0.72 g or 1.40 g2 creatinine per litre urine and where n=10 determinations in each case. Inter-day repeatability: Standard deviation (rel.) sw =1.5% or 1.4%.
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Creatinine in urine
Creatinine in urine Application
Determination in urine
Analytical principle
Photometry in microtitre plates (Jaffe method)
Completed in
November 2007
Summary The method described here permits the determination of creatinine in urine on a miniaturised scale by means of a photometric microplate reader. The urine samples that have been diluted in a ratio of 1:50 are applied to a microtitre plate and picric acid in alkali is added. After a reaction time of 45 minutes, the absorbance of the orange-red creatinine picrate reaction product (see Fig. 1) is measured at the absorbance maximum of 492 nm and at a temperature of 36 8C using a photometric microplate reader.
Fig. 1. Jaff reaction for the photometric determination of creatinine [1±4].
Calibration is carried out using aqueous creatinine standard solutions that are treated in the same manner as the samples by adding picric acid in alkaline solution and measured by photometry. Intra-assay repeatability:
Standard deviation (rel.)
sw = 1.6% or 1.6% 2.1% or 2.5%
Confidence interval
u = 3.6% or 3.6% 4.7% or 5.7%
The MAK-Collection for Occupational Health and Safety Part IV: Biomonitoring Methods, Vol. 12. DFG, Deutsche Forschungsgemeinschaft Copyright ° 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32538-2
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0.79 g or 1.71 g 1 0.72 g or 1.40 g 2 creatinine per litre urine and where n = 10 determinations in each case at concentrations of
Inter-day repeatability:
Standard deviation (rel.)
sw = 1.5% or 1.4% 1.4% or 2.2% Confidence interval u = 3.9% or 3.6% 3.6% or 5.7% at concentrations of 0.80 g or 1.72 g 1 0.72 g or 1.40 g 2 creatinine per litre urine and where n = 6 determinations in each case
Accuracy:
Recovery rate
Detection limit:
£ 0.125 g creatinine per litre urine
r = 107.9% or 102.6% 93.7% or 84.6% at theoretical concentrations of 0.72 g or 1.66 g 1 0.78 g or 1.63 g 2 creatinine per litre urine and where n = 8 determinations in each case
Creatinine Creatinine is a by-product in the metabolism of proteins and it is formed in the metabolism of the muscles from creatine and creatine phosphate. Creatinine mainly undergoes glomerular filtration in the kidney and is almost completely excreted via the renal route. On average, adults of normal body weight aged between 30 and 60 years excrete from 1.0 to 1.6 g of creatinine per day. In the case of healthy persons the physiological formation of creatinine is largely proportional to their muscle mass, which explains why creatinine excretion is generally lower in women than in men. Children exhibit a daily excretion of creatinine that is relatively strongly dependent on their age. In addition to age and gender, creatinine excretion is also particularly influenced by the consumption of meat and the intake of certain medication, such as opiates and diuretics. Urine production can exhibit relatively strong fluctuations, depending on the intake or loss of fluid and the consumption of coffee, alcohol or medication. In contrast, the excretion of creatinine throughout the day generally remains relatively constant with only slight diurnal fluctuations. For this reason the creatinine concentration in urine can be taken as a measure of the dilution of the urine [5±8]. The concentration of creatinine in urine often serves as a reference value for the analysis of working materials and their metabolites in urine if these materials, like creatinine, undergo glomerular filtration in the kidney. In this way diurnal variations 1 2
Duotrol¾ control urine Quantimetrix¾ control urine
171
Creatinine in urine
in the dilution of the urine can be compensated when exposure to xenobiotics is assessed. However, linking concentrations of hazardous substances in urine to the creatinine concentration does not make sense in every case and the above-mentioned factors that influence creatinine excretion also have to be taken into consideration. If xenobiotics are reabsorbed to a significant extent in the tubular region of the kidney, their concentrations cannot be assumed to be directly proportional to that of creatinine [5, 6]. In highly diluted or very concentrated urine samples too, using the creatinine content as a reference value does not lead to valid results for substance concentrations. Therefore, on principle, the Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area advises against calculating the concentration of hazardous substances or metabolites in urine with respect to the creatinine concentration. Nevertheless, the creatinine concentration should be measured in each urine sample that is to be tested for hazardous substances. It aids physicians in their assessment of the measured result. In the case of creatinine concentrations of less than 0.5 g/L and of more than 2.5 g/L the results obtained for the hazardous substances or their metabolites should not be taken into account in the reported findings [5]. At present 13 parameters, for which a relationship with creatinine