Urea Nitrogen Lab .No.3

Lab .No.3
Urea Nitrogen

Enzymatic determination of urea in human urine serum and plasma
Urase _modified Berthelot reaction

_Summary and Explanation :
Urea end product of aminoacid catabolism is synthesis in the liver (ureogenesis ) and for 90% eliminated in the urine
Urea in serum and urine fluctuates in healthy subjects as it depend on the renal function the nitrogen intake from food the endogenous protein catabolism and the state of hydration and diuresis
Afell in serum urea levels to under 2 mmol/ l in adults and adecease in urine urea  can be observed in terminal stages of sever hepatic insufficiency togother with hyperammonemia
Nitrogen retention syndrome is a more common occurrence combined with an increase is serum urea in acute and chronic renal insufficiencies where serum urea concentration can reach up to 60 mmol/l as high serum urea levels are not specific to renal disorders and can also be observed in cause of protein _rich diets geart disease  hehydration diuretic treatment nitrogen hypercatabolism (burns infection fever corticoid therapy ) and in elderly subjects .

Principle
Urea _ kit S enable end point enzymatic determination of urea concentration (Urease _modified Berthelot reaction ) in human urine serum or plasma .
Urease hydrolyzes urea by producing ammonium :

Urea  +H2O  = 2NH3  +CO2

In alkaline medium the ammonium ions react with the saliclate and hypochlorite to form agreen colored indophenol (2,2 _ dicarboxylindophenol ) .the reaction is catabolyzed by the sodium nitroprusside .

NH4  + Salicylate  + Hypochlorite  =  Indophenol.

The color intensity is proportional to the urea concentration in the sample . the possible presence of heavy metals inhibite the formation of indophenol .EDTA lifts  
 The inhibition .

Content of the kit
Reagent 1 (R1) : Urea
                (R2) : Urease
                (R3) : Phosphate buffer PH 8
                          Sodium Salicylate
                           Sodium nitroprusside
                           EDTA
                (R4) :Soium hydroxide (NaoH)
                         Sodium hypochlorite (Nac10)


1_ serum or plasma collected in EDTA ,heparine or sodium floride
2_ 24 hour _old urine samples collected in a vial containing 10 ml Hcl 6N to avoid bacterial urease action
Dilute 1:100 in distilled water.

Stability of serum or plasma
_ 4days at 2_8 c
_3 days at 20_25c
_ 3 months at -25 -,+6C


4 days at 2_8 C

Relevant interferents
Presence of the following factors have been found to significant influence this assay
1_hemolysis after spiking sample with hemoglobin ,up to 204 mmol/l
2_ lipemia after spiking samples with lipid ,up to7 mmol/l
3_ bilirubinemia after spiking samples with bilirubinemia up to 855 mmol/l
It is recommended not to use samples that appear to be hemolyzed ,lipemic and if possible to collect anew sample

Clibiration
Use Reagent 1

Instructions for use manual method
Preparation of working solution :
Dispense one vial of reagent 2 in to one vial of reagent 3 and mix by turning upside down several times

Stability in orginal vial
2 month at 2_8 C




Procedure :



 
sample    standard    Reagent blank           
-
10 micro litter
1ml    10 micro litter
-
1ml    -
-
1ml    Standard
Sample
Working solution     


Mix
Incubate for 5 min. at 20_25c

 
200 micro litter    200 micro litter    200 micro litter    Reagent   4     

Mix

Incubate for10 min. at 20_25c
Perform photometry.(wave length = 580nm)

_ Color intensity is stable in the dark :2 hour at 20_25 C

_ Calibiration stability perfom a calibration for each series of tests

_ Result and interpretation
Interpretation of the test results should be made taking in to consideration the patients history and if necessary the results of any other tests perfomed

Calculation
Sample concetratuin = A sample /A standaed x n
N= concentration of standerd
If urine samples are tested multiply yhe result obtained by the dilution fector

_Conversion factor
Mmol /l x 0.060 =g/l
Mmol/l x 6 = mg/dl
g/l x 16.67 = mmol/l
mg /dl x 0.167 = mmol/l

Range of expected values

 
Mg /dl    G /l    Mmol /l    Serum or plasma      
6 _18    0.06 _0.18    1.00_ 3.00    Infants      
15 _33    0.15 _0.33    2.50 _ 5.50    Chidren      
15 _45    0.15 _ 0.45    2 .50 _ 7 .50    Adults     

Urine :
250 _500 mmol/24h
15 _ 30 gm/24h