Praktika Creatinine and Clearance 2014

7/24/2019 Praktika Creatinine and Clearance 2014

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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)


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1. Clearance of endogenous creatinine

The kidneys areorgans that serve several essential regulatory roles in mostanimals,including

vertebrates and someinvertebrates.They servehomeostatic functions such as the regulation of

electrolytes,maintenance ofacidbase balance,and regulation ofblood pressure (via maintaining

salt and water balance).In chemical terms, creatinine is a cyclic derivative of creatine (cyclic imide of N-

methylguanidineacetate 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 creatinineconcentration in urine is 100x higher than in serum. Creatinine excretion in adults is normally 9-

16 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 Uxconcentration of substance X in urine [mol/l]

GFR= ---------- Pxconcentration of substance X in plasma [mol/l]

Px Vxdiuresis 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] Clcrclearance of creatinine

Clcr= ---------------------------------------------- Pcrplasmatic concentration of creatinine

44.5 x Pcr [mol/l]
http://en.wikipedia.org/wiki/Organ_(anatomy)http://en.wikipedia.org/wiki/Animalshttp://en.wikipedia.org/wiki/Vertebratehttp://en.wikipedia.org/wiki/Invertebratehttp://en.wikipedia.org/wiki/Homeostasishttp://en.wikipedia.org/wiki/Electrolytehttp://en.wikipedia.org/wiki/Acid%E2%80%93base_homeostasishttp://en.wikipedia.org/wiki/Acid%E2%80%93base_homeostasishttp://en.wikipedia.org/wiki/Acid%E2%80%93base_homeostasishttp://en.wikipedia.org/wiki/Blood_pressurehttp://en.wikipedia.org/wiki/Blood_pressurehttp://en.wikipedia.org/wiki/Acid%E2%80%93base_homeostasishttp://en.wikipedia.org/wiki/Electrolytehttp://en.wikipedia.org/wiki/Homeostasishttp://en.wikipedia.org/wiki/Invertebratehttp://en.wikipedia.org/wiki/Vertebratehttp://en.wikipedia.org/wiki/Animalshttp://en.wikipedia.org/wiki/Organ_(anatomy)


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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 1.32.8 ml/s/1.73 m2(1.73 m2average

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 m2has

been referred as renal insufficiency, under 0.15 ml/s/1.73 m2as 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 concentrationReagents (ml) Sample S Sample U Standard Blank

Deproteinized serum 0.25 - - -

Diluted urine (50x) - 0.25 - -

Standard K - - 0.25 -

Distilled water 0.5 0.5 0.5 0.75

Trichloracetic acid 0.25 0.25 0.25 0.25

Mix and let incubate at laboratory temperature for 5 min

Picric acid 0.5 0.5 0.5 0.5

Sodium hydroxide 0.5 0.5 0.5 0.5

e. Mix the reaction mixture thoroughly.


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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 Mean Concentration

Standard K *

S mol/l

U mol/l

Concentration of the standard will be given

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:

Vp = GFR = (ml/s)

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 = (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).

cu.Vu

cp

cux Vux 1.73

cpSA


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Table 3. Specifications ofpatients characteristicsfor calculation of clearance

PatientA.

Male

B.

Female

C.

Male

D.

Female

E.

Male

F.

Female

Diuresis* (ml/24h)

Weight (kg)

Height (cm)* Diuresis will be given

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

Reference range Your result

Creatininemia (mol/l)females

males

5397

61115

Creatinine excretion in urine (mmol/24h)females

males

7.515

918

GFR (ml/s)females

males

1.582.68

1.632.80

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Praktika Creatinine and Clearance 2014