in urine has been established, are shown in the List of BAT Values [9]. In particular, organic solvents are included in this group. In contrast, it has proved inapplicable to relate creatinine levels to the concentration of methanol and other polar organic solvents that also undergo tubular filtration. The use of creatinine as a reference value can be recommended in spontaneous urine samples under the following conditions [5]: ± in the case of substances for which the advantage of reference to creatinine has been confirmed, ± for substances that undergo glomerular filtration, ± for biomonitoring at workplaces subject to heat, ± in the case of large fluctuations in the intake of fluids and urine production. Authors: M. Blaszkewicz, K. Liesenhoff-Henze Examiners: G. Scherer , H.-U. Kåfferlein
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Creatinine in urine Application
Determination in urine
Analytical principle
Photometry in microtitre plates (Jaffe method)
Completed in
November 2007
Contents 1 2 2.1 2.2 2.3 2.4 3 3.1 4 4.1 5 5.1 5.2 6 6.1 6.2 7 8 9 9.1 9.2 9.3 9.4 10 11 12
General principles Equipment, chemicals and solutions Equipment Chemicals Solutions Calibration standards Specimen collection and sample preparation Sample preparation Operational parameters UV spectrometric operational conditions Analytical determination Sample application Photometric measurement of the samples Calibration Manual calibration Software-assisted calibration Calculation of the analytical results Standardisation and quality control Evaluation of the method Precision Accuracy Detection limit Sources of error Discussion of the method References Appendix
The MAK-Collection for Occupational Health and Safety Part IV: Biomonitoring Methods, Vol. 12. DFG, Deutsche Forschungsgemeinschaft Copyright ° 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32538-2
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1 General principles The urine samples that have been diluted in a ratio of 1 : 50 are applied to a microtitre plate and picric acid in alkali is added. After a reaction time of 45 minutes, the absorbance of the orange-red creatinine picrate reaction product is measured at the absorbance maximum of 492 nm and at a temperature of 36 8C using a photometric microplate reader. Calibration is carried out using aqueous creatinine standard solutions that are treated like the samples by adding picric acid in alkaline solution and measured by photometry.
2 Equipment, chemicals and solutions 2.1 Equipment Photometric microplate reader with evaluation software (e.g. Tecan GENios with Magellan software) Transparent 96-well microtitre plate for absorbance measurements with cover (e.g. Costar 96-Well Cell Culture Cluster) Analytical balance (e.g. Mettler AE 163) 10 mL, 100 mL, 250 mL, and 1000 mL Volumetric flasks Hand dispenser for adding a volume of 200 lL (e.g. Multipipette from Eppendorf) Variable piston pipette for volumes between 20 and 1000 lL (e.g. from Eppendorf) 10 mL Volumetric pipettes and 2 mL graduated pipettes made of glass (e.g. from Brand) 20 mL Glass beaker Aluminium foil If required, 10 mL polyethylene tubes Urine collection vessels (e.g. Sarstedt, No. 77.577) 1.5 mL Centrifuge tubes (e.g. from Eppendorf) Microcentrifuge (e.g. Eppendorf Centrifuge 5415 D) Stopwatch Cotton cloth
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2.2 Chemicals Hydrochloric acid, 32%, p. a. (e.g. Merck No. 1.00319.1000) Bidistilled water Picric acid (2,4,6-trinitrophenol), p. a. (e.g. Sigma-Aldrich No. 197378) Alternatively, 1% aqueous picric acid solution (e.g. Sigma-Aldrich No. 319287) Sodium hydroxide, p. a. (e.g. Merck No. 1.06498.0500) Creatinine hydrochloride, p. a. (e.g. Sigma-Aldrich No. C6257) Duotrol¾ Urine Liquid Level 1 (No. 107151) and Level 2 (No. 107152) from BIOMED Labordiagnostik GmbH (Combined Order No. 107164 ) Quantimetrix¾ Level 1 (No. Q 1431-31) and Level 2 (No. Q 1432-31) from Labor + Technik Methanol, p. a. (e.g. Merck No. 1.06009.1000) 2.3 Solutions 0.1 M Hydrochloric acid: 11.4 mL of 32% hydrochloric acid are transferred to a 1000 mL volumetric flask using a 10 mL volumetric pipette and a 2 mL graduated pipette made of glass. The flask is subsequently filled to its nominal volume with bidistilled water. 4.365 mM picric acid solution: 100 mg picric acid are weighed into a 100 mL volumetric flask and pre-dissolved with approx. 10 mL bidistilled water. The volumetric flask is subsequently filled to its nominal volume with distilled water. This solution is stable for approximately 10 weeks when stored at 4 8C. 0.25 M Sodium hydroxide: 2.5 g NaOH pellets are weighed into a 250 mL volumetric flask and pre-dissolved with approx. 100 mL bidistilled water. The volumetric flask is subsequently filled to its nominal volume with distilled water. Picric acid working solution: 10 mL volumetric pipettes are used to transfer 10 mL of the 4.365 mM picric acid solution and 10 mL of the 0.25 M sodium hydroxide solution to a 20 mL glass beaker. The resulting picric acid working solution in alkali is protected from light by wrapping the glass beaker in aluminium foil. The solution must always be prepared freshly, as it is stable for at most 2 hours.
