An Experimental Determination of L-Lactate Concentration in Plasma - Lab Report Example

New Experimental Design - An Experimental Determination of L-Lactate Concentration in Plasma Using Lactic Dehydrogenase Reagents to be used in the assay: 1. 0.5 M Glycine buffer containing O.5M hydrazine hydrate, adjusted to pH 9.0, 2.5ml 2. 20mM NAD+ , 0.1ml 3. LDH, 12 μg in 0.2ml (i.e., 60 μg/ml) 4. 4.4 mM DL-Lactate Standard 5. Plasma (separated from erythrocytes within 15 min of collection to prevent leakage of lactate from damaged red cells) Method 1.1 Calibration curve with standard L- lactate Dilution of starting standard DL-lactate solution (4.4 mM equivalent to 2.2 mM L-lactate) to obtain working standards containing L-lactate in the concentration range 0.25 to 2.2mM was done as follows: Serial No. (Sl. #) Standard solution concentration* (mM) Dilution of Standard solution: Standard (x ml) + water (y ml) Working standard concentration* (mM) 1 2.2 x = 10.00 ; y = 0.00 2.20 2 2.2 x = 10.00 ; y = 1.00 2.00 3 2.0 x = 8.75 ; y = 1.25 1.75 4 2.0 x = 7.50 ; y = 2.50 1.50 5 2.0 x = 6.25 ; y = 3.75 1.25 6 2.0 x = 5.00 ; y = 5.00 1.00 7 1.0 x = 7.50 ; y = 2.50 0.75 8 1.0 x = 5.00 ; y = 5.00 0.50 9 1.0 x = 2.50 ; y = 7.50 0.25 * Refers to the concentration of L-Lactate present in the DL-Lactate solution The undiluted starting standard provided 2.2mM L-lactate. The rest of the working standards containing L-lactate concentrations of 2.00, 1.75, 1.50, 1.25, 1.00, 0.75, 0.50, 0.25 mM were obtained by combining 10.00ml, 7.50ml, 6.25ml, 5.00ml, 7.50ml, 5.00ml, and 2.50ml of the starting DL- lactate standard (4.4 mM) with 1.00, 2.50, 3.75, 5.00, 2.50, 5.00 and 7.50ml of distilled water, respectively. Blank solution contained no lactate. 1.2 Assay Procedure The various constituents and their volumes to be added to the reaction tubes are as follows: Tube # Glycine –hydrazine buffer, pH 9.0 20 mM NAD+ Working Standard (From Table 1, Sl. # ) (ml) L-Lactate concn. (mM) LDH Plasma (ml) Water (ml) 1 (Blank) 2.6 ml 0.1 ml -- 0.00 0.2 ml -- 0.1 ml 2 (Blank) 2.6 ml 0.1 ml -- 0.00 0.2 ml -- 0.1 ml 3 2.6 ml 0.1 ml #9, 0.1 ml 0.25 0.2 ml -- -- 4 2.6 ml 0.1 ml #9, 0.1 ml 0.25 0.2 ml -- -- 5 2.6 ml 0.1 ml #8, 0.1 ml 0.50 0.2 ml -- -- 6 2.6 ml 0.1 ml #8, 0.1 ml 0.50 0.2 ml -- -- 7 2.6 ml 0.1 ml #7, 0.1 ml 0.75 0.2 ml -- -- 8 2.6 ml 0.1 ml #7, 0.1 ml 0.75 0.2 ml -- -- 9 2.6 ml 0.1 ml #6, 0.1 ml 1.00 0.2 ml -- -- 10 2.6 ml 0.1 ml #6, 0.1 ml 1.00 0.2 ml -- -- 11 2.6 ml 0.1 ml #5, 0.1 ml 1.25 0.2 ml -- -- 12 2.6 ml 0.1 ml #5, 0.1 ml 1.25 0.2 ml -- -- 13 2.6 ml 0.1 ml #4, 0.1 ml 1.50 0.2 ml -- -- 14 2.6 ml 0.1 ml #4, 0.1 ml 1.50 0.2 ml -- -- 15 2.6 ml 0.1 ml #3, 0.1 ml 1.75 0.2 ml -- -- 16 2.6 ml 0.1 ml #3, 0.1 ml 1.75 0.2 ml -- -- 17 2.6 ml 0.1 ml #2, 0.1 ml 2.00 0.2 ml -- -- 18 2.6 ml 0.1 ml #2, 0.1 ml 2.00 0.2 ml -- -- 19 2.6 ml 0.1 ml #1, 0.1 ml 2.20 0.2 ml -- -- 20 2.6 ml 0.1 ml #1, 0.1 ml 2.20 0.2 ml -- -- 21 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- 22 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- 23 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- 1. The blank and assays #1-23 were set up as follows: 2.6 ml of buffer, 0.1 ml of NAD+ and 0.2 ml of LDH solution were pipetted out into spectrophotometer tubes marked 1 to 23 and brought to 37oC by placing in a water bath for 5 min. 2. The tubes were next placed in the spectrophotometer previously adjusted to 340 nm and the absorbance values at zero time were recorded. 3. The tubes were transferred again to the water bath at 37oC and the reaction was started by adding 0.1 ml of the individual working standards containing different L-lactate concentrations to tubes 3 to 20. The tubes were covered with a piece of Parafilm and the contents mixed well. 4. To tubes #21, 22 and 23, 0.1ml of plasma was added in place of the standard lactate. 