This document summarizes phosphate distribution and regulation in the body. It discusses: 1) Phosphate is found throughout the body, mostly in bone and associated with proteins in blood. Serum phosphate is normally 3-4 mg/dL, with some bound to proteins and some free. 2) Phosphate functions include bone formation, energy storage in ATP, enzyme cofactors, and acid-base buffering. 3) Parathyroid hormone and calcitriol regulate phosphate levels by increasing renal excretion and intestinal absorption, respectively, to maintain normal serum levels. 4) The document also describes causes and consequences of hypo- and hyperphosphatemia, and the principle of the ANSA method for measuring

1. Estimation of serum total phosphate by ANSA method
Amit Jha
Lecturer
UCMS, Bhairahawa

2. Distribution of phosphate in body
• Found in every cell of body.
• Total body phosphate content= 700gm (25mole)
– (both in organic & inorganic form)
Mostly in combination with Ca in bone & teeth
10% is in association with protein, Lipid & Carbohydrate in muscle & blood
Remaining is distributed in various chemical compound.

3. Distribution of phosphate in blood
• Plasma contain both inorganic & organic phosphate.
• Phosphate: intracellular ion
• [Phosphate] of whole blood =40mg/dl
• Serum phosphate= 3-4 mg/dl
» Protein bound [10%]
» Complexed with Na, Ca, Mg [35%]
» Free [55%]

4. Functions of phosphate
• Bone & teeth formation.
• ATP [energy currency of cell] [ATP → ADP + Pi]
– Involved in many energy intensive physiological
functions.
– Muscle contraction
– Neurological functions
– Electrolyte transport
• Essential for absorption & metabolism of carbohydrate.

5. Functions of phosphate
• Essential component of cyclic nucleotides. (cAMP, NADP)
• Present in many coenzymes (TPP, NADP, PLP).
– Thus req. for activity of many enzymes.
• Phosphate is essential for formation of:-
» Phosphoproteins
» Phospholipids
» Nucleic Acids

6. Functions of phosphate
• Acts as buffer. [H2PO4
- &HPO4
--]
H2PO4
- : HPO4
--
In acidosis 1:1
At pH 7.4 1:4
In alkalosis 1:9

7. Regulation of phosphate level
• Role of PTH
– ↑es Phosphate excretion via kidney (phosphaturic
effect)
• Role of Calcitriol
– ↑es intestinal Phosphate absorption
• Role of calcitonin
– ↑es phosphate excretion via kidney.
– ↓es bone resorption &↑es bone mineralization
[phosphate deposition into bone]

8. ↓ in Plasma Phosphate
↓
Stimulates 1-α-hydroxylase
↓
↑es Calcitriol
↓
↑es Intestinal absorption of Phosphate
↓
Plasma Phosphate ↑es towards normal

9. ↑ in Plasma Phosphate
↓
Stimulates PTH
↓
↑es Phosphate excretion via kidney
↓
Plasma Phosphate ↓es towards Normal

10. Principle: Inorganic Phosphorus by
(ANSA Method)
Phosphorus + Ammonium molybdate
Acidic pH
Ammonium Phosphomolybdate
Heteropoly molybdenum Blue (680 nm)
1-Amino 2-Naphthol, 4- Sulfonic acid (ANSA)

11. Procedure:
PPF: 1 ml Serum + 4 ml 10 % TCA,
mixed and centrifuge
Blank Standard Test
Filtrate ---- ---- 2.5 ml
Standard (4 mg/dl) ---- 2.5 ml ----
D/W 2.5 ml ---- ----
Molybdate solution 0.5 ml 0.5 ml 0.5 ml
ANSA 0.2 ml 0.2 ml 0.2 ml
Mix well and allow to stand at 10 minutes and add 1.5 ml of D/W to each tube. Mix
and read OD at 680 nm (Red filter)
Serum Phosphorus = OD T – OD B X 4 mg/dl
OD S – OD B

12. • HYPOPHOSPHATEMIA
– [Phosphate] : < 2.5 mg/dL (0.8 mmol/L).
– Mechanisms causing hypophosphatemia:
» Inadequate intake
» ↓ed intestinal absorption
» ↑ed excretion
» Shift from ECF to ICF

13. • Inadequate intake & ↓ed intestinal absorption
13
Long-term low phosphate intake in setting of
sudden ↑ in intracellular phosphate requirements
eg: refeeding syndrome
Intestinal malabsorption
Vit. D def. ↓ Calbindin  ↓ intestinal absorption
Ingestion of Al/Mg antacids Binds to dietry phosphate ↓ absorption

14. 14
• ↑ed excretion
Hyperparathyroidism ↑ in PTH  inhibit phosphate reabsorption in PCT
Forced saline diuresis Inhibitory effect on PCT transport processes
Fanconi syndrome

15. • Renal Phosphate wasting
15

16. • Shift from ECF to ICF
16
Carbohydrate diet Entry of PO4 & Glucose into cell
Insulin therapy Entry of PO4 & Glucose into cell
Short-term ↑ in cellular demand eg: Hungry bone syndrome
Acute respiratory alkalosis Activation of PFK  ↑ed glycolysis

17. • Hyperphosphatemia
 [Phosphate] : > 4.0 mg/dL
– Mechanisms causing hyperphosphatemia:
• ↑ed phosphate intake
• ↓ed phosphate excretion
• Shift from ICF to ECF

18. • ↑ed intake
– IV or oral administration
– Phosphate containing laxative

19. • ↓ed renal phosphate excretion
19
Acute or chronic renal failure ↓ GFR → ↓ excretion
Hypoparathyroidism ↓ PTH → ↓ excretion

20. • Shifts from ICF to ECF
20
Transcellular shift Cell lysis
•Lactic acidosis •Rhabdomyolysis
•DKA •Leukemia
•Respiratory acidosis •Lymphoma
•Cytotoxic therapy