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Creatinine in urine
2.4 Calibration standards Creatinine stock solution (1 g/L): 132.2 mg creatinine hydrochloride (MW: 149.58 g/mol) are weighed exactly into a 100 mL volumetric flask. The flask is subsequently filled to its nominal volume with 0.1 M hydrochloric acid. The stock solution should be stored at a temperature of 2 to 8 8C, and is stable for at least 10 weeks under these conditions. Calibration standards in water: The creatinine stock solution (1 g/L) is diluted with bidistilled water in 10 mL volumetric flasks according to the pipetting scheme shown in Table 1. The calibration standards are stored in sealed volumetric flasks or 10 mL polyethylene tubes. The prepared standards are stable for at least 10 weeks when stored at a temperature of + 4 8C. Table 1. Pipetting scheme for the preparation of the calibration standard solutions in water. Volume of the creatinine stock solution [lL]
Final volume of the calibration standard solution [mL]
Concentration of the calibration standard solution [mg/L]
Equivalent concentration in the analytical samples [g/L]
25 50 100 250 400 500 700
10 10 10 10 10 10 10
2.5 5.0 10.0 25.0 40.0 50.0 70.0
0.125 0.250 0.500 1.250 2.000 2.500 3.500
The resulting calibration standard solutions are analysed directly as described in Sections 4 and 5 in the same manner as the diluted urine samples. As the urine samples, in contrast to the calibration solutions, are diluted 1:50 before measurement (see Section 3.1), the creatinine content in the calibration standards is equivalent to concentration levels of 0.125 to 3.500 g/L in the samples.
3 Specimen collection and sample preparation The urine samples collected in urine vessels can be stored for at most one day in the refrigerator or they can be stored deep-frozen at ±20 8C until they are analysed at a later date.
Creatinine in urine
176
3.1 Sample preparation Any urine samples that were previously frozen are thawed. The samples are shaken briefly, and 20 lL of each of the urine samples that have been brought to room temperature are placed in a 1.5 mL centrifuge tube. Then 980 lL of bidistilled water are added (dilution 1 : 50), and the tubes are sealed and shaken briefly. They are subsequently centrifuged for 5 minutes at 6000 rpm in order to remove any suspended particles. If required, the diluted urine samples can be frozen at ±20 8C and then used at a later date after they have been thawed.
4 Operational parameters The photometric measurement of the samples is performed on a microtitre plate using a microplate reader. 4.1 UV spectrometric operational conditions Measurement type:
Absorbance
Measured wavelength:
492 nm
Measurement direction:
Column by column
Number of measurements: 50 per sample Pre-shaking of the plate:
Duration: Mode: Intensity:
Settlement phase:
10 sec
Temperature:
36 8C Ô 0.5 8C
60 sec Circular Normal
5 Analytical determination The urine samples treated as described in Section 3.1 are determined in duplicate. Two urine control samples of different concentrations (see Section 8) are included in each analytical series for the purpose of quality control. Bidistilled water is included as a reagent blank value. The quality control samples, the calibration standards and the reagent blank are analysed in a duplicate determination in the same manner as the samples.
