"Serum Phosphorus estimation"

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