5. A blank consisting of 2.6 ml of glycine-hydrazine buffer, 0.1ml of NAD+ , 0.1ml of distilled water instead of lactate and 0.2 ml of LDH was performed in duplicate in tubes #1 and 2. 6. Absorbance readings were noted at the end of 30, 35 and 40 min. 1.3 Data analysis The corrected absorbance for the standards and plasma sample were obtained by subtracting the individual zero-time values from all the duplicate readings, finding the average values and subtracting the mean blank value from them. A standard graph of L-lactate versus absorbance was drawn by plotting the L-lactate concentrations (0.25 to 2.2 mM) on the X-axis and the corresponding absorbance values (corrected for blank absorption) on the Y-axis. The unknown concentration of lactate in the plasma sample was determined based on its absorbance value with reference to the standard graph. Experimental Lab Report Introduction Life depends on innumerable biochemical reactions that are catalysed by enzymes. Many of the more accessible enzymes in blood as well as urine have diagnostic clinical applications. They have become useful as aids for diagnosing and monitoring several diseases. Small amounts of different enzymes are usually present in the blood as a result of normal cell turnover. However, the concentration of specific enzymes in blood may rise as a result of tissue damage or due to increased cellular proliferation as in cancer. It is generally preferred that the enzyme assays are simple to perform and are easily measured, e.g., by following the oxidation or reduction of nicotinamide adenine dinucleotide coenzyme or a colorimetric end-point. According to General Practice Notebook1, some of the enzymes commonly used for clinical diagnosis are: (1) alkaline phosphate for the diagnosis of bone disorders; (2) Creatine Phosphokinase (CPK or CK) which catalyses the reversible transfer of phosphate groups between creatine and phosphocreatine. CPK is largely found in skeletal muscle and heart muscle and is a marker for acute myocardial infarction (heart damage) since CK levels rise following myocardial infarction and in myocarditis, or skeletal muscle damage and muscular dystrophies. (3) Aminotransferase enzymes are involved in with amino acid metabolism. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are two aminotransferases that are clinically important. AST and ALT are released when liver cells are damaged as in viral hepatitis. AST is also elevated in myocardial infarction. Glutamic-Oxaloacetic Transaminase (GOT) is another aminotransferase that is used to diagnose myocardial infarction. Glutamic-Pyruvic Transaminase (GPT) is found in the highest quantities in liver. When liver cells are damaged, GOT and GPT levels rise quite early in the disease thereby constituting early markers for hepatitis. (4) Lipase is an enzyme that the pancreas secretes into the duodenum. Acute pancreatitis causes an elevation in lipase in the blood. (5) Acid phosphatase is an enzyme that hydrolyses phosphate esters. It is present in the prostate, liver, erythrocytes, platelets and bone in different isoforms. Clinically, the isoform present in the prostate has been found to be the most useful. (6) Lactate dehydrogenase (LD or LDH) is found in the highest concentrations in heart, skeletal muscle, liver, kidney and red blood cells albeit in different isoforms. LD1 and LD2 are predominant in the heart, red blood cells and kidney ; while, LD4 and LD5 are predominant in the liver and skeletal muscle. Total LDH measurement is not useful as it lacks tissue