177
Creatinine in urine
5.1 Sample application A pipette is used to place 50 lL of each sample carefully into a separate well of the microtitre plate. After all the samples have been placed in the wells, a hand dispenser is used to add 200 lL of the picric acid working solution to each well, starting at position A1 and proceeding vertically column by column. Table 7 in the appendix shows an example of a template for a microtitre plate with 96 wells. The batch is closed with a lid, carefully shaken manually and stored at room temperature protected from light (e.g. in aluminium foil). 5.2 Photometric measurement of the samples After a reaction time of 45 minutes, the samples are measured within a period of 3 minutes by photometry using a plate reader at a temperature of 36 8C. Before the actual measurement, the underside of the plate must be cleaned with a cloth soaked in ethanol. In the case of creatinine concentrations outside the calibration range (>3.5 g/L) the urine samples that have been diluted 1:50 are diluted again 1:2 with bidistilled water and measured anew.
6 Calibration The calibration standards prepared as described in Section 2.4 are measured directly as stipulated in Section 5 as part of a daily measurement series. The calibration can be carried out manually or with the aid of the reader software. The calibration curve is linear over a measurement range of at least 0.125 to 3.50 g of creatinine per litre urine. 6.1 Manual calibration The absorbances of the relevant calibration standards are measured in duplicate, then corrected by the blank value, and the mean is calculated and plotted as a function of the corresponding concentrations (taking the previous dilution of the urine samples of 1:50 into account, i.e. 0.125 to 3.50 g/L). The measured absorbances were between 0.1 and 0.9 for the calibration solutions and approx. 0.08 for the blank value. 6.2 Software-assisted calibration Certain parameters must be previously entered into the evaluation program in order to carry out software-assisted evaluation. This includes entry of the standard concentrations, the duplicate determination of the standards and samples, the dilution fac-
Creatinine in urine
178
tors, the blank value correction and the calculation of the calibration function passing through the zero point of the co-ordinates. The program calculates the calibration function and also the creatinine concentration in the samples. The linear calibration function must be checked for each batch.
7 Calculation of the analytical results The mean absorbance of the urine sample calculated from the duplicate determination is evaluated either manually or using the software of the reader from the linear calibration function obtained as described in Section 6. In the case of manual evaluation, the mean absorbance of the sample minus the mean absorbance of the blind value is divided by the gradient of the regression functions.
8 Standardisation and quality control Quality control of the analytical results is carried out as stipulated in the guidelines of the Bundesårztekammer [German Medical Association] [10] and in the special preliminary remarks to this series. Two control urine samples of different concentrations (Level 1 for the lower and Level 2 for the higher concentration range) are included in each measurement series for continuous control of the precision and accuracy of the method. In this case commercially available control material can be used. Alternatively, it is also possible to prepare control urine samples in the laboratory. In this case the expected value of the quality control material must be ascertained in a pre-analytical period (one analysis of the control material on each of 15 different days) [10±12]. Tables 2 and 3 show the expected values and the tolerance ranges given by the manufacturers of the control urine samples that were used by the authors. Table 2. Theoretical values and tolerance ranges for creatinine in Duotrol¾ control urine samples (Level 1 and Level 2) for various measurement devices as stated in the manufacturer's instructions for measurement according to Jaff. Measurement device
Method
Beckmann DadeDimension Olympus AU Roche Hitachi Roche Integra
Jaff Jaff Jaff Jaff Jaff
Mean value
Duotrol¾ Level 1
Duotrol¾ Level 2
Theoretical value [g/L]
Tolerance range [g/L]
Theoretical value [g/L]
Tolerance range [g/L]
0.77 0.69 0.74 0.69 0.73
0.59±0.96 0.52±0.86 0.56±0.91 0.52±0.86 0.56±0.91
1.69 1.60 1.68 1.70 1.63
1.28±2.10 1.22±1.98 1.28±2.08 1.29±2.10 1.24±2.02
0.72
0.55±0.90
1.66
1.26±2.06
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Creatinine in urine
Table 3. Theoretical values and tolerance ranges for creatinine in Quantimetrix¾ control urine samples (Level 1 and Level 2) for various measurement devices as stated in the manufacturer's instructions for measurement according to Jaff. Quantimetrix¾ Level 1
Quantimetrix¾ Level 2
Theoretical value [g/L]
Tolerance range [g/L]
Theoretical value [g/L]
Tolerance range [g/L]
Jaff Jaff
0.81 0.81
0.64±0.97 0.61±0.97
1.69 1.67
1.35±2.02 1.33±2.00
Jaff Jaff
0.74 0.85
0.59±0.88 0.68±1.02
1.59 1.72
1.27±1.90 1.37±2.06
Jaff Jaff Jaff
0.68 0.79 0.79
0.54±0.81 0.63±0.94 0.63±0.94
1.60 1.61 1.56
1.28±1.92 1.28±1.93 1.24±1.87
0.78
0.62±0.93
1.63
1.30±1.96
Measurement device
Method
Abbott Aeroset Beckmann Synchron DadeDimension Olympus AU 400/640 Ortho Vitros Roche Hitachi Roche Integra Mean value
As the manufacturer of the control material did not supply any data on control values for the measurement device used by the authors (Tecan GENios), the mean values of the theoretical values and tolerance ranges stated for the other measurement devices were calculated. All the measurements of the quality control samples during the measurement phases were always within the control ranges calculated in this way.
