Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

2. Pipette out 25, 50, 75, 100, 125, 150, 175, and 200μl of 4 mg % calcium standard
   into the tubes labelled as S 1 to S 6. Add 50μl undiluted serum and urine samples
   into tubes Ts and TU.


3. Then make thefinal volume of all the tubes 200μl by adding distilled water. Add
   2 ml cresolphthalein complexone reagent to all the tubes and mix well.


4. Then add 2.5 ml diethylamine buffer to all the tubes, mix well, and measure
   absorbance at 575 nm.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 TS TU

Calcium standard

(μl)

• 25 50 75 100 125 150 175 200 ––

Calcium amount

(μg)

• 123456 78––

Serum/urine (μl) ––––––– 50 50

Distilled water

(μl)

200 175 150 125 100 75 50 25 – 150 150

Cresolphthalein

complexone

reagent (ml)

2 222222 2222

Diethylamine

buffer (ml)

2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Mix well and measure absorbance at 575 nm

21.7 Calculations

Plot a graph by taking calcium amount at x-axis vs absorbance at y-axis, and
extrapolate calcium concentration of given serum/urine sample from the graph.
The calcium levels in serum and urine can also be calculated by using equation:

In serum¼

ODof testamount of standardðÞμg 100

ODof standardvolume of sample 1000

¼xmg=dl

In urine¼

OD of testamount of standardðÞμg 1000

OD of standardvolume of sample 1000

¼xmg=L

21.8 Clinical Significance...............................

The normal value of calcium in serum is 9–11 mg/dl and 100–300 mg/24 h urine.
Extracellular calcium provides calcium ions for maintenance of intracellular cal-
cium, bone mineralization, blood coagulation, and maintenance of plasma

21.8 Clinical Significance 87