The electrolytes calcium and phosphate

have many roles in:

•the structure and function of bones,
•the function of membranes,
•the activation of hundreds of enzymes
involved in genetic regulation, muscles
contraction, blood coagulation, and energy
use.
The clinical importance: Ca² and PO4 are
used for diagnosing:
•parathyroid dysfunction,
•hypercalcemia of malignancies,
•monitoring surgery, critical care and
 Skin Food
0,3mmol/24h 25mmol/24h

Bone 99%
25000mmol
Exchange Absorption
500mmol/24h 12mmol/24h
ECF
22,5mmol Plasma
9 mmol GUT
Formation
7,5mmol/24h
Secretion
Resorption
6mmol/24h
7,5mmol/24h

Reabsorption Glomerular
234mmol/24h Filtration
240mmol/24h

Faeces
19mmol/24h
Distribution
of calcium
Urine 6mmol/24h
BONE CALCIUM
Bone consists of osteoid, on which is deposited
inorganic hydrated calcium salts (hydroxyapatite).
(Ca10(PO4)6(OH)2)

Osteoid synthesis by osteoblasts requires

adequate calcium, phosphate, ALP and this
process is controlled by hormones (GH and
Cytokines).

Bone provides an important reservoir of calcium

and phosphate.

The amount of intracellular calcium is relatively

low, (5000 -10000) lower than blood.
 PLASMA CALCIUM
Total calcium
2.3 – 2.6mmol/l

Nondiffusible
Diffusible (bound to protein)
54% 46%

Free ions Complexed

(physiologically active) (bicarbonate, citrate, phosphate)
47% 7%
Plasma calcium
In alkalosis, there is increase in calcium binding to
albumin and calcium complex formation. As a result, the
concentration of ionized calcium falls, and this may
produces clinical signs of hypocalcaemia although total
plasma calcium concentration is unchanged.

In an acute acidosis, the reverse effect is observed, the

ionized calcium concentration is increased.

Measurement of total plasma calcium is satisfactory for

most clinical purposes because of speed with which
results are available. (blood transfusion, surgery with
extracorporeal bypass.
Plasma calcium

Changes in plasma albumin concentration will

affect total calcium concentration, leading to
misinterpretation of results in both
hypoproteinaemic and hyperproteinaemic cases.

A common cause of apparent hyperproteinaemia,

and hence hypercalcaemia, is venous stasis during
blood sampling.

The increase in ‫ץ‬-globulin in patient with myeloma

can also increase the total calcium, however,
hypercalcaemia is due to secretion of calcium-
mobilizing substances by the tumour cells.
Plasma calcium
For albumin < 40 g/l :
Corrected calcium =Ca + 0.02 X (40 – alb)
mmol/l

For albumin > 40 g/l :

Corrected calcium =Ca – 0.02 X (alb – 45)
mmol/l
Reference ranges for calcium
mmol/l mg/dl
Total calcium
Child 2.2-2.7 8.8-10.7
adult 2.1-2.6 8.4-10.2

Ionized calcium
At birth 1.3-1.6 5.2-6.4
Neonate 1.2-1.5 4.8-5.9
Child 1.2-1.4 4.8-5.5
adult 1.2-1.3 4.6-5.3
Physiologic functions of calcium
ions

The flow of calcium ions into the

myocardial cells, helps control cardiac
contraction and rhythm by binding to
contractile proteins (troponin, calmodulin).

Calcium ions also serve as second

messengers in controlling the secretion of
many enzymes (insulin, aldosterne,
vasopressin, renin).
PTH: Parathyroid gland

hypercalcemia – + hypocalcaemia

PTH SECRETION

Bone kidney
Promotes:
Stimulate:
Ca² absorption
Osteoclastic activity
HPO4 excretion
(release Ca²,HPO4)
1-α-hydrolase activation
Calcium homeostasis
 Calictriol: Circulating 25-OH vit D
(INACTIVE)

1-α- hydroxylase +

1,25(OH)2 vit D

intestine Kidney

vitD promotes: vitD promotes:

intestinal absorption renal reabsorption
of Ca² and PO4 of Ca² and HPO4
Calcium homeostasis
Calcitonin:

 This polypeptide hormone, produced by C-

cells of the thyroid gland in response to a
marked hypercalcemia.

