Estimation of blood Urea
It is done by DAM (Diacetyl monoxime method).
Principle: Urea reacts with diacetyl under acidic conditions to form a
coloured chromogen called Diazene yellow.
O O
║ ║
Diacetyl (CH3 ─ C ─ C ─ CH3) is unstable compound, so we use diacetyl
monooxime (CH3COC═NOHCH3).
As the colour developed is faint and unstable, so ferric alum or
thiosemicarbazide is used to intensify and stabilize the colour.
H+
CH3 ─ CO ─ C ─ CH3 + H2O CH3 ─ C ─ C ─ CH3 + NH2OH
o
│ (100 C) ║ ║ (Hydroxylamine)
NOH O O
(Diacetyl Mono-oxime) (Diacetyl)
CH3 ─ C ─ C ─ CH3 + NH2CONH2 ---------------------- Diazine (yellow)
║ ║ (urea)
O O
(Diacetyl)
Preparation of PFF:
Take 0.2 ml of oxalated blood, 1.9 ml of 5% zinc sulphate and 1.9 ml of
0.3 N sodium hydroxide solution. Centrifuge for 5 minutes. Supernatant is
PFF.
Procedure:
Take 3 test tubes and label them as test (T), standard (S) and blank (B).
T S B
PFF 2 ml - -
Working standard - 2ml -
(0.025 mg/ml)
Distilled water - - 2ml
DAM 2ml 2ml 2ml
Acid Reagent 2ml 2ml 2ml
Keep all the test tubes in boiling water bath for 5 minutes and then take OD
at 420 nm.
Calculations:
OD of test
Blood urea (mg %) = ------------------ x Conc. of standard
OD of standard
1 ml of standard solution has urea = 0.025 mg
2ml of standard solution has urea = 0.05 mg
As volume of blood in 4ml of PFF = 0.2 ml
So, volume of blood in 2ml of PFF = 0.1 ml
OD of test
Therefore conc. of urea in 0.1 ml of blood is = -------------------- x 0.05
OD of standard
OD of Test x 0.05
And conc. of urea in 100 ml of blood will be = ---------------------- x 100
OD of Standard x 0.1
OD of test
i.e. Blood urea (mg%) = -------------------- x 50
OD of Standard
Urea is a waste product, derived from the catabolism of either exogenous
(dietary) or endogenous (tissue) proteins.
Protein
↓ Proteolysis, principally enzymatic
Amino acids
↓ Transamination and oxidative deamination
Ammonia
↓ Enzymatic synthesis in the ‘urea cycle’
Urea
Urea cycle enzymes
CO2 + NH3 Urea Excreted
Liver
More than 90% of urea is excreted through kidneys, remaining is excreted
through GIT and skin.
In a normal kidney, 40% to 70% of highly diffusible urea reenters the
plasma through the renal tubules. This back diffusion of urea is inversely
related to the urine flow rate as when the rate of urine flow decreases, there
is enough time for reabsorption of urea by tubules.
Normal blood urea level = 15- 45 mg%
Advantages:
It is a very simple and accurate method.
Disadvantages:
Linearity is only up to 100 mg%, after that the sample has to be
diluted.
Time to keep the tubes in boiling water bath should depend on the
quantity of urea, as formation of coloured complex occurs at boiling
temperature, but we are boiling all the tubes for 5 minutes.
Importance of blood urea estimation:
For assessment of kidney function since the route of excretion of urea is
through the kidneys.
Other methods of urea estimation:
Various methods are in use
A. Indirect methods (Enzymatic): Urea is hydrolysed by urease to
ammonia which is measured.
Urease
NH2CONH2 + 2H2O -------------------- 2NH4+ + CO32-
1. Nessler’s method: Ammonia generated with help of enzyme urease is
made to react with Nessler’s reagent ( potassium mercuric iodide) giving
rise to a brown coloured compound, which is read at 450 nm. The enzyme
acts optimally at 55oC and pH 7-8 and is inhibited by ammonia and
fluoride.
Disadvantages:
Turbidity
Colur instability
Non- linear calibration
Susceptibility to contamination with ammonia from lab and
endogenous ammonia in the specimen.
2. Berthelot reaction: In this, ammonia reacts with phenol in the presence
of hypochlorite in alkaline medium to form an indophenol. Nitroprusside
acts as a catalyst increasing the rate of the reaction, the intensity and
reproducibility. The absorbance of the dissociated indophenol, a blue
chromogen, is measured at 560 nm and related to concentration of ammonia
formed.
Advantages:
Colour stability
linearity
Disadvantages:
Interference from hemolysis, bilirubin, lipemic serum and ammonia
in reagents or atmosphere giving rise to falsely high values. So it
must be removed by adsorption on permutt it or sodium aluminium
silicate.
