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Republic Algeria.

Djillali Liab University School of Medicine Department of Phar

TP # 01 Determination of plasma creatinine.
Produced by: • GHERMI Mohamed. • CHAREF KHODJA Meriem. • TAHRI Mahmoud. • BETTAY
EB Mohamed Amine. 4th year pharmacy G05. Academic Year: 2008 / 2009
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Crea kreas comes from the Greek meaning flesh. A measurement of creatinine gives
information on two points: the renal function and muscle mass. Creatinine is a
chemical molecule generated by muscle metabolism. Creatinine is produced from cr
eatine, a molecule of major importance for energy production in muscles. About 2
% of body creatine converted to creatinine every day. Creatinine is transported
through the blood to the kidneys. For a given subject, the plasma and the amount
of creatinine excreted daily in urine laboratory parameters are remarkably fixe
d. For these reasons, the value of the creatinine clearance is semiological fund
amental significance in the study of renal failure. The clearance of creatinine
is independent of diuresis, it directly measures the glomerular filtration. The
plasma is independent of dietary protein intake and reflects the muscle mass of
the subject and its own metabolism. Elimination is only urine, so any change in
the clearance information directly on the functional status of the kidney.
1 - Background physiological
Has Metabolic Origin: Creatinine comes from the dehydration of creatine, which i
s itself present in striated muscle where it allows the storage form of ATP and
phosphagen creatine phosphate in a reaction catalyzed by creatine kinase (CK). b
-Behavior creatinine level of creatinine undergoing nephron glomerular filtratio
n, it is not subsequently reabsorbed or excreted in the tubule. His clearance me
asures the volume of glomerular filtrate formed per second. Clearance = 2 ml / s
econd per 1.73 m2 of body surface area (see tables Dubois).
2 - Formation of creatine phosphate:
The creatine phosphate is synthesized in cells from amino acids. In its structur
e we find the molecule of glycine, which nitrogen is substituted by the nucleus
guanidinium of arginine and a methyl from methionine. The phosphate-rich and bin
ding energy comes from ATP.
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3 - Formation of creatinine
The metabolism of creatine and therefore that of creatinine can be summarized in
this explanatory scheme.
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III-Methods of exploration:
1 - Determination of blood creatinine
Whole blood contains creatine and creatinine. Creatine is found primarily in red
blood cells at low levels, while creatinine is equally distributed between bloo
d cells and plasma. The total concentration of creatinine in the blood is remark
ably constant in a given subject, depending on muscle mass. It does not depend o
n the plan, or exercise, or even other biological influences. It is the blood co
nstituent whose rate is the fixed. Creatinine blood hardly varies in kidney dama
ge. The rate varies in adults, from 50 to 105 pmol / l.
2 - Clearance
It establishes the relationship between the quantity of the substance provided b
y the plasma in the kidney and the quantity of the substance excreted by the kid
ney. This is the coefficient of plasma treatment or number of milliliters of pla
sma completely purified by the kidney in unit time. This theoretical volume is e
xpressed in SI units in ml / second. It is useful to manipulate simply clearance
s, retaining only 24 hours correspond to 1,440 minutes and 86,400 seconds. C = U
V / PC = clearance = volume of plasma completely purified. U = urinary concentra
tion per liter. P = plasma concentration per liter. V = volume of urine emitted
in one second (or minute). So just for the determination of creatinine in plasma
and urine, to express the results in the same unit, known diuresis (urine outpu
t) per second, to calculate clearance. It is essential during the test (3 h 24 h
or more) to accurately measure urine output and to drink lots of the subject to
have a diuresis greater than 1.5 1 / 24 h. The normal value is 2 ml / s. In add
ition the formula C = UV / P is valid for the normal adult subject whose body su
rface is close to 1.73 m2.€In children and infants must take into account the co
rrected area Se: Ce = C x 1.73 / Se Se is obtained from tables that allow Dubois
, adding a right size and weight, obtain the desired body surface to the interse
ction with the line of body surface. This clearance is the glomerular filtration
rate GFR is an essential part in the study of renal function.
