Measuring Metabolites in Plasma Samples by Flame Photometry and Spectrophotometry - Research Paper Example

Measuring Metabolites in plasma samples by flame photometry and spectrophotometry of The concentration of plasma urea and creatinine was determined using spectrophotometry at 540 and 500nm respectively. The concentration of urea for both sample A and B were found to lie within the reference interval. However, the plasma creatinine concentration was found to be above the reference intervals. Based on the measurements it was determined that Sample A was from a healthy individual whereas sample B was from the sick individual probably diagnosed with renal impairment. It is worth noting that both these parameters have shortfalls and cannot be exclusively used to diagnose disease but can be used for preliminary prognosis. Introduction Creatinine is a waste product not known possess any physiological function and normally excreted unchanged. It is a heterocyclic nitrogenous compound (2‐amino‐1‐methyl‐5H‐imidazol‐4‐one) with Molecular Weight of 113. It is produced in the muscle from creatine and consequently excreted by the kidneys through both glomerular filtration and active secretion. Dietary sources, especially cooked meat contribute approximately 10% creatinine. Urine creatinine content can be used to assess renal function instead of urea since it is not considerably influenced by non-renal factors such as protein turnover and hydration state. A component of ratios with other analytes can be used to assess their excretion (e.g. calcium: creatinine ratio). Serum creatinine measurement is used as a test of renal function. The progression and treatment of acute kidney injury and chronic kidney disease (CKD) can be monitored using serum creatinine (Marshall 2012, pg. 2). Serum creatine can also be used in combination with measurements of urine creatinine to generate the creatinine clearance, which is an indicator of the glomerular filtration rate (GFR). However, imprecision in difficulty of collecting accurate timed urine specimens hinder reproducibility. Together with demographic information; age, race, sex it can be used to estimate GFR (eGFR). A (slightly) elevated [creatinine] may not indicate impaired renal function (Husdan & Rapoport 1968). Surplus nitrogen in the body is removed through The formation of urea and its eventual excretion by the kidneys (~75%). As an end product of metabolism urea does not take part in synthetic reactions within the body. The contribution of urea to the medullary hypertonicity In the kidneys determines the kidneys ability to generate concentrated urine. Even though Urea is recycled by this process, it does not undergo any metabolic conversion. Traditionally urea is measured as an index of renal function with limited value in this application. Creatinine measurement and glomerular filtration rate (eGFR) are more robust indices (Marshall 2012). The dependence of plasma urea on the rate of formation-nitrogen turnover and the rate of its excretion is a limitation in its use. Approximately 10% is lost in sweat and the gut, even though, the kidneys are the major excretory pathway. Reabsorption also makes the excreted urea an inaccurate reflector of GFR Urea measurement should not be used as a general test of renal function however in certain Circumstances related to renal disease it is useful; suspected prerenal renal failure due to fluid depletion or cardiac failure, where plasma urea will tend to rise before plasma creatinine concentration. Plasma urea concentration is also useful in monitoring the effects of renal replacement treatment, the prediction of the severity of acute pancreatitis and inherited metabolic disease more definitive diagnostic tests may be required to diagnose these conditions further since Plasma urea concentration] tends to be low in patients with urea cycle disorders. Electrolytes are charge possessing chemicals that interact with water (sodium) or cellular impulses (potassium). Potassium which a primary intercellular electrolyte modulates cellular signalling in the cell controlling vesicles and channels. Hyperkalaemia is a condition the plasma potassium content is high and tissue that is exposed to the plasma is damaged. Acute cases involve damage to the heart muscle that and thereby induce myocardial infarction. Urea and electrolytes are also commonly referred to as U & E’s and are important markers for Kidney function and electrolyte level. Apart from the diagnosis of renal diseases, U & E’s can be used to in the determination of cardiac risk and dehydration (Basten 2010, pg. 47-49). The aim of this experiment is to find the amount of potassium (K+), urea and creatinine in the plasma samples of two individuals using a flame photometer and a spectrophotometer. One of the individuals is normal and while the other has disease that will be identified. Methods 1) Measurement of creatinine by Jaffe’s method 2 ml of saturated picric acid solution (1.5% w/v) and 1ml of NaOH (10% w/v) were added to each of four test tubes. To test tube 1 which was labelled as the blank about 40μl of distilled water. To test tube 2 labelled as the standard about 40μl of creatinine standard solution (0.05 mg/ml) was added. 