M.Sc. Medical Biochemistry
Urea is the major end product of protein metabolism In
Its formed in liver.
Urea biosynthesis occurs in four stages:
(2) oxidative deamination of
(3) ammonia transport,
(4) reactions of the urea
ammonia is produced in various ways by tissues are rapidly
removed from circulation by the liver and converted to urea.
This is essential, since ammonia is toxic to the central nervous
Urea is water soluble ,conversion of ammonia to urea is a kind
This is the importance of urea excretion and its estimation.
Any defect in any of the enzyme function of urea cycle can lead
to various metabolic disorders.
More than 90% of urea is excreted through the
kidneys(glomeruli), with losses through GIT and skin in minor
Determination of blood urea is important not only in many
diseases of kidney but in a wide range of conds which r not
Causes of increase in serum urea
Causes r divided in pre-renal , renal and post-renal
pyloric n intestinal obstruction with vomiting
sever n prolonged diarrhea
chrn intestinal obstruction without vomiting
ulcerative colitis with severe chloride loss
congenital cystic kidney
calcium deposition in kidney in hyperthyroidism n
enlargement of prostate
stones in urinary tract
stricture of the urethra
tumors of the bladder affecting the ureter
Cirrhosis of liver
Acute yellow atrophy of liver
Diacetyl monoxime method
when urea is heated with diacetyl(,containing
two adjacent carbonyl group) under acidic
condition yellow coloured compound is
formed.the OD of the colour developed can be
read at 420 nm.intensity of the color depends
on conc of urea in serum.
additions BLANK STANDARD TEST
Protein free filtrate _ _ 1ml
Working standard _ 1ml _
Distilled water 1ml _ _
DAM reagent 0.4ml 0.4ml 0.4ml
Acid mixture 1.6ml 1.6ml 1.6ml
OD of the test × conc of standard × 100
OD of the standard
vol of the test
BUN(blood urea nitrigen):
= blood urea level× 28/60
Normal value - 5 -20 mg/dl
urease hydrolyses urea to ammonia and CO2.the
ammonia formed further reacts with a phenolic chromogen and
hypo chloride to form a green colored complex. Intensity of
colour is directly proportional to the amount of urea present in
addition blank standard test
1.0 ml 1.0ml 1.0ml
Distilled water 0.01ml - -
Urea standard - 0.01ml -
sample - - 0.01ml
0.2ml 0.2ml 0.2ml
The hypobromite method
the vol of nitrogen liberated by the action of
hypobromite on the urea present in the urine is measured. the
action is usually though not accurately, expressed
URINE - 15 to 30mg/day.
Urea clearance is less than GFR.
Maximum urea clearance:
=U × V/P
U=mg of urea /ml of urine
p=mg of urea/ml of plasma
v=mg of urine excreted /minute
normal value - 75 ml /mint
Standard urea clearance:
u × √v/p
normal value - 54ml/mint
Values below 75% is abnormal
Values fall progressively with increasing renal failure
Clearance values r the early indicator then plasma value
creatinine clearance is more preferred
Neither urea clearance nor blood urea r used as index of kidney
Creatine constitutes about 0.5% of total muscle weight.
Its synthesized from 3 amino acids Glycine , Arginine &
(occurs in mitochondia of kidney n pancreas)
Guanidoacetic acid ornithine
SAM (in liver)
Creatine creatine phosphate (ck is pesent in muscle brain n liver)
CREATINE creatine kinase PHOSPHOCREATINE
Stored creatine phosphate which is a high energy compound,
stored in the muscle, serves as an immediate store of
Inter conversion of phosphocreatine & creatine is an indicator
of metabolic process of muscle contraction.
A particular proportion of muscle creatine is converted to
creatinine spontaneously everyday.
So amount of creatinine produced in body is related to muscle
Conc. Of creatinine in blood is mostly constant.
Diet may influence the value with high meat intake.
Its freely filtrated by glomerulus.
Its an indicator of renal function.
Increased conc of creatine
Causes of increase in serum Creatinine
Causes are divided in pre-renal , renal and post-renal
Pyloric & intestinal obstruction with vomiting
Severe n prolonged diarrhea
Chronic intestinal obstruction without vomiting
Ulcerative colitis with severe chloride loss
Enlargement of prostate
Stones in urinary tract
Stricture of the urethra
Tumors of the bladder effecting the ureter
Increased conc of creatinine
Conditions r same as increased urea conc.
But in renal causes creatinine increases more than the
Jaffe`s method (in urine)
creatinine in urine reacts with picric acid in
presence of NaOH to give orange colored compound.creatinine-
picrate color read at 500 nm.
3ml - -
- 3ml -
Diluted urine - - 3ml
Picric acid 1ml 1ml 1ml
0.75 N NaOH 1ml 1ml 1ml
=gms of creatinine /lt
Normal value – 1 - 1.5gm/lt
Modified jaffe`s reaction:
cretinine reacts with alkaline picrate to
produce a reddish color. Absorbance of the color is directly
proportional to creatinine conc in plasma n its measured in
addition blank standard sample
- 1000ùl 1000ùl
Standard - 100ùl -
serum - - 100ùl
=T/S ×conc of standard(mg/lt)(2mg/dl)
normal value – 0.7 -1.4mg/dl (male)
0.6 – 1.2 mg/dl (female)
U=urinary creatinine conc.
p=plasma creatinine conc
v=urine flow in ml/min
85-125 ml/min (male)
80-115 ml/min (female)
decreased creatinine clearance is an sensitive indicator of
UPTO 75% is nornal.
Early detection of renal impairment
Long term monitoring of renal patients
Creatinine clearance is altered by body muscle mass