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Otherwise the same GFR can be calculated by the formula Cockcrott and Gault (197
6). Creatinine clearance = K x Weight (kg) x [140-age (years)] / creatinine (nmo
l / L) K = 1.05 in women = 1.25 K in humans creatinine clearance (male) = Weight
(kg) x [140-age (years)] / creatinine (mg / l) x 7.2 creatinine clearance (fema
le) = 0.85 x Weight (kg) x [140-age (years)] / Creatinine ( mg / l) x 7.2.
Conditions for using this formula: Age: between 18 and 110 years between 35 and
weight 120 kg serum creatinine 6 and 70 mg / l IV
Assay methods:
The serum or plasma can be used interchangeably. Samples of serum or plasma or u
rine can be stored for several days to protect from evaporation.
• Samples:
Venous blood: Materials needed: Withers, needle and syringe, disinfectant, cotto
n, plaster, pipes with or without anticoagulant, gloves. Procedure: The sample i
s usually at the elbow, it can also be achieved in case of problems on the dorsu
m of the hand. Provide first tubes needed, possibly to the anticoagulant. Please
bring a syringe to the volume corresponding to the total analysis, counting 5 m
l per tube. Bind the patient's arm, disinfect the skin, loosen the first time th
e plunger of the syringe, then back to its original position. Abouchi the syring
e needle and puncture the vein. Pull the plunger, remove the needle first, throw
in a disposable container for consumable and fill the tubes, always starting wi
th the dry tube, immediately seal all tubes and mix thoroughly those containing
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Never fill tubes with a syringe which was not removed the needle (risk of aeroso
ls, splashes and brutal expulsion of the needle).
Materials needed: A bottle for analysis of urine or if a closed container clean
and dry. Procedure: To measure the creatinine clearance or proteinuria expressed
as mg / day, we practice a urine sample for 24 hours. It is preferable that the
patient remains a day and two nights near the laboratory, or makes this collect
ion at home, if he was well explained. In the morning, making patients urinate i
n the latrine. It should drink normally during the test. Then collect in a large
jar (at least 3 liters) of urine every day, night and finally those early morni
ng awakening. Stopper the jar and shake. Accurately measure the volume in ml gra
duated cylinder with a plastic 2000 ml. Then proceed to a determination of urina
ry creatinine.
For the determination of creatinine, several methods have been developed, from a
1 - Enzymatic Methods:
a-enzymatic method for reading UV: Creatinine
é é
Creatine + ATP
> Creatine (P) + ADP
Phospho-enol-pyruvate + ADP Pyruvate + NADH.H +> Lactate + NAD +.
> Pyruvate + ATP
The decreasing kinetics of disappearance followed the NADH.H + 340nm is proporti
onal to the initial amount of creatinine present. b-action of a specific oxidase
> Sarcosine H2O2 +> H2O + colored chromogen
H2O2 + colorless chromogen
Rmrq: Chromogenic colorless aminophenazone + 4-dichlorophenoxyacetic acid 3-5 2
hydroxybenzene sulfonic. Chromogenic color: quinone-imine red complex (absorbs a
t 520 nm).
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2 - Method by reflectometry:
This is true of equipment in chemistry on solid support. Example: plate "Vitra"
of enzymatic creatinine assay on a plate. The plate contains a series of layers
to achieve a cascade of enzymatic reactions. Creatinine is first hydrolyzed to c
reatine: Creatine Creatinine + H20 + H20 + H20 + 02 Sarcosine H202 + leuco creat
inine concentration. Creatine is then hydrolyzed to sarcosine, which is oxidized
to produce hydrogen peroxide (hydrogen peroxide). Hydrogen peroxide in the pres
ence of peroxidase, oxidizes a leuco which stains, and the speed of color is the
n measured kinetics by reflectometry.€The reaction rate is proportional to the c
oncentration of creatinine present in the serum. Rmrq: These previous methods th
at are very specific enormously expensive which probably limits their general ap
plication in the laboratory of biochemistry.
