A Lab Determination of Serum Creatinine. Methods and Results

1.0. Introduction

Creatinine is considered to be a waste product of phosphocreatine and creatine. Thus, it is among the common analytes in clinical chemistry, apart from glucose. There are a number of methods used to analyze creatinine; these methods have evolved from the first reaction described by Jaffe in 1880s. Scientists, over the years, have progressed Jaffe’s method through various phases. This method employed deproteinized blood and to improve its specificity, it isolated creatinine from other common substances that interfered with the process through absorption on the aluminium silicates, followed by its extraction into the alkaline picrate after decanting (Slot 1965). Performing Jaffe’s method at alkaline pH and neutral pH are among other strategies that are used to improve specificity. The only reaction at the neutral pH in this method is that of interfering substances; this result in a more accurate outcome (Husdan and Rapoport 1968).

Using Jaffe’s reaction method, when creatinine is exposed to the picric acid which is in an alkaline substance, it quantitatively produces an orange color. An experiment performed by Toora and Rajagopal (2002) indicated that after an incubation duration of approximately 15 minutes, at a normal temperature the orange color developed. Furthermore, the experiment also found out that it only requires a protein free of serum urine, one percent acid and approximately 0.75N NaOH for the orange color to develop. 1.0, 0.75 and 0.25N NaoH are the only alkaline measurements that produce the maximum orange color in PFF, standard and urine serum respectively. Moreover, the experiment als noted that the standard creatinine solution is prepared in 0.1N HCI and a PFF of serum is made by mixing the creatinine solution with a fresh tungstic acid. Thus, directly diluted sample of urine is required for measurement of creatinine.

2.0. Methods

2.1. Materials

This practical comprised of three experiments that applied Jaffe’s reaction method to determine serum creatinine. The first experiment was performed to prepare a standard curve for creatinine. The second experiment was performed to determine creatinine in ‘unknowns’ and the third experiment was performed to show data handling of the clinical data set.

The required solutions included 0.75M NaoH, saturated picric solution and the standard creatinine solution of 1mg/100ml.

2.2. Procedure

2.2.1. Experiment 1

The first step of this experiment was to use the standard solution of creatinine to prepare a series of standard solutions to cover the between 0mg/100ml to 1mg/100ml. in the second step, 1.0ml of the picric acid solution was added to 3ml if each standard solution in a test tube; 1.0ml of the NaoH was added to the resulting solution. The final solution was mixed thoroughly and left for a duration of 15 minutes to allow the orange color to develop. The third step of the experiment was to prepare blank using distilled water in place of the standard solution. Thereafter, the absorbance of the solution was measured at 500nm. The final step in this experiment was to prepare a calibration curve from the recorded, plotting all the points for the concentration and standard deviation as the error bars.

2.2.2. Experiment 2

This experiment determined creatinine in ‘unknowns.’ The major step involved was to test the 4 serum samples, from A to D for creatinine and quantitate the creatinine with calibration curve prepared in experiment 1. The samples used were diluted where appropriate so that the final absorbance of the reaction mixture is less than 1.0 absorbance unit.

2.2.3. Experiment 3

This experiment was on handling a clinical dataset. It required the researcher to find the percentage of each group of male and female, and the highest or lowest GFR values according to sex.

3.0. Results

3.1. Experiment 1

The data obtained from this experiment is summarized in table 1 below:

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Table 1: Absorbance

Table 1 shows that the rate of absorbance varies in every category according to the concentration of the solution. For instance, at a concentration of 0.25 mg/100ml, category 1 has an absorption rate of

0.175, category 2 has a rate of 0.162 and category 3 has a rate of 0.156. At a concentration of 0.5mg/100ml, the rates of absorption are 0.46, 0.302 and 0.316 for category 1 to 3 respectively. For a concentration of 0.75, the rates of absorption are 0.483, 0.509 and 0.503 for category 1 to 3 respectively. Lastly, for a concentration of 1mg/100ml, the rates of absorption are 0.664, 0.669 and 0.67 for category 1 to 3 respectively. Thus, the calibration curve for the data in table (1) is shown in figure 1 below:

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Figure 1: Creatinine calibration curve

Figure (1) indicates that the rate of absorption for all the categories was increasing with increase in the concentration.

3.2. Experiment 2

The results obtained in this experiment are shown in table (2) below:

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Table 2: Experiment 2

From the table above, under category 1, patients in group A have an absorption rate of 0.636, in category 2 the absorption rate is 0.63 and category 3 the absorption rate is 0.63. In group B, patients have absorption rates of 0.605, 0.583 and 0.608 for category 1 to 3 respectively. For group C, the absorption rates are 0.203, 0.199 and 0.202 for category 1 to 3 respectively. For patients in group D, the absorption rates are 0.33, 0.33 and 0.336 for category 1 to 3 respectively.

3.3. Experiment 3

The results for this experiment are as follows:

a. There are 17 patients in group A, 3 patients in B, 10 patients in C and 14 patients in group D

b. 70.6% are females whereas 29.41% are females in group A. Group B have 66.67% female and 33.33% males. Group C has 50% females and 40% males. Group D has 57.1% females and 71.4% males.

c. Patient number 541 has the highest GFR value in males (1.52) and patient number 494 has the highest GFR value in females (1.24).

GFR for patient number 10 can be estimated using the following formula: GFR = 166 × (Scr/0.7)-0.329 × (0.993)Age

Age = 29

Scr = 0.58 mg/100ml

Therefore, GFR = (0.58/0.7)-0.329 × (0.993)29 = 0.867756 mL/min/1.73 m2

4.0. Conclusion

The results from experiment show that the rate of absorption was increasing with increase in concentration of the standard solution. However, the curve in figure 1 depicts that the rate of absorption in category 1 has a greater deviation. At a concentration of 0.5mg/100ml, the rate of absorption was quite constant up to 0.75mg/100ml where it again rose as the concentration changed to 1mg/100ml; rate of absorption in category 1 and 2 rose uniformly.

The second experiment shows absorbance of patients in with different ages. The rate of absorbance is relatively high in group A (60 years old), followed by group B (102 years old), group D (86 years) and finally group C (56 years). Lastly, experiment 3 shows that majority of patients with kidney diseases are between 56 and 86 years. Males have the highest value of GFR as compared to females; highest male is 1.52 whereas the highest female is 1.24.

References

Heinegård, D. and Tiderström, G., 1973. Determination of serum creatinine by a direct

colorimetric method. Clinica chimica acta, 43 (3), pp.305-310

Husdan, H. and Rapoport, A., 1968. Estimation of creatinine by the jaffe reaction a comparison

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Liu, W.S., Chung, Y.T., Yang, C.Y., Lin, C.C., Tsai, K.H., Yang, W.C., Chen, T.W., Lai, Y.T.,

Li, S.Y. and Liu, T.Y., 2012. Serum Creatinine Determined by Jaffe, Enzymatic Method, and Isotope Dilution‐Liquid Chromatography‐Mass Spectrometry in Patients Under Hemodialysis. Journal of clinical laboratory analysis, 26 (3), pp.206-214.

Slot, C., 1965. Plasma creatinine determination a new and specific Jaffe reaction

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Taussky, H.H., 1954. A microcolorimetric determination of creatine in urine by the Jaffe

reaction. Journal of Biological Chemistry, 208 (2), pp.853-862

Toora, B. D. and Rajagopal, G. 2002. Measurement of creatinine by Jaffe's reaction—

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