9 Evaluation of the method 9.1 Precision The two commercially available control urine samples (in each case Level 1 and Level 2) were used to determine the intra-assay repeatability. Ten determinations resulted in the intra-assay repeatability shown in Table 4.
Table 4. Intra-assay repeatability for the determination of creatinine in urine (n = 10). Control material
Mean value [g/L]
Standard deviation (rel.) sw [%]
Confidence interval u [%]
Duotrol¾ Level 1 Duotrol¾ Level 2 Quantimetrix¾ Level 1 Quantimetrix¾ Level 2
0.79 1.71 0.72 1.40
1.6 1.6 2.1 2.5
3.6 3.6 4.7 5.7
Creatinine in urine
180
Table 5. Inter-day repeatability for the determination of creatinine in urine (n = 6). Control material
Mean value [g/L]
Standard deviation (rel.) sw [%]
Confidence interval u [%]
Duotrol¾ Level 1 Duotrol¾ Level 2 Quantimetrix¾ Level 1 Quantimetrix¾ Level 2
0.80 1.72 0.72 1.40
1.5 1.4 1.4 2.2
3.9 3.6 3.6 5.7
The inter-day repeatability was also determined using the same control urine samples. The analyses were performed on six different days. The calculated precision is shown in Table 5. 9.2 Accuracy The accuracy of the method was checked by performing eight series of measurements in the course of one day on the control urine samples used. The recovery rates found, based on the theoretical concentration of the control urine samples, are shown in Table 6. Table 6. Recovery rates for creatinine, determined in four commercially available control urine samples (n = 8). Control material
Theoretical concentration * [g/L]
Recovery r [%]
Range [%]
Duotrol¾ Level 1 Duotrol¾ Level 2 Quantimetrix¾ Level 1 Quantimetrix¾ Level 2
0.72 1.66 0.78 1.63
107.9 102.6 93.7 84.6
107±110 101±104 92±96 81±87
* As the manufacturer of the microplate reader cannot provide any data regarding the theoretical values, the mean value of the stated theoretical values for other measurement devices was calculated in each case (cf. Table 2 and Table 3).
9.3 Detection limit The detection limit was not determined directly, but it is 0.125 g creatinine per litre urine or lower. Thus even creatinine concentrations in the pathological range are detectable well below the normal limit of 0.36 g/L, based on an average adult urine production of 1350 mL/day [13].
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Creatinine in urine
9.4 Sources of error The course of the Jaff colour reaction between picric acid and creatinine in alkali depends on the temperature, the reaction time and the concentrations of picric acid and sodium hydroxide. Therefore it is decisive to keep the above-mentioned parameters constant in order to ensure the reproducibility of the reaction. This applies in particular to the temperature of the measurement device, which should be kept constant at 36 8C (setting range 36.0 Ô 0.5 8C). Otherwise unforeseen measurement fluctuations may occur within a measurement series. Picric acid has only limited stability in alkaline solution. The picric acid/sodium hydroxide solution used is only stable for about two hours and should not be used after this period of time. In addition, the solution is sensitive to light and should therefore be kept in the dark. This also applies to the prepared microtitre plate during incubation. On principle, when working with microtitre plates it is essential to ensure that no liquid splashes out of the cavities while pipetting, thus leading to contamination of other samples. To prevent this a freely moving hand dispenser is recommended for pipetting the picric acid working solution. Soiling (e.g. fingerprints) on the underside of the microtitre plate may lead to considerable interference during measurement. In extreme cases this may render the plate unreadable for the measurement device. Therefore the underside of the plate must be kept clean and should be wiped with a cloth soaked in ethanol before measurement.