> Creatine.
> Sarcosine + Urea. > Glycine + formaldehyde + H202> proportional to the stainin
3 - Jaffe kinetic colorimetric method:
Described for the first time in 1886 in an alkaline solution, creatinine reacts
with picrate to form a yellow-red product. Creatinine + picric acid
> Yellow-red complex
The rate of formation of pigment (color intensity) is directly proportional to t
he concentration of creatinine in the sample. It is determined by the increase i
n absorbance at 512 nm. This method has been thoroughly described its advantages
: simplicity of determination and low cost of reagents. The main drawback of the
Jaffe method is its lack of specificity. Up to 20% of the color generated by as
sessments of serum or plasma may originate from endogenous substances other than
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creatinine, creatinine called chromogenic not. The protein, glucose, ascorbic ac
id, cephalosporins and α-keto cids such s ceto cet te nd pyruv te re p rt o
f these nonspecific chromogens re cting to the J ffe method. Depending on their
concentr tion, these compounds c n c use n overestim tion of 18-20 micromol / L
(0.2-0.4 mg / dL) the concentr tion of cre tinine. At the s me time, incre sing
the concentr tion of bilirubin m sk the development of color, giving the result
s of cre tinine erroneously low. Sever l commonly used drugs c n lso ffect the
results of the ev lu tion. Since its first pplic tion, m ny modific tions of t
he J ffe re ction h ve been described on the composition of the re gent nd the
me surement procedure. Rmrq: Sever l people h ve s id th t neither the J ffe met
hod, or the enzym tic method does not provide ccept ble results in the clinic l
setting. According to Thom s Hostetter, MD, MPH t the N tion l Kidney Dise se
Educ tion Progr m t the N tion l Institutes of He lth (USA): "the record of ser
um cre tinine results m y v ry from 30% for l bor tories certified qu lity. Know
ledge of the methodology used to me sure cre tinine is therefore essenti l th t
methodologic l interference c n signific ntly lter the results nd thus influen
ce tre tment decisions.
Norm l V lues:
1 - cre tinine:
♂: 1 to 1.8 g/24H. (9-16 mmoles/24H) ♀: 0.8 to 1.2 g/24H. (7 to 10.5 mmoles/24H)
2 - Cre tinine:
♂: 6.8 to 13 mg / l. (60-115 micromol / l), ♀: 5.65 to 11.3 mg / l. (50-100 micr
omol / l). Child: 3.5 to 7.5 mg / l. (31-66 micromol / l). New born / Inf nt: 2.
3 to 5.6 mg / l. (20-50 micromol / l).
3 - Cre tinine cle r nce:
≈ 120 ml / min / 1.73 m2. The blood of dults nd young people p rticul rly musc
ul r cre tinine m y cont in more th n the ver ge popul tion. In contr st, the b
lood of the elderly m y cont in less cre tine th n norm l. A person with only on
e kidney m y h ve norm l cre tinine of 160 micromol / L (1.8 mg / dL).
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VI Applic tion (TP) "S mple 92":
The method used is th t of st ining J ffe.
1 - Prep r tion of re gents:
• Picric cid (17.5 mmol / l): MM = 229.1 g / mol. V = 100 ml. M = X. X = 17.5 *
229.1 * 10-3 / 10. X = 0.400925 g. • N OH (0.29 mol / l): MM = 40 g / mol. V =
100 ml. M = Y. Y = 0.29 * 40/10. Y = 1.16 g.