40μl of plasma A was added to test tube 3 labelled as test 1. Another 40μl of plasma B was added to test tube 4 (test 2). The contents of each test tube were Mixed and incubated at 37°C for 30 minutes. The reactions were then carefully transferred to cuvettes. A spectrophotometer set to 520 nm and zeroed with the blank in test tube 1 was used to measure absorbance of each of the solutions and the values recorded in the data sheet. The concentration of creatinine in each plasma sample was calculated using the following equation. = (absorbance of test / absorbance of std) * conc of std 2) Measurement of urea using a colorimetric reaction To each of the four glass test tubes provided 3 ml of reagent A be added. Thereafter 15μl of water was added to test tube 1 (the blank), 15μl of urea standard solution was added to test tube 2 (the standard), 15μl of plasma A added to test tube 3 (test 1) and 15μl of plasma B To test tube 4 (test 2). The contents of each test-tube were Mixed and incubated for 15 minutes at 37°C. The spectrophotometer was set to 500 nm and zeroed with the blank. Each of the remaining samples solutions was transferred into a cuvette, and their respective absorbance at 500 nm recorded. The concentration of urea in the plasma was calculated using the following equation = (absorbance of test / absorbance of std) * conc of std 3) Measurement of K+ by flame photometry A standard curve that covered the concentration of K+ from 0 – 0.5 mM was prepared. Dilute each of the plasma samples was diluted 1 in 50 with water (10ml of each). Both the standards and samples were read on the flame photometer, and their emission values recorded in a table. Results The plasma creatinine and urea was measured using spectrophotometry at 520 and 500nm respectively. The average creatinine in plasma was found to be 0.153mg/ml with a standard deviation of 0.0118 and 0.0273mg/ml with a standard deviation of 0.01122 for group A and B samples respectively. Table 1 Concentration of creatinine in plasma (mg/ml) Group   A     B   Mean 0.015333967 0.027250500 SD 0.011774500 0.012160437 SEM 0.002775276 0.002866242 N 18            18    P value and statistical significance:  The two-tailed P value for the two sample means was calculated to be 0.0052 which implies that the difference between concentration of creatinine in Group A and B is very statistically significant (Ci= -0.011916533 at 95% confidence interval, t = 2.9868, df = 34, standard error of difference = 0.004). For the urea concentration in plasma, the average concentration was measured to be 0.2309 with standard deviation of 0.0819 and 1.94587 with standard deviation of 0.6355171 for Group A and B respectively. Table 2 Descriptive statistics for Urea in plasma (mg/ml) Group   A     B   Mean 0.2308705 1.9458700 SD 0.0818862 0.6355171 SEM 0.0183103 0.1421059 N 20          20    The difference means of the two samples was calculated using the t test and assuming equal variances. The two-tailed P value was calculated to be less than 0.0001 implying that the difference in ureaplasma concentration is extremely statistically significant (Ci: -1.7149995, 95% confidence interval, t = 11.9695, df = 38, standard error of difference = 0.143) Discussion, From the results of the measurements, it can be seen that Sample A represents the reference samples from the creatinine range between 0.007 and 0.015mg/ml (Marshall 2012, pg. 3). Sample 2 has creatinine values above the reference intervals in adults. However, it is also observed that the plasma urea is within the reference interval both for sample A and B. Urea reference interval should typically range between 0.15 and 4.68mg/ml. The diagnosis made is impaired renal function based on the plasma creatinine content Ashley & Morlidge 2008, pg. 21). Creatinine is commonly measured in patients who are suspected of having or being at Risk of, impaired renal function due shock, nephrotoxic drug exposures dehydration and intrinsic renal disease. However, about 10% of the total difference can be accounted in diet. However wide the reference intervals for creatinine are; intra‐individual variations are relatively little. This broad reference interval in addition to the Inverse Correlation of creatinine to glomerular filtration rate (GFR) make it an insensitive test in the detection of early or mild renal impairment. The High creatinine values could be due to renal failure (both acute and chronic) and also high muscle mass. Alternatively over hydration and low muscle mass contribute to low creatinine values. The simultaneous measurement of Urea and creatinine is a common practice though the urea is an inferior indicator of renal function as it is influenced by the rate of nitrogen turnover. However, plasma urea has been found to increase faster typically in instances of dehydration that leads to pre-renal failure than creatinine (Association for Clinical Biochemistry 2012, pg. 4). References Husdan, H., & Rapoport, A. (1968). Estimation of creatinine by the jaffe reaction a comparison of three methods. Clinical chemistry, 14(3), 222-238 Marshall. W (2012). Creatinine (serum, plasma). Copyright Association for Clinical Biochemistry. Retrived from http://www.acb.org.uk/Nat%20Lab%20Med%20Hbk/Creatinine.pdf Basten G. (2010). Introduction to clinical Biochemistry. Interpreting Blood Results. Dr. Graham Basten & Ventus publishing Aps pg. 48-50 Ashley, C. (2004). Renal failure–how drugs can damage the kidney. Hospital Pharmacist, 11, 48-53. Ashley, C., & Morlidge, C. (Eds.). (2008). Introduction to renal therapeutics. Pharmaceutical Press. Read More