10 Discussion of the method This analytical method is based on the Jaff colour reaction [1] in which the active methylene group of the creatinine reacts with the C3 atom of picric acid [3±4] and thus forms an orange-red reaction product [2±4] (see Fig. 1). However, this colour reaction between picric acid and creatinine in alkaline solution is not specific to this substance. As a general rule, reducing compounds or compounds with a methylene group activated by ±NO2, ±CONH2, ±CH2=CH2-, ±COOR or ±N=N- can also form coloured products. However, no interfering reactions are caused by glucose, fructose, maltose, hydroxylamine or ascorbic acid. On the other hand aminoacetone, d-aminolevulinic acid and aminoxyacetic acid exhibit a colour reaction with picric acid [2]. However, the concentrations of the above-mentioned chromogens are very low in urine [14], so the interference caused by them can be regarded as insignificant. Thus, for example, the concentration of d-aminolevulinic acid in urine is lower by a factor of 100 to 1000 compared with that of creatinine. In addition to the method described here, the non-miniaturised Jaff reaction is classically used for the routine analysis of creatinine in urine [15±17]. The particular advantage of this miniaturised batch method using a microplate reader lies in the high number of samples that can be determined in a very short period. Moreover, it is easier to keep the reaction conditions stable, as all the samples are analysed within a few minutes. In this way the risk of temporal measurement fluctuations is minimised.
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182
Furthermore, it should be mentioned that commercially available determination kits for creatinine (e.g. from Rolf Greiner Biochemica GmbH) can be used above all in the clinical area. Commercially available control materials, for which data on the method and instrumental theoretical values and tolerance ranges (approx. Ô 20% deviation from the theoretical value) were provided by the manufacturer, were used for method validation. As no comparative values were available for the measurement system used by the authors, the mean values of the data given for the other devices were calculated and used as a basis for assessment of the accuracy. All the measured values for three of the examined control urine samples were close to the theoretical value and were clearly within the mean tolerance ranges. In the case of one control urine sample individual measured values were obtained that were slightly less than the lower limit for the maximum deviation from the theoretical value of 12% specified by the Bundesårztekammer [German Medical Association] [10]. The authors made the control material available to one examiner who confirmed the observed deviation. In no case was the mean tolerance range according to the manufacturer's data exceeded in either of the laboratories. The method described here permits reliable and accurate determination of creatinine in urine in the physiologically relevant concentration range. The examiners were able to reproduce the reliability criteria of the method without problems. In one case the Infinite M200 plate reader from Tecan was used. With regard to reduction of the potential danger, one examiner recommends the use of an aqueous picric acid solution (1%) instead of the solid substance for preparation of the alkaline picric acid working solution. Instruments used: Tecan GENiosTM microplate reader with Magellan software.
11 References [1] M. Jaff: Ûber den Niederschlag welchen Pikrinsåure in normalen Harn erzeugt und çber eine neue Reaktion des Kreatinins. Hoppe-Seylers Z. Physiol. Chem. 10, 391±400 (1886). [2] B. Kakac, Z. J. Vejdelek: Handbuch der photometrischen Analyse organischer Verbindungen, Reaktion mit 2,4,6-Trinitrophenol (Pikrinsåure), 275±276. Verlag-Chemie, Weinheim (1974). [3] B. Kakac, Z. J. Vejdelek: Handbuch der photometrischen Analyse organischer Verbindungen, 2nd supplementary volume, Reaktion mit 2,4,6-Trinitrophenol (Pikrinsåure) und Alkalien (Basen), 113±114. Verlag-Chemie, Weinheim (1983). [4] A. R. Butler: The Jaff reaction. Identification of the coloured species. Clin Chim Acta. 59, 227±232 (1975). [5] M. Weihrauch, B. Schulze, K.-H. Schaller, G. Lehnert: Kreatinin als Bezugsgræûe fçr Stoffkonzentrationen im Harn. In: H. Drexler, H. Greim (eds.): Biologische Arbeitsstoff-Toleranz-Werte (BAT-Werte), Expositionsåquivalente fçr krebserzeugende Arbeitsstoffe (EKA) und Biologische Leitwerte (BLW). Deutsche Forschungsgemeinschaft ± Arbeitsmedizinisch-toxikologische Begrçndungen, Spezielle Vorbemerkungen Vol. 1, 21±31, 9th issue, Wiley-VCH (2000). [6] Kommission Humanbiomonitoring des Umweltbundesamtes: Normierung von Stoffgehalten in Urin ± Kreatinin. Bundesgesundhbl. 616±-618 (2005). [7] B. M. Brenner, F. C. Rector (eds.): The Kidney. W. B. Saunders, Philadelphia, PA (1986).