specificity. However, LD1 and LD5 are predominant in heart and liver, respectively. Apart from enzyme activity measurements in the blood, some enzymes are also used to estimate the concentration of specific metabolites in the blood. For example, the enzyme glucose oxidase is used to monitor blood glucose levels in diabetes. Another metabolite in the blood that is enzymatically measured is lactate. Lactate is a byproduct of anaerobic metabolism, whose levels are elevated in the hypoperfusion state (that is, when there is a decreased blood flow through an organ) because pyruvate cannot enter the Krebs cycle owing to insufficient oxygen supply and it is reverted to lactate. Hypoperfusion may result in permanent cellular dysfunction and death. Indeed, a considerable number of deaths in the ICU are secondary to multiple organ failure which is an end result of persistent hypoperfusion. Blood lactate constitutes a widely used marker of hypoperfusion. Also, since clinically lactate can be a measure of illness severity in circulatory shock, lactate measurements are routinely incorporated into treatment protocols 2. The aim of this experiment was to determine L-lactate concentration in plasma using the enzyme, lactic dehydrogenase. (Word count: 593) Method 1.1 A standard curve of L-lactate was established to estimate the concentration of lactic acid in plasma by diluting the stock solution with water as shown in Table 1. Table 1. Dilution of starting standard DL-lactate solution (4.4 mM equivalent to 2.2 mM L-lactate) to obtain working standards containing L-lactate in the concentration range 0.25 to 2.2mM. Serial No. (Sl. #) Standard solution concentration* (mM) Dilution of Standard solution: Standard (x ml) + water (y ml) Working standard concentration* (mM) 1 2.2 x = 10.00 ; y = 0.00 2.20 2 2.2 x = 10.00 ; y = 1.00 2.00 3 2.0 x = 8.75 ; y = 1.25 1.75 4 2.0 x = 7.50 ; y = 2.50 1.50 5 2.0 x = 6.25 ; y = 3.75 1.25 6 2.0 x = 5.00 ; y = 5.00 1.00 7 1.0 x = 7.50 ; y = 2.50 0.75 8 1.0 x = 5.00 ; y = 5.00 0.50 9 1.0 x = 2.50 ; y = 7.50 0.25 * Refers to the concentration of L-Lactate present in the DL-Lactate solution 1.2 Assay mixture The reaction mixtures for assay, including the blank, were prepared in tubes numbered 1 to 23 whose constituents were as shown in Table 2. Table 2. Table showing the composition of the various assay tubes. Tube # Glycine –hydrazine buffer, pH 9.0 20 mM NAD+ Working Standard (Sl. # in Table 1, ml) L-Lactate concn. (mM) LDH Plasma (ml) Water (ml) 1 (Blank) 2.6 ml 0.1 ml -- 0.00 0.2 ml -- 0.1 ml 2 (Blank) 2.6 ml 0.1 ml -- 0.00 0.2 ml -- 0.1 ml 3 2.6 ml 0.1 ml #9, 0.1 ml 0.25 0.2 ml -- -- 4 2.6 ml 0.1 ml #9, 0.1 ml 0.25 0.2 ml -- -- 5 2.6 ml 0.1 ml #8, 0.1 ml 0.50 0.2 ml -- -- 6 2.6 ml 0.1 ml #8, 0.1 ml 0.50 0.2 ml -- -- 7 2.6 ml 0.1 ml #7, 0.1 ml 0.75 0.2 ml -- -- 8 2.6 ml 0.1 ml #7, 0.1 ml 0.75 0.2 ml -- -- 9 2.6 ml 0.1 ml #6, 0.1 ml 1.00 0.2 ml -- -- 10 2.6 ml 0.1 ml #6, 0.1 ml 1.00 0.2 ml -- -- 11 2.6 ml 0.1 ml #5, 0.1 ml 1.25 0.2 ml -- -- 12 2.6 ml 0.1 ml #5, 0.1 ml 1.25 0.2 ml -- -- 13 2.6 ml 0.1 ml #4, 0.1 ml 1.50 0.2 ml -- -- 14 2.6 ml 0.1 ml #4, 0.1 ml 1.50 0.2 ml -- -- 15 2.6 ml 0.1 ml #3, 0.1 ml 1.75 0.2 ml -- -- 16 2.6 ml 0.1 ml #3, 0.1 ml 1.75 0.2 ml -- -- 17 2.6 ml 0.1 ml #2, 0.1 ml 2.00 0.2 ml -- -- 18 2.6 ml 0.1 ml #2, 0.1 ml 2.00 0.2 ml -- -- 19 2.6 ml 0.1 ml #1, 0.1 ml 2.20 0.2 ml -- -- 20 2.6 ml 0.1 ml #1, 0.1 ml 2.20 0.2 ml -- -- 21 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- 22 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- 23 2.6 ml 0.1 ml -- -- 0.2 ml 0.1 ml -- Assay Procedure: 2.6 ml of buffer, 0.1 ml of NAD+ and 0.2 ml of LDH solution are pipetted into tubes marked 1 to 23 and brought to 