Calcitonin reduces calcium by inhibiting

osteoclast activity and inhibiting the action
of both PTH and vitamin D.
 Hypocalcaemia:
Causes
Clinical features
Artefactual: blood collected into EDTA or
oxalate.
• Behavioural
Associated with low PTH: disturbance.
•Hypoparathyroidism. • Numbness and
•Hypomagnesaemia. paraesthesae.
•Hungry bone syndrome. • Muscle cramps and
•Neonatal hypocalcaemia. spasms.
• Laryngeal stridor.
• Convulsions.
Associated with high PTH:
• Cataracts chronic.
•Deficiency and disorders of vitamin D.
•Acute pancreatitis.
• Basal ganglia
calcification.
•High PO4 intake.
• Papilloedema.
•Massive transfusion with citrated blood.
•Acute rhabdomyolysis.
• Prolonged QT on ECG.
Hypercalcemia:
CAUSES Clinical features
COMMON:  Weakness, tiredness, weight
Malignant disease. loss.
Primary hyperthyroidism.  Mental changes.
 Abdominal pain.
LESS COMMON:  Anorexia, nausea and
Thyrotoxicosis. vomiting.
VitD intoxication.  Polyuria, dehydration and
Sarcoidosis. renal failure.
Renal transplantation.  Short QT on ECG.
 Cardiac arrhythmias and
UNCOMMON: hypertension.
Milk-alkali syndrome.  Corneal calcification,
tuberculosis. vascular calcification.
Acute adrenal failure.
Laboratory testing of calcium
 Methods:
1. Atomic absorption spectroscopy.
2. Ion selective electrode (ionized calcium).
3. Titrimetric method.
4. Colorimetric methods :
I. Precipitation method.
II. Ortho-cresolphthalein complexone,
Arsenazo III.
5. Turbidometric method ( for urine).
Laboratory testing of calcium
1. Atomic absorption spectroscopy:
This method involves introducing a dilute sample
into an air-acetylene flame and measuring the
absorption of light at 422.5nm.

2. Ion selective electrode (ionized

calcium):
These electrodes use membranes impregnated
with molecules (ionophores) that selectively bind
calcium ions. An electric potential develops across
the membrane that is related to ionized calcium
concentration.
Laboratory testing of calcium
3. Titrimetric method:
 A diluted specimen is titrated against
EDTA which removes calcium from the
titrating fluid and changes the colour of an
indicator (calcon dye from purple to blue)
as response to the presence or absence of
calcium.
 Calculations:
Serum calcium conc. = Tt / Tstd X conc. Of std
Tt: volume of titrant EDTA needed for the test serum.
Tstd: volume of titrant EDTA needed for the standard.
 Laboratory testing of calcium
4. Colorimetric methods:
I. Precipitation method:
 Calcium is precipitated from the
specimen as calcium chloranilate by adding
a saturated solution of sodium chloranilate.
 The precipitate is washed (with isopropyl
alcohol) and then treated with EDTA, which
binds the calcium and releases the
cholranilic acid (purple in colour) which can
be read at 520nm against an EDTA blank .
Laboratory testing of calcium
4. Colorimetric methods:
II. Ortho-cresolphthalein complexone, Arsenazo III:
This method is based on the complexometric
reaction between calcium and the dye ortho-
cresolphthalein complexone. Another method
uses the dye Arsenazo III.

CALCIUM + Arsenazo III ALK calcium-Arsenazo complex (purple)

The colored complex is absorbed at 650nm.

Laboratory testing of
calcium
5. Turbidometric method ( for urine):
 When Sulkowitch reagent (oxalate
buffered with acetate) is added to urine, it
will produce a white precipitate of calcium
oxalate without co-precipitation of other
urinary constituents.

 This semiquantitative test should only

be used for quick screening.
 Laboratory testing of calcium
Decreased ionized Ca
Measure serum Mg
If acute, is the patient:
• Receiving citrated blood?
• A neonate (1-3day)?
• Recovering from surgery?
• Having pancreatitis?
• Having drugs?

