Pi Dr H.

Khouja

Inorganic Phosphate (PHOSPHORUS) (Pi)

Physiology and Function

• Total body Pi ~ 650g in adults
• Major site of accumulation is the bone and teeth 80-85%
• Intracellular 14%
• Blood & ECF 1%
• Circulating Pi show circadian variations
o Highest levels in the late morning
o Lowest levels in the evening
• Major functions
o Structural (bone)
o Energy ATP, ADP, GTP, GDP, AMP, Creatine phosphate
o Intermediary metabolism G-6-P; F-6-P
o Carbohydrate and lipid metabolism
o Regulation of Ca levels
o Regulation of acid-base balance
o In nucleic acids
o Cell membrane
• Dietary sources: milk, meat (esp fish)
• Maximal absorption in the jejunum (small intestine) [favors acidic pH]
• At an alkaline pH, Ca & Pi form insoluble complex
• Phosphate in blood
• Organic phosphates ATP, ADP, G-6-P….etc. [10X more concentrated than Pi]
• Inorganic phosphate (Pi) physiologically active
o At pH 7.40 1 (H3PO4-) : 4 (H2PO42-)
o HPO43- negligible amounts.

Clinical Correlations
• Hyperphosphataemia
o Hypervitaminosis D -Hypoparathyroidism -Renal failure
o Acromegaly -Diabetic acidosis -Intestinal
obstruction
o Non-pathological bone regrowth during healing of fracture

• Hypophosphataemia
o Prolonged vomiting -Prolonged diarrhea
o Vitamin D deficiency rickets -Osteomalacia
o Hyperparathyroidism
o Fanconi Syndrome (defect in the reabsorption of Pi & other metabolites by
the renal tubules)
o Malnutrition
o Renal tubular acidosis
o Treatment of diabetic acidosis
o Extended I.V infusion of dextrose 5%
o Ingestion of phosphate-binding antacids
o Therapy with
 Acetazolamide
 Insulin

1
Pi Dr H. Khouja

 Adrenalin (epineherin)
o Non-pathological immediately after a meal due to its mobilization into
cells for energy requirements & intermediary metabolism

Determination of Phosphate

Specimen :
• Fasting blood serum. Separate serum as soon as clot forms. Analyze immediately or
freeze at -20oC if not assayed soon.
o If freezing is delayed, organic phosphate may dissociate to Pi thus falsely
increasing Pi
o Avoid haemolysis  false increase in Pi
o Never collect after a meal  decrease in Pi
o Check for any treatment or drug intake
• Urine 24hr sample

Methodology
Principle:
All the methods depend on the specific reaction of Pi with ammonium molybdate in acid medium.
The resulting complex mixture is then reacted with a reducing agent which usually produce a
coloured product which is read in the spectrophotometer. Colour intensity is directly proportional to
the concentration of Pi.

Pi + (NH4)6Mo7O24 ------{H2SO4}--→ (NH4)3[PO4(MoO3)]12 (PHOSPHOMOLYBDATE)

(NH4)3[PO4(MoO3)12 + Fe2+ → molybdenum blue (Absorbance read at 660nm)

Absorbance α Pi Concentration

Notes:-
 There are several reducing agents available such as; ascorbic acid; stannous
chloride, Fe2+, malachite green (wave length and colour are different for each reducing agent)
 The pH of the reaction must be maintained at an acidic pH to prevent
dissociation of organic phosphate into Pi and falsely increase the result
 Protein precipitation is quite important as it eliminates interference and
matrix effects
 The addition of the reducing agent to the phosphomolybdate complex must
be rapid to prevent any dissociation of the phosphomolybdate and give false decreased results

• Normal range Adults 0.90 – 1.45 mmol/L

Children 1.45 – 2.09 mmol/L