**2.9.1 Principle**

The phosphate ion reacts with Molybdate to produce Phosphomolybdate, which is finally reduced to a molybdenum blue, which is photo metrically measured.

### **2.9.2 Technique**

1. Three test tubes were labeled as: Blank –BL

Standard –ST

$$\text{Sample} \quad \text{-SA}$$


Inorganic phosphate (mg/dl) = SA O.D 4 ST O.D 

O.D = Optic Density

Normal value of serum phosphate was taken as 2.4- 4.5 mg/dl.

The Prevalence of Renal Osteodystrophy in

in the presence of alkaline oxidizing agents.

**2.11.1 Princile** 

**2.11.2 Technique** 

**2.12.1 Principle** 

bone iso-enzyme.

**2.13.1 Procedure** 

acid for 2 days.

**2.13 Bone histology (post mortem)** 

Chronic Renal Failure Patients in Urban Niger Delta of Nigeria 53

4- amino antipyrine gives a red purple colour with compounds containing a phenolic group

2mls of buffer substrate was measured into each of the 2 test tubes and placed in water bath at 370C for a few minutes. To one of the test tube (patients' test tube) was added 0.lml of serum and the tubes were incubated for exactly 15 minutes. They were removed from the bath and 0.8ml of 0.5M sodium hydroxide and 1.2ml of 0.5M (the blank) was added to both tubes, 1ml of amino- antipyrine reagent and 1ml of potassium ferricyanide were added. For the standard, 1ml of buffer and 1ml of phenol standard containing 0.01mg of phenol was taken. For the standard blank, 1.1ml of buffer and 1ml of water was taken to both tubes, and then sodium hydroxide, bicarbonate amino-antipyrine and ferricyanide was added as above. It was read with a spectrophotometer set at 520 millimicrons. Serum alkaline

phosphatase (King- Amstrong unit per 100mls) was derived from the formula: Serum alkaline phosphatase = Reading of unknown - Reading of blank x 10

Total serum alkaline phosphatase values of 25-95 IU/L was taken as normal

**2.12 Determination of bone specific alkaline phospatse** 

spectrophotometer set at 520 millimicron wavelength.

1. The autopsy specimen biopsy was taken from the pelvic bone.

ascending grades of alcohol. (70%, 90%, 100%) respectively.

Reading of standard - Reading of standard blank

This was done by curve-fitting of inhibition kinetic as popularized by (Statland et al, 1972).

After the determination of the total serum alkaline phosphatase as described above, the serum is heated for 13 minutes at 560c to inactivate the bone- type isoenzyme. The sera are now read as in the determination of the total serum alkaline phosphatase using a

The concentration of serum alkaline phosphatase excluding the bone isoenzyme was determined as was done for total alkaline phosphatase using a spectrophotometer set at 520millimicron wavelength. The bone isoenzyme was determined by subtracting this value from the total alkaline phosphatase. Values of bone specific alkaline phosphatase level greater than 50% of total serum alkaline phosphatase shows significant contribution from

2. Decalcification: The piece of bone biopsied was decalcified by emersion in 10% nitric

3. Dehydration: the decalcified bone was dehydrated by passing the bone through

#### **2.10 Measurement of total serum calcium**

The Cresolphtalein- Complexone method (CPC) was used for the determination of total serum calcium. This is a standard Colorimetric method. This like most calcium assays measures the total serum calcium although it is only the free calcium which constitutes 50- 65% of total calcium that is biologically active.

#### **2.10.1 Principle**

CPC forms a violet colored complex with calcium. The absorbance of the colour produced was measured in a colorimeter using a Spectrophotometer at 575nM wavelength measured in a colorimeter using a spectrophotometer at 575nM wave length. Interference from magnesium was reduced by including 8-hydroxquinoline in the working CPC reagent. The Ethanediol in the reagent suppresses the ionization of the 0- Cresolphtalein and helps to give a clear solution. A correction was made when the patient's serum albumin level was below 40g/l using this formula:

Corrected serum albumin = <sup>2</sup> albumin g /l Ca <sup>40</sup> 40 
