabdomyolysis
○ An increase in the release of phosphate
from the cells binding with the calcium
ions in the circulation
● Pseudohypoparathyroidism
○ End-organ resistance so the target
tissues do not response to the PTH
MAGNESIUM
● 4th most abundant cation in the body
● 2nd most abundant intracellular ion
FUNCTIONS
● Cofactor of cellular enzymes
● DNA replication and transcription
● Cellular energy metabolism
● Membrane stabilization
● Ion transport
● Nerve conduction
DISTRIBUTION
● 53% = bone
● 46% = muscle, other organs and soft tissues
● <1% = serum, RBCs
○ ⅓ bound to proteins (albumin)
○ ⅔
▪ 61% free/ionized state
(physiologically active)
▪ 5% complexed to other ions
(PO4-, citrate)

REGULATION
● Intestinal absorption
● Kidneys
○ Major organ that is responsible for the
overall regulation of Mg++ (reabsorb or
excrete)
○ Renal absorption:
▪ 25-50% in PCT(Proximal
Convoluted Tubule)
HYPOCALCEMIA ▪ 50-60% in Ascending limb
● Primary Hypoparathyroidism ▪ 2-5% in DCT(Distal Convoluted
○ Decrease in PTH, decrease absorption of Tubule)
Ca ions Note: Majority are present in the ascending limb in
● Hypomagnesemia
○ Inhibition of PTH secretion
contrast to other ions which are absorbed in the PCT
● Hypermagnesemia ● PTH increases in renal and intestinal Mg++
● Hypoalbuminemia reabsorption
○ Primarily bound to albumin, decrease in ○ but changes in ionized Ca++ have a
albumin decreases Ca greater effect on PTH secretion
● Acute pancreatitis ● Aldosterone and Thyroxine increase renal
○ Increase of lipase, lipase release Free excretion of Mg++
FA, binds to Ca, leading to hypocalcemia
● Vitamin D deficiency
○ Lead to a decrease in absorption of
calcium ions
● Renal disease

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

METHODS OF DETERMINATION SPECIMEN

● Non hemolyzed serum or lithium heparin
Total Ca++ determination plasma - preferred
● EDTA, oxalate, and citrate
● Colorimetric method
○ Not recommended because it will bind
○ The principle is the same as with
Mg++ causing falsely decreased ion
calcium determination. Magnesium ion
concentration.
binds with a dye to form a colored
● 24-hour urine
complex and the colored complex is the
○ Preferred because of diurnal variation
one read spectrophotometrically.
○ The increase in absorbance is directly
proportional with the added PROCEDURES
concentration 1. Prepare and label cuvettes properly
○ Calmagite method 2. Prepare the working reagent by mixing equal
▪ Mg++ + Calmagite → reddish volumes of color reagent and buffer
violet complex (read at 532 nm) 3. Follow the micropipetting scheme in the table
○ Formazan dye below:
▪ Mg++ + Formazan dye →
Pipette into RB S CN CP U
colored complex (read at 660 cuvettes
nm) Standard - 0.01 mL - - -
○ Methylthymol blue or Magon or solution
Xylidyl blue Control - - 0.01 mL - -
normal
▪ Mg++ + Methylthymol blue → supernatant
colored complex Control - - - 0.01 mL -
▪ This method is used in the pathologic
experiment in the laboratory supernatant
manual. Unknown - - - - 0.01 mL
supernatant
● Atomic Absorption Spectrophotometer Working 1.0 1.0 mL 1.0 mL 1.0 mL 1.0 mL
○ reference method reagent mL
Mix and incubate for 10mins at 37C. Measure the
Ionized Mg++ determination absorbance of the sample and the standard against the
reagent blank within 60 minutes
● Ion Selective Electrode
CALCULATION FOR MAGNESIUM
MATERIALS/INSTRUMENTATION
● Automatic pipettor DETERMINATION
● Pipette tips
(𝐴 𝑠𝑎𝑚𝑝𝑙𝑒)
● Tissue 𝐶 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 (𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
𝑖𝑛 𝑚𝐸𝑞/𝐿 𝑜𝑟 𝑚𝑚𝑜𝑙/𝐿
● Parafilm
● Specimen (controls & unknowns - serum or Conversion factor: multiply by 0.5 by mEq/L = mmol
plasma) ● Reference Range:
● Spectrophotometer set at 520 nm and 37C ○ Serum
● Cuvettes ■ 0.63-1.0 mmol/L
● Centrifuge machine ■ 1.26-2.10 mEq/L
● Reagents
○ Magnesium Reagent Kit CLINICAL SIGNIFICANCE
■ potassium carbonate
■ GEDTA (Glycoletherdiamine -
HYPERMAGNESEMIA
N, N, N’. N’ - tetraacetic acid)
● Acute/chronic renal failure
■ xylidyl blue ● Hypothyroidism
■ activators ● Hypoaldosteronism
○ Buffer & Stabilizer ● Hypopituitarism
■ TRIS buffer (pH 11.0) ○ There’s a decrease in the
■ Sodium azide thyroid stimulating
○ Standard: Magnesium (1.03 Decreased hormone therefore leading
mmol/L or 2.5 mg/dL; may vary) excretion to hyperthyroidism

Thyroxine and aldosterone increases

renal excretion of magnesium ions.

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

When there is a decrease of these ● Digitalis (interferes with Mg

hormones, these will lead to a decrease reabsorption)
in the excretion of magnesium ions Miscellaneous ● Excess lactation
leading to hypermagnesemia. ● Pregnancy
Increased ● Antacids
intake ● Enemas
(Medications ● Cathartics
that contain ● Therapeutics
magnesium ○ eclampsia
ions) ○ cardiac arrhythmia
● Dehydration
○ Reversible
Miscellaneous ● Bone carcinoma
● Bone metastases
○ both causing increased
bone loss leading to
increased Mg
HYPOMAGNESEMIA
Reduce intake ● Poor diet
● Prolonged magnesium
○ deficient IV therapy
○ chronic alcoholism
Decreased ● Malabsorption syndrome
absorption ● Surgical resection of small
intestine
● Nasogastric suction
● Pancreatitis
● Vomiting
● Diarrhea
● Laxative abuse
● Neonatal hypomagnesemia
● Primary Deficiency
○ selective
malabsorption of the
Mg ion in the intestine
● Chronic congenital
hypomagnesemia
○ defect in the transport
protein for magnesium
Renal
● Tubular disorder
● Glomerulonephritis
● Pyelonephritis

Leads to increased excretion of Mg ions

Endocrine
● Hyperparathyroidism
● Hyperaldosteronism
● Hyperthyroidism
● Hypercalcemia
Increased Leads to increased renal excretion of Mg
excretion ions

● Diabetic ketoacidosis
○ Mg ions are lost in the
urine along w/
glucosuria
Drug Induced
● Diuretics
● Antibiotics
● Cyclosporin (inhibits renal
absorption of Mg ions)

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