2 - H ndling: Working Solution S mple AB
2ml 2ml 200μl
St llion
For every time we me sure the bsorb nce t 512nm to 30'' nd 90'', the concentr
tion of cre tinine w s obt ined by the following equ tion: C (s mple) = [St llio
DO A 30''B
0.506 0.524 C (s mple) =
0.582 0.524 0.536 0.524
OD 90''
0.536 0.582 [20]
C = 38.66 mg / l
3 - Interpret tion of results:
The concentr tion obt ined (38.66 mg / l) is f r superior to the usu l st nd rds
of between 6.8 to 13 mg / L in m les nd 5.65 to 11.3 mg / L in fem les, this h
ypercré tininémie c n be expl ined by sever l p thologic l processes which prim
rily ffect ren l function.
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4 - Wh t to do when hypercré tininémie:
In this ch pter we restrict ourselves to the discovery of n incipient ren l f i
lure.€ -Circumst nce of discovery: Outside of few noisy complic tions often mi
sle ding (edem syndrome or cute incre se in blood pressure often directing the
p tient to c rdiologist) or l te ( nemi , bnorm l c lcium phosph te), chroni
c ren l f ilure m nifested by mild symptoms clinics. This is p rticul rly true f
or ren l debut nte whose ch r cter symptom tic, indolent, re re dily seen s s
ynonymous with "not serious" by both p tients nd pr ctitioners. Yet this is the
beginning st ge th t ther peutic options re more numerous nd more effective i
n ltering the course of progressive kidney dise se. b-Di gnostic (+) ren l f il
ure: The interpret tion of pl sm cre tinine rem ins n import nt screening nd
di gnosis of ren l f ilure. The incre se in pl sm cre tinine is l te sign nd
insensitive to detect incipient lter tion of ren l function. In pr ctice, the
"norm l" pl sm cre tinine only define the G ussi n distribution of pl sm cre t
inine me sured in the popul tion (me n ± 2 SD), but do not indic te the threshol
d v lues defining ren l f ilure. To r ise the ssessment of ren l function from
pl sm cre tinine, it is recommended lw ys using cle r nce estim ted by the Coc
kcroft nd G ult (CoCr).
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Another benefit of cre tinine corrected by Cockcroft is th t ch nges in cle r nc
e re estim ted in the s me direction nd re roughly correl ted to ch nges in g
lomerul r filtr tion r te (interest + + + for sc l ble monitoring of ren l funct
ion in the s me person in the bsence of ch nge in muscle m ss). The interpret t
ion of the CoCr is: A CoCr> 90 ml / min. corresponds to norm l ren l function
CoCr <60 ml / min. reflects moder te imp irment. The interpret tion of CoCr
between 90 nd 60 ml / min. must t ke into ccount the signs ssoci ted with ren
l nd Age: A CoCr between 90 nd 60 ml / min. ssoci ted with biologic l signs
(proteinuri , hem turi ), r diologic l or histologic l kidney corresponds to n
incipient ren l f ilure. A CoCr between 90 nd 60 ml / min. without ny other si
gns of kidney dise se m y correspond to norm l ren l function or low incipient n
ephrop thy. Th t's evolution under supervision th t will decide between the two
situ tions. This situ tion is p rticul rly common in the elderly (+ influences +
of ge in the Cockcroft), nd we spoke openly of ging "physiologic l" of ren l
function. This notion is mist ken, however, bec use the decre se in glomerul r
filtr tion nd cle r nce by Cockcroft is not system tic with ge (one third of i
ndividu ls rem in st ble) nd is usu lly ssoci ted with hypertension nd / or l
nd therom tous (ren l v scul r minimum likely). c-IR Differenti l Di gnosis vs
. chronic. To ffirm the cute chronic ren l f ilure, there re three criteri :
Criterion n mnestic: history of ren l dise se, high doses of cre tinine old Cri
terion ultr sound: kidney size is reduced in the IRC: <10 cm ultr sound (or <3 v
ertebr e on sn pshot of ASP). Fin lly, biologic l criteri : two nom lies poin
t to IRC normochromic normocytic nemi regener tive (second ry f ilure to pr
oduce erythropoietin) Hypoc lcemi (low ctive vit min D (c lcitriol) by f iling
ren l hydroxyl tion).
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