CREATININE AND CLEARANCE

Key words: creatine, creatinine, cystatin C, glomerular filtration (GFR), tubular secretion,
reabsorption, c1earance, excretion,
Reagents:
1. Serum sample
2. Urine sample
3. Standard K
4. Trichloroacetic acid (1.22 mol/l) !CAUSTIC AGENT!
5. Picric acid (0.04 mol/l)
6. Sodium hydroxide (0.75 mol/l)

1. Clearance of endogenous creatinine

The kidneys are organs that serve several essential regulatory roles in most animals, including
vertebrates and some invertebrates. They serve homeostatic functions such as the regulation of
electrolytes, maintenance of acidbase balance, and regulation of blood pressure (via maintaining
salt and water balance).
In chemical terms, creatinine is a cyclic derivative of creatine (cyclic imide of Nmethylguanidineacetate acid) and is a break-down product of creatinephosphate in muscle
(creatine dehydration). Physiological reference ranges for serum creatinine are 70-125 mol/l for
men (50-105 mol/l for women, respectively). Concentration of creatinine in plasma depends on
body weight and muscle mass and its level is stable without marked fluctuation. Lower creatinine
concentration is characteristic for children, persons with low muscle mass, patients with
myodystrophy as well as bedridden patients. Rise in blood creatinine level is associated with
marked damage of nephrons. The food intake or overweight do not influence the plasmatic
concentration.
Creatinine is filtered out of the blood by the kidneys (90% glomerular filtration and 10%
proximal tubular secretion). There is little-to-no tubular reabsorption of creatinine. The creatinine
concentration in urine is 100x higher than in serum. Creatinine excretion in adults is normally 916 mmol/day (1-1.8 g/day).
Measurement of glomerular filtration rate (GFR) is an essential test reflecting the ability
of kidneys to maintain the composition of body fluids. Clearance of endogenous creatinine test
provides important information about the glomerular filtration (the effectiveness of kidneys in
performing their excretory function). Clearance of substance is defined as the volume of plasma
from which a measured amount of substance can be completely eliminated into the urine per unit
of time (second).
Creatinine concentration in serum rises (>150 mol/l) during acute and chronic renal
disorders. Reduction of creatinine clearance under 0.5 ml/min has been referred as a renal
insufficiency, under 0.15 ml/min as a renal failure. In order to estimate GFR from measuring
clearance of any substance, it is necessary that this substance is freely filterable and unaltered
once it reaches the tubules (no secretion, reabsorption, synthesis or degradation). Therefore, the
excretion of such a substance reflects the rate of glomerular filtration. The example of such a
substance is inulin, a polysaccharide produced by the plants.
To calculate the clearance of a substance X, we need to known its concentration in urine and
plasma, the volume of urine per day (diuresis [ml/s] the total volume of urine in ml / 86400s):
Ux.Vx
GFR= ---------Px

Ux concentration of substance X in urine [mol/l]

Px concentration of substance X in plasma [mol/l]
Vx diuresis per 24 hours [ml/s]

A number of formulae have been proposed to estimate GFR, the Cockroft-Gault formula is
widely used:
(140 - Age [years]) x bodyweight [kg]
Clcr = ---------------------------------------------44.5 x Pcr [mol/l]

Clcr clearance of creatinine

Pcr plasmatic concentration of creatinine

This formula expects weight to be measured in kilograms and creatinine to be measured in mg/dL.
The resulting value is multiplied by a constant of 0.85 in females. This formula is useful because
the calculations are simple and the diuresis is not necessary.
The plasmatic concentration of creatinine together with the clearance of endogenous
creatinine are commonly used clinical blood tests to monitor the severity of renal failure.
Clearance of endogenous creatinine in adults is normally 1.3 2.8 ml/s/1.73 m2 (1.73 m2 average
body surface area for male). For extremely obese/slim patients, the clearance needs to be
corrected for the body surface area. Reduction of creatinine clearance under 0.5 ml/s/1.73 m2 has
been referred as renal insufficiency, under 0.15 ml/s/1.73 m2 as renal failure.
Currently, a small basic protein, cystatin C, is used as a biomarker of renal function.
Cystatin C has a low molecular weight (approximately 13.3 kDa) and is an endogenous product of
the organism. Its plasmatic concentration is stable and does not depend on patient age, gender or
body weight. It is removed from the blood exclusively by glomerular filtration in the kidneys.
Plasmatic level of cystatin C is more precise test of kidney function (as represented by the
glomerular filtration rate, GFR) than plasmatic creatinine level. Moreover, it reveals the renal
insufficiency earlier than the creatinine. The plasmatic concentration reflects the glomerular
filtration rate, while the urine concentration helps us to monitor the impairment of proximal
tubular secretion.
1.1. Estimation of creatinine concentration in serum and urine
Principle:
Creatinine clearance test is based on Jaffe assay (developed in 1886). In Jaffe assay
creatinine reacts in deproteinized serum with picric acid in an alkaline solution resulting in
formation of orange-red product of creatinine picrate suitable for photometric determination.
Jaffe assay could be a source of error reading because reaction is not specific for the
creatinine only, but picric acid reacts also with other noncreatinine chromogens with active
methylene group, including ascorbate, pyruvate, acetone and glucose. Currently, alternatives of
Jaffe assay, based on enzymatic assays, have been used.
Procedure:
a. Take two patient's samples described as follows: S = serum, U = urine
b. DILUTE URINE 50x, RESULT OF YOUR ANALYSIS MULTIPLY BY 50!
c. For serum, urine and standard samples proceed the estimation three times, for a blank
sample only once.
d. Pipette reagents into test tubes (marked by appropriate number) according to Table 1.