Mg normal Mg low:
Mg high:
Parathyroid gland PTH release is inhibited
are suppressed
Measure serum PTH

PTH elevated: PTH low/normal:

CRF (PO4 high) Primary or post surgical
Vit D defect (PO4 low) hypoparathyroidism
Laboratory testing of calcium
Is there is:
Increased ionized Ca • Renal failure?
• Drugs (vita D, Li, thiazides)?
• Hyperthyroidism?
• Low cortisol?
Measure serum PO4 and PTH

• PTH high;PO4 normal or high: possible renal failure.

• PTH normal or high; PO4low: hyperthyroidism.
• PTH normal or low: malignancy, Sarcoidosis.
• PTH low; PO4 low: dietary excess of Ca.
Phosphate
Distribution:
About 80% of the(700-800) gm of body phosphate is
contained in bone, in the form of hydroxyapatite.

Blood phosphate is either absorbed from dietary

sources or reabsorbed from bone.

Most phosphate is found within cells, and the transport

of glucose into cells is accompanied by an influx of
phosphate, which is used in the synthesis of
phosphorlyted compounds.
Plasma phosphate

Plasma phosphate
12mg/dl

Inorganic
(phosphorus) Organic
25% 75%

Free ions Bound to anions

22.5% 2.5%
Biochemical functions
Compounds of phosphorus are in all
cells participating many processes:
As component of: nucleotides(DNA,RNA),
phospholipids and most enzymes.

As biochemical energy reservoirs: ATP,

creatine phosphate and phosphoenol
pyruvate.
Homeostasis:
The kidney plays an important role in the
regulation of phosphate concentration by
several factors including:
acid –base status (normally by reabsorb
90% of phosphate filtered at the
glomerulus),
Vitamin D (promotes renal reabsorption,
and intestinal absorption, of phosphate),
PTH inhibit renal reabsorption of
phosphate.
Reference ranges
mmol/l mg/dl
S.Phosphate:
Newborn 1.8-3.1 5.5-9.5
Infant 1.5-2.1 4.5-6.5
Child 1.5-1.8 4.5-5.5
Adult male 0.7-1.2 2.3-3.7
Adult female 0.9-1.3 2.8-4.1
Hypophosatemia:
Clinical features:
Causes: Muscles weakness,
Vitamin D deficiency, Respiratory and
Primary myocardial
hyperparathyroidism, insufficiency,
Nutrition with Hepatocellular
inadequate phosphate, damage.
Diabetic ketoacidosis Convulsions.
(recovery phase),
Renal tubular disease,
Respiratory alkalosis.
 Hyperphosphatemia:
Causes:
Renal failure,
Hypoparathyroidism,
Acromegaly,
Excessive phosphate intake,
Vitamin D intoxication,
Laboratory testing of phosphate

1. Colorimetric method:
Determinations are usually made only of inorganic
phosphorus after removing the protein by
precipitation with trichloroacetic acid (TCA).

Phosphate has been measured by methods in

which molybdate react with phosphate to form
complex molecules of phosphomolybdate. Several
reducing agents have been developed:
 Laboratory testing of
phosphate
Fiske and Subbarow used1-amino-2-naphthol-
4-sulfonic acid (ANS), to form molybdenum
blue with high absorptivity at 660 nm.

 Stannous chloride and ferrous ammonium

sulfate are also been used.

 Garber and Miller selected a method uses

semidine HCl as a reducing agent.
 Laboratory testing of
phosphate
2. Spectrophotometeric method:
(without protein precipitation)

Inorganic H+2SO4 + Ammonium Phosphomolybdate

phosphorus molybdate complex

The absorbance at 340 nm is directly proportional to

the amount of inorganic phosphorus concentration.
 Low serum PO4

low Urinary PO4 excretion high

aldistribution GI losses Renal losses

Serum Ca²
Low or
normal increased

Diuretics; hyperparathyroidism
Renal tubular defects;
Recovery from burns;
Inadequate vit D.
 High serum PO4

<25 ml/min GFR >30-40 ml/min

Renal failure Urinary PO4

(acute or chronic)

Increased Normal
(>1500mg/day) (<1500mg/day)

Increased PO4 intake;

Cell destruction; Hypoparathyroidism;
Cellular redistribution. Other endocrine disorders