37oC by placing in a water bath for 5 min. The tubes were next placed in the spectrophotometer previously adjusted to the absorption maxima of NADH that is, 340 nm and the absorbance values at zero time were recorded. The tubes were transferred again to the water bath at 37oC and the reaction is started by adding 0.1 ml of the individual working standards to tubes 3 to 20, the tubes were covered with pieces of Parafilm, the solutions were mixed well by inverting the tubes closed with a finger over the Parafilm, and the absorbance readings were noted at the end of 30 min, 35 min and 40 min to ensure that the oxidation of lactate was complete. Along with the standards, a blank reaction was run in duplicate with 0.1 ml water in the place of lactate. Similarly, to tubes #21, #22 and #23, 0.1ml of plasma was added in place of the standard lactate and the assay was conducted as before. Results The absorbance values obtained with the standards and the plasma sample in triplicate were tabulated (Table 3). Table 3. Absorbance values of standard L-lactate (concentration range: 0.25 mM to 2.2 mM), plasma and blank reaction with Lactic dehydrogenase in glycine-hydrazine buffer, pH 9.0 taken at 0, 30, 35 and 40 min of incubation. Tube (mM L-lactate) A340 T = 0 T = 30min T=35min T= 40min 1 (Blank) 0.040 0.060 0.060 0.060 2 (Blank) 0.040 0.070 0.080 0.080 3 (0.25) 0.020 0.105 0.110 0.110 4 0.030 0.122 0.125 0.125 5 (0.5) 0.010 0.145 0.157 0.157 6 0.010 0.156 0.163 0.163 7 (0.75) 0.050 0.236 0.250 0.250 8 0.030 0.244 0.250 0.250 9 (1.00) 0.010 0.260 0.280 0.280 10 0.020 0.271 0.280 0.280 11 (1.25) 0.010 0.310 0.321 0.321 12 0.050 0.355 0.375 0.375 13 (1.50) 0.030 0.380 0.400 0.400 14 0.010 0.341 0.350 0.350 15 (1.75) 0.030 0.401 0.410 0.410 16 0.040 0.447 0.451 0.451 17 (2.00) 0.020 0.421 0.439 0.439 18 0.040 0.441 0.461 0.461 19 (2.20) 0.020 0.469 0.475 0.475 20 0.040 0.459 0.475 0.475 21 (Plasma) 0.050 0.377 0.390 0.390 22 (Plasma) 0.020 0.366 0.390 0.390 23 (Plasma) 0.030 0.382 0.394 0.394 As seen from Table 3, the oxidation of lactate was incomplete until 30 min of reaction time since the absorbance was rising, indicating that NADH was still being formed. All the lactate was converted to pyruvate only at 35 min of incubation as shown by the absorbance value reaching a plateau. Table 4. Corrected Absorbance values obtained by subtracting the respective zero time (T=0) values from the individual maximum absorbance values, calculating the mean, and subtracting the mean blank values. Tube A340 at T = 0 Maximum A340 Mean A340 [(Maximum A340) – T0] /N* Corrected A340 (Mean A340 -Blank) 1 (Blank) 0.040 0.060 0.030 - - 2 (Blank) 0.040 0.080 3 (0.25) 0.020 0.110 0.092 0.062 4 0.030 0.125 5 (0.5) 0.010 0.157 0.150 0.120 6 0.010 0.163 7 (0.75) 0.050 0.250 0.210 0.180 8 0.030 0.250 9 (1.00) 0.010 0.280 0.265 0.235 10 0.020 0.280 11 (1.25) 0.010 0.321 0.318 0.288 12 0.050 0.375 13 (1.50) 0.030 0.400 0.355 0.325 14 0.010 0.350 15 (1.75) 0.030 0.410 0.395 0.365 16 0.040 0.451 17 (2.00) 0.020 0.439 0.420 0.390 18 0.040 0.461 19 (2.20) 0.020 0.475 0.445 0.415 20 0.040 0.475 21 (Plasma) 0.050 0.390 0.358 0.328 22 (Plasma) 0.020 0.390 23 (Plasma) 0.030 0.394 * N = number of replicate assays As seen from Table 4, the absorbance values showed a rising trend throughout the selected range of L-lactate (0.25 to 2.2 mM) that was used to set up the calibration curve. This indicates that the coenzyme NAD+ was available in sufficient amounts for the enzyme to maintain a steady reaction rate until all the lactate provided had been oxidised. Calibration Curve A calibration curve of L-lactate versus absorbance was drawn by plotting the L-lactate concentrations (0.25 to 2.2 mM) on