3. Urease/ Glutamate dehydrogenase method: In this, glutamate
production from ammonia and 2-oxoglutarate is measured by absorbance
change at 340nm, owing to concomitant conversion of NADH to NAD+.
Each molecule of urea hydrolysed causes the production of 2 molecules of
NAD+. Rate of decrease in absorbance at 340 nm (Due to decrease in conc.
of NADH) is monitored. It is used in autoanalyzer.
B. Direct methods:
Urea is reacting directly with reagent as in DAM method.
C. Other methods:
Stick tests (semiquantative)
1. Urastrat strip: It is a bedside test and not very accurate. There are
several bands of reagents on the strip. The end of the strip is dipped in the
serum which rises by capillary action. Reaction occurs, liberating ammonia,
which is trapped by indicator band of bromocresol green to give colour
whose intensity is proportional to urea concentration.
2. Azostix: It is used for whole blood. Reagents contain urease enzyme and
bromothymol blue as indicator. Urea is broken down in blood sample by
urease to release ammonia which changes the colour of indicator from
yellow to green to blue in the solution.
Clinical Significance:
Increased urea levels in blood: Increased blood urea levels above normal
is termed Azotemia. When this is associated with clinical symptoms, it is
called Uremia.
Causes can be divided into:
1. Pre-renal:
Increased breakdown of tissue protein e.g. in fever and other toxic
states.
Dehydration leading to low perfusion of kidneys and therefore
decreased filtration of urea. e.g. in cases of
- Burns
- Diarrhoea
- Vomiting
- Severe hemorrhage
- Diabetic ketoacidosis
2. Renal :
Glomerulonephritis- acute and chronic
Chronic renal failure
Chronic pyelonephritis: is characterized by renal inflammation and
fibrosis induced by recurrent or persistent renal infection, vesicoureteral
reflux, or other causes of urinary tract obstruction.
Poisoning affecting kidneys
Nephrotoxic drugs e.g. gentamycin, cephaloridine, sulfonamides etc.
Diabetic and gouty nephropathy
3. Post- renal:
Stone anywhere in the urinary tract
Malignancy
Stricture of the urinary tract: A stricture restricts the flow of urine from
the bladder and can cause a variety of medical problems in the urinary tract.
Prostatic hypertrophy in elderly
Because backflow to kidney increases and ultimately nephron gets destroyed.
Increased backflow also leads to reduction in effective filtration pressure.
Clinical utility of blood urea lies in its measurement in conjunction with
Creatinine and in distinguishing prerenal from postrenal causes.
Normal urea nitrogen/ Creatinine ratio = 12-20
(Blood urea = Blood urea nitrogen (BUN) x 2.14)
Estimation of urine urea
Sample: 24 hours urine sample/ random urine sample
Method: Same as in estimation of blood urea.
As urine has much high level of urea than serum and linearing of DAM
method is still 100 mg% only, the sample is pre diluted 20 times. Hence, the
results obtained are multiplied by 20 to get the urine urea sample.
Urine urea(mg/dl) x 24 hr urine volume(ml)
24 hour urea(g/day) = ------------------------------------------------------ ww
100000
Normal range = 12.8- 36.4 g/day
UREA CLEARANCE:
Volume of plasma cleared off urea by both the kidneys in one minute.
It is calculated by the formula:
uv
Cm = ----
p
Where Cm = maximal urea clearance
u = urine urea (mg%)
v = flow of urine (ml/min)
p = blood urea (mg%)
The above formula is applicable if the output of urine is equal to or more than
2ml per minute. This is referred as maximal urea clearance.
Normal value is = 60-95 ml/min (average = 75 ml/min).
It has been observed that urea clearance drastically changes when volume of
urine is less than 2ml/minute, so standard urea clearance (Cs) is calculated by
the formula:
u √v
Cs = -----
p
√v is taken to bring the urine volume close to 1 ml/min.
Normal standard urea clearance = 40-65ml/min (average = 54 ml/min).
Interpretation:
When clearance is less than 70% of the normal, it indicates renal damage.
Grading of renal function based on urea clearance:
Over 70 Normal
70-50 Mild deficit
49-20 Moderate deficit
Below 20 Severe deficit
Below 5 Uremic coma
Nitrogen Balance:
It is the difference between the total nitrogen absorbed in the form of
amino acids and the total nitrogen lost (mainly as urea in mammals) by the
body. Normal adults are in nitrogen equilibrium i.e. intake and output are
almost equal. Children and pregnant females have a positive balance with intake
exceeding loss. Dietary protein deficiency, cancer cachexia and post-surgical
patients have a negative balance due to loss exceeding intake. Intake of poor
quality proteins (with low biological value) that are deficient in essential amino
acids also leads to negative nitrogen balance.