Table 1. Reaction mixes for estimation of creatinine concentration

Reagents (ml)
Sample S
Sample U
Standard
Deproteinized serum
0.25
Diluted urine (50x)
0.25
Standard K
0.25
Distilled water
0.5
0.5
0.5
Trichloracetic acid
0.25
0.25
0.25
Mix and let incubate at laboratory temperature for 5 min
Picric acid
0.5
0.5
0.5
Sodium hydroxide
0.5
0.5
0.5
e. Mix the reaction mixture thoroughly.

Blank
0.75
0.25
0.5
0.5

f. Read absorbance of both samples and standard after 20 minutes exactly, (use blank as zero
standard) at 505 nm.
g. Write measured values in the table 2.
h. Calculate mean values for each sample and standard, put it in the table 2.
i. Calculate the concentrations of creatinine in serum cP (mol/l) and in urine
cU (mol/l) Do not forget to multiply your result by dilution of urine - 50x!
j. Write results in the table 2.
Table 2. results of estimation of creatinine concentration
Sample No:
Absorbance
Standard K
S
U
Concentration of the standard will be given

Mean

Concentration
*
mol/l
mol/l

1.2. Calculation of clearance

Principle:
The amount of creatinine (n) (in mol/l), filtered through glomerular membrane into the
glomerular filtrate in 1 second is equal to concentration of creatinine in filtrate (mol/ml)
multiplied by volume of filtrate (ml) produced in 1 second. Creatinine is a low-molecular
substance and its concentration in the filtrate is the same as that one in plasma (cp). Therefore, the
volume of the formed filtrate must be the same as the volume of plasma (VP) from which
creatinine is completely eliminated:
n = Cp Vp (mol/l)
Next, the same amount of creatinine (n) from the glomerular filtrate is per second
excreted into the final urine. Therefore, it is equal to concentration of creatinine in final urine (cp)
multiplied by volume of final urine formed per second (Vp,):
n = cu Vu (mol/l)
Combining of above could perform calculation of GFR given formulas:
cu.Vu
Vp = GFR =
(ml/s)
cp
The measured creatinine clearance should always be corrected to an average adult
surface area of 1.73 m2. Use of this correction factor allows for normalization of the muscle mass
difference in individuals. Determination of surface area (SA) can be taken from following
formula:
SA = 0.167 m . l
Where 0.167 is empirically estimated correction factor, m is body weight (kg) and l is
height (cm).
Formula for determination of corrected creatinine clearance (GFRcor) is:
GFRcor =

cu x Vu x 1.73
cp SA

(ml/s)

Concentration of creatinine in urine and serum, diuresis, body weight and height has to
be known for the calculation of clearance (Table 3).

Table 3. Specifications of patients characteristics for calculation of clearance

A.
B.
C.
D.
E.
Patient
Male
Female
Male
Female
Male
Diuresis* (ml/24h)
Weight (kg)
Height (cm)
* Diuresis will be given

F.
Female

Procedure:
a. Calculate creatinine clearance and excretion of creatinine in urine of given patient.
Use your results from task 1.1.
GFRcor = ............... ml/s
Excretion = ............... mmol/24 h

Summary of your results

Creatininemia (mol/l)
Creatinine excretion in urine (mmol/24h)
GFR (ml/s)

Reference range
females
53 97
males
61 115
females
7.5 15
males
9 18
females
1.58 2.68
males
1.63 2.80

Your result