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 0,3mmol/24h

Food 25mmol/24h

Bone 99% 25000mmol
Exchange 500mmol/24h Formation 7,5mmol/24h Resorption 7,5mmol/24h Reabsorption 234mmol/24h Secretion 6mmol/24h ECF 22,5mmol Absorption 12mmol/24h
Plasma 9 mmol

GUT

Glomerular Filtration 240mmol/24h

Distribution of calcium
Urine 6mmol/24h

Faeces 19mmol/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

Diffusible 54%

Nondiffusible (bound to protein) 46%

Free ions
(physiologically active) 47%

Complexed
(bicarbonate, citrate, phosphate) 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. and hence hypercalcaemia, is venous stasis during blood sampling. can also increase the total calcium, however, hypercalcaemia is due to secretion of calciummobilizing substances by the tumour cells.

A common cause of apparent hyperproteinaemia,

The increase in ‫-ץ‬globulin in patient with myeloma

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 adult Ionized calcium At birth Neonate Child adult 2.2-2.7 2.1-2.6 1.3-1.6 1.2-1.5 1.2-1.4 1.2-1.3 8.8-10.7 8.4-10.2 5.2-6.4 4.8-5.9 4.8-5.5 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
Stimulate: Osteoclastic activity (release Ca²,HPO4)

kidney
Promotes: Ca² absorption HPO4 excretion 1-α-hydrolase activation

Calcium homeostasis
 Calictriol:

Circulating 25-OH vit D
(INACTIVE)

1-α- hydroxylase

+
1,25(OH)2 vit D

intestine vitD promotes: intestinal absorption of Ca² and PO4

Kidney vitD promotes: renal reabsorption of Ca² and HPO4

Calcium homeostasis
Calcitonin:

This polypeptide hormone, produced by Ccells 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
Artefactual: blood collected into EDTA or oxalate. Associated with low PTH: •Hypoparathyroidism. •Hypomagnesaemia. •Hungry bone syndrome. •Neonatal hypocalcaemia. Associated with high PTH: •Deficiency and disorders of vitamin D. •Acute pancreatitis. •High PO4 intake. •Massive transfusion with citrated blood. •Acute rhabdomyolysis.

Clinical features
• Behavioural disturbance. • Numbness and paraesthesae. • Muscle cramps and spasms. • Laryngeal stridor. • Convulsions. • Cataracts chronic. • Basal ganglia calcification. • Papilloedema. • Prolonged QT on ECG.

Hypercalcemia:
CAUSES Clinical features
 Weakness, tiredness, weight

COMMON: Malignant disease. Primary hyperthyroidism. LESS COMMON: Thyrotoxicosis. VitD intoxication. Sarcoidosis. Renal transplantation. UNCOMMON: Milk-alkali syndrome. tuberculosis. Acute adrenal failure.

loss.  Mental changes.  Abdominal pain.  Anorexia, nausea and vomiting.  Polyuria, dehydration and renal failure.  Short QT on ECG.  Cardiac arrhythmias and hypertension.  Corneal calcification, vascular calcification.

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 orthocresolphthalein 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
If acute, is the patient: • Receiving citrated blood? • A neonate (1-3day)? • Recovering from surgery? • Having pancreatitis? • Having drugs? Mg low: PTH release is inhibited

Measure serum Mg
Mg high: Parathyroid gland are suppressed Mg normal

Measure serum PTH
PTH elevated: CRF (PO4 high) Vit D defect (PO4 low) PTH low/normal: Primary or post surgical hypoparathyroidism

Laboratory testing of calcium
Increased ionized Ca
Is there is: • 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.

Distribution: Phosphate
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) 25%

Organic 75%

Free ions 22.5%

Bound to anions 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 S.Phosphate:
Newborn Infant Child Adult male Adult female 1.8-3.1 1.5-2.1 1.5-1.8 0.7-1.2 0.9-1.3 5.5-9.5 4.5-6.5 4.5-5.5 2.3-3.7 2.8-4.1

mg/dl

Hypophosatemia:
Causes: Vitamin D deficiency, Primary hyperparathyroidism, Nutrition with inadequate phosphate, Diabetic ketoacidosis (recovery phase), Renal tubular disease, Respiratory alkalosis. Clinical features: Muscles weakness, Respiratory and myocardial insufficiency, Hepatocellular damage. Convulsions.

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 H2SO4 + + phosphorus Ammonium molybdate Phosphomolybdate complex

The absorbance at 340 nm is directly proportional to the amount of inorganic phosphorus concentration.

Low serum PO4 low Urinary PO4 excretion GI losses Low or normal high Renal losses Serum Ca² increased

aldistribution

Diuretics; Renal tubular defects; Recovery from burns; Inadequate vit D.

hyperparathyroidism

High serum PO4 <25 ml/min GFR >30-40 ml/min

Renal failure (acute or chronic) Increased (>1500mg/day)

Urinary PO4 Normal (<1500mg/day)

Increased PO4 intake; Cell destruction; Cellular redistribution.

Hypoparathyroidism; Other endocrine disorders