the X-axis and the corresponding absorbance values corrected for blank absorption (i.e., values shown in the last column, Table 4) on the Y-axis. Fig. 1 depicts the calibration curve. Fig. 1. Estimation of plasma L-lactate concentration from the calibration curve of known L-lactate concentrations plotted against respective absorbance values The concentrations in the range of 0.25 to 2.2 mM L-lactate showed a linear relationship with the measured absorbance values (Fig. 1). The concentration of L-lactate corresponding to the absorbance (0.328 Au) of the experiment with plasma was obtained from the X-axis as 1.6 mM. Discussion and Conclusion Lactic dehydrogenase catalyses the oxidation of L-lactate in the presence of the coenzyme NAD+ to produce pyruvate. This reaction is made use of to determine the concentration of lactate in blood which is an indicator of the anoxic state (hypoperfusion) of cells. There is a need for continuous measurement of blood lactate as a reliable end point indicator of resuscitation. However, the conventional photometric assays are rather slow, which is a drawback, and these might be replaced in the near future by biosensors that use lactate oxidase (instead of LDH) and electrochemical detection. The normal values of L-lactate in plasma3 range between 0.5 and 2.2 mM. Lactate concentrations greater than 4 mM call for concern and necessitate immediate hospitalisation of the patient. The calibration curve in the present experiment was set up with a concentration range of 0.25 to 2.2 mM L-lactate. In the presence of adequate amounts of LDH as determined by the Pilot study, and NAD+, the reaction Lactate  Pyruvate took about 35 min to reach completion. Therefore, it is important to confirm that the entire quantity of lactate present in the reaction mixture has been converted to pyruvate by allowing the incubation to continue and measuring the absorbance until no more increase is registered. The value obtained for the plasma sample in the current experiment was 1.6 mM which was within the normal range. In the clinical biochemical lab, blood samples with lactate concentrations higher than normal are commonplace. To estimate higher lactate values in pathological samples, an appropriately higher range of L-lactate standards will have to be used. This would also necessitate using greater amounts of the coenzyme NAD+ as well as the enzyme, since the rate of the reaction may be reduced due to substrate inhibition if the amount of substrate is very high while the amount of enzyme present is inadequate. The standard curve was expected to pass through the origin since both the zero-time reading and blank absorbance value had been subtracted from the absorbance of the individual standard reaction mixtures. However, an offset (intercept value 0.04 Au) was observed (Fig. 1). This might have been caused by experimental errors such as (1) pipetting error while using the Gilson pipette, (2) loss of reaction mixture due to adherence to the Parafilm, and (3) a minor discrepancy in the spectrophotometer readings. Future experiments should take enough care to minimise these errors. References 1. General Practice Notebook (2010). Diagnostic Enzymes. General Practice Notebook – a UK Medical Reference on the World Wide Web. Accessed on 19 July 2010 from http://www.gpnotebook.co.uk/simplepage.cfm?ID=470155322 2. Meregalli, A., Oliveira, RP. and Friedman, G. 2004. Occult hypoperfusion is associated with increased mortality in hemodynamically stable, high-risk, surgical patients. Critical Care, 8: R60-R65. Accessed on 19 July 2010 from http://ccforum.com/content/8/2/R60 3. http://www.nlm.nih.gov/medlineplus/ency/article/003507.htm Read More