This document provides information on determining plasma creatinine levels, including:
1) Creatinine is produced from creatine in muscles and transported through the blood to the kidneys, making creatinine levels an indicator of muscle mass and renal function.
2) There are several methods for determining creatinine levels, including enzymatic, reflectometry, and Jaffe kinetic colorimetric methods.
3) The Jaffe method is simple and inexpensive but lacks specificity, as other substances can also produce color that is measured. Precise measurements require accounting for these interfering substances.
This document provides information on determining plasma creatinine levels, including:
1) Creatinine is produced from creatine in muscles and transported through the blood to the kidneys, making creatinine levels an indicator of muscle mass and renal function.
2) There are several methods for determining creatinine levels, including enzymatic, reflectometry, and Jaffe kinetic colorimetric methods.
3) The Jaffe method is simple and inexpensive but lacks specificity, as other substances can also produce color that is measured. Precise measurements require accounting for these interfering substances.
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This document provides information on determining plasma creatinine levels, including:
1) Creatinine is produced from creatine in muscles and transported through the blood to the kidneys, making creatinine levels an indicator of muscle mass and renal function.
2) There are several methods for determining creatinine levels, including enzymatic, reflectometry, and Jaffe kinetic colorimetric methods.
3) The Jaffe method is simple and inexpensive but lacks specificity, as other substances can also produce color that is measured. Precise measurements require accounting for these interfering substances.
Copyright:
Attribution Non-Commercial (BY-NC)
Available Formats
Download as TXT, PDF, TXT or read online from Scribd
Djillali Liab University School of Medicine Department of Phar
macy 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 TP N 01 DETERMINATION OF PLASMA CREATININE I- Definition: 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. II-Metabolism: 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. LDU - Pharmacy 1 TP N 01 DETERMINATION OF PLASMA CREATININE 3 - Formation of creatinine The metabolism of creatine and therefore that of creatinine can be summarized in this explanatory scheme. 2 LDU - Pharmacy TP N 01 DETERMINATION OF PLASMA CREATININE 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. 3 UDL - Pharmacy TP N 01 DETERMINATION OF PLASMA CREATININE 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 anticoagulant. LDU 4 - Pharmacy TP N 01 DETERMINATION OF PLASMA CREATININE Never fill tubes with a syringe which was not removed the needle (risk of aeroso ls, splashes and brutal expulsion of the needle). - Urine 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 mongst: 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 Creatinine e > 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). 5 LDU - Pharmacy TP N 01 DETERMINATION OF PLASMA CREATININE 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. e > Creatine. e > Sarcosine + Urea. > Glycine + formaldehyde + H202> proportional to the stainin g 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 6 LDU - Pharmacy TP N 01 DETERMINATION OF PLASMA CREATININE 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. V 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). 7 LDU - Ph rm cy TP N 01 DETERMINATION OF PLASMA CREATININE 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 200μl 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 n] 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. 8 LDU - Ph rm cy TP N 01 DETERMINATION OF PLASMA CREATININE 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). 9 LDU - Ph rm cy TP N 01 DETERMINATION OF PLASMA CREATININE 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). 10 LDU - Ph rm cy