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[8] J. Wallach: Interpretation of Diagnostic Tests. Little, Brown & Co., Boston, MA (1986). [9] DFG Deutsche Forschungsgemeinschaft: List of MAK and BAT Values 2009: Maximum Concentrations and Biological Tolerance Values at the Workplace, Report 45, Wiley-VCH, Weinheim (2009). [10] Bundesårztekammer: Richtlinie der Bundesårztekammer zur Qualitåtssicherung quantitativer laboratoriumsmedizinischer Untersuchungen. Dt. Ørztebl. 105, A341±A355 (2008). [11] J. Angerer and G. Lehnert: Anforderungen an arbeitsmedizinisch-toxikologische Analysen ± Stand der Technik. Dt. Ørztebl. 37, C1753±C1760 (1997). [12] J. Angerer, T. Gæen and G. Lehnert: Mindestanforderungen an die Qualitåt von umweltmedizinisch-toxikologischen Analysen. Umweltmed. Forsch. Prax. 3, 307±312 (1998). [13] L. Thomas: Labor und Diagnose. 3rd edition, p. 1444. Die Medizinische Verlagsgesellschaft, Marburg (1988). [14] Wissenschaftliche Tabellen Geigy: Teilband Kærperflçssigkeiten, Harn, 8th edition, 51±97 (1972). [15] J. A. Owen, B. Iggo, F. J. Scandrett, and C. P. Stewart: The determination of creatinine in plasma or serum and in urine: A critical examination. Biochem. J. 58, 426 (1954). [16] H. Bartels and M. Bæhmer: Eine Mikromethode zur Kreatininbestimmung. Clin Chim Acta. 32, 81±85 (1971). [17] K. Larsen: Creatinine assay by a reaction-kinetic principle. Clinica. Chimica. Acta 41, 209 (1972).
Authors: M. Blaszkewicz, K. Liesenhoff-Henze Examiners: G. Scherer , H.-U. Kåfferlein
Std. 2 5 mg/L
Std. 3 10 mg/L
Std. 4 25 mg/L
Std. 5 40 mg/L
Std. 6 50 mg/L
Std. 7 70 mg/L
C
D
E
F
G
H
Std. 7 70 mg/L
Std. 6 50 mg/L
Std. 5 40 mg/L
Std. 4 25 mg/L
Std. 3 10 mg/L
Std. 2 5 mg/L
Std. 1 2.5 mg/L
Sample 8
Sample 7
Sample 6
Sample 5
Sample 4
Sample 3
Sample 2
Sample 1
3
Sample 8
Sample 7
Sample 6
Sample 5
Sample 4
Sample 3
Sample 2
Sample 1
4
Sample 16
Sample 15
Sample 14
Sample 13
Sample 12
Sample 11
Sample 10
Sample 9
5
Sample 16
Sample 15
Sample 14
Sample 13
Sample 12
Sample 11
Sample 10
Sample 9
6
CoDu 1, CoDu 2: Control urine from Duotrol¾ Levels 1 and 2. CoQ 1, CoQ 2: Control urine from Quantimetrix¾ Levels 1 and 2.
Std. 1 2.5 mg/L
Blank
Blank
B
2
1
Sample 24
Sample 23
Sample 22
Sample 21
Sample 20
Sample 19
Sample 18
Sample 17
7
Table 7. Example of a template for a 96-well microtitre plate (Tecan GENios).
12 Appendix
Sample 24
Sample 23
Sample 22
Sample 21
Sample 20
Sample 19
Sample 18
Sample 17
8
Sample 32
Sample 31
Sample 30
Sample 29
Sample 28
Sample 27
Sample 26
Sample 25
9
Sample 32
Sample 31
Sample 30
Sample 29
Sample 28
Sample 27
Sample 26
Sample 25
10
CoQ 2
CoQ 1
CoDu 2
CoDu 1
Sample 36
Sample 35
Sample 34
Sample 33
11
CoQ 2
CoQ 1
CoDu 2
CoDu 1
Sample 36
Sample 35
Sample 34
Sample 33
12
Creatinine in urine 184
