Assay of Plasma Lactate Concentration - Lab Report Example

Introduction Lactic acid dehydrogenase (LDH, L-lactate: NAD oxidoreductase, EC 1 27) is an enzyme that catalyses the reversible oxidation of lactate to pyruvate in the presence of NAD+ as the oxidant. The enzyme assay is based on the conversion of lactate to pyruvate in the presence of the coenzyme NAD+ which, in turn, gets reduced to NADH, a compound that has strong absorption at 340nm. Thus, the conversion of lactate to pyruvate or vice versa will be accompanied by an increase/decrease in the absorption of the reaction mixture at 340nm1 on account of formation/consumption of NADH. The formation of NADH is followed by measuring the change in absorption at 340nm using UV spectrophotometry. Absorbance of a compound being proportional to its concentration according to the Beer-Lambert Law, the rate of the reaction can be calculated. Also, using a calibration curve of absorbance versus concentration, the amount of lactate present in an unknown solution such as plasma can be obtained. The aim of the experiment was to design a pilot study that could be further developed into an assay protocol for the evaluation of plasma lactate levels using the catalytic reaction of lactate dehydrogenase. (191 words) Method 1.1 Assay of LDH activity Oxidation of NADH and effect of enzyme concentration The effect of the enzyme concentration on the rate of the reaction was determined by running two reactions (Reactions 1 & 2, Table 1) with 0.1ml and 0.2ml LDH solution containing 0.3 μg and 0.6 μg of the enzyme protein, respectively. A micropipette was used to transfer the enzyme to the spectrophotometer tube. Pyruvate and NADH were the substrates used. The reaction was conducted in glycine buffer at pH 9. The absorbance was measured spectrophotometrically at 15-second intervals at 340nm until the absorbance stabilised. 1.2 Reversibility of the reaction Reduction of NAD+ LDH assay was repeated in the reverse direction (Reaction 3, Table 2) using lactate and NAD+ as the substrates. A higher concentration of enzyme i.e., 0.2ml of LDH equivalent to 6 μg of enzyme protein was used in this reaction. The reaction was started by adding 0.1ml of 1M lithium lactate and the absorbance was noted every 15 secs. Reversibility of the reaction; Effect of pyruvate Reaction 4 (Table 2) was a continuation of Reaction 3. After equilibrium had been attained in Reaction 3, the reaction was again reversed by adding 0.1M pyruvate to the reaction mixture and the absorbance values measured until a new equilibrium had been reached (Fig. 2). This reaction was performed to study which reaction would be more suitable. Effect of hydrazine Reaction 5 (Table 2) was performed exactly as Reaction 3 but using glycine-hydrazine buffer at pH 9.0. The result obtained is depicted in Fig.2. Results Experiment 1.1 : Effect of enzyme concentration in LDH activity assay The results of this experiment conducted with 0.1 (Reaction 1) and 0.2 (Reaction 2) are shown in Table 1. Table 1 – Table showing the absorbance (Au) readings of Reactions 1 and 2 at 15 second intervals at 340nm Time (in seconds) Reaction 1 (with 0.1ml LDH) Absorbance at 340nm (Au) Reaction 2 (with 0.2ml LDH) Absorbance at 340nm (Au) 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 0.592 0.522 0.5 0.476 0.451 0.426 0.401 0.375 0.35 0.327 0.303 0.28 0.257 0.235 0.213 0.192 0.171 0.152 0.135 0.12 0.107 0.094 0.088 0.083 0.08 0.605 0.471 0.426 0.378 0.328 0.28 0.232 0.189 0.148 0.113 0.09 0.078 0.074 0.072 0.071 0.071 The absorbance changes observed with 0.1ml and 0.2ml enzyme are plotted in the graph (Fig.1). Since the reduction of pyruvate by LDH was studied here, the concurrent oxidation of the coenzyme NADH is observed as a decrease in the absorbance at 340 nm. It is seen from the graph that the rate of reaction is faster with the higher concentration of the enzyme with the oxidation of NADH reaching a plateau around 3 min (180 sec) while the reaction goes beyond 5min (300 sec) when less enzyme is used. Fig. 1. Graph showing NADH consumption or lactate formation from pyruvate with time catalysed by lactate dehydrogenase. A: 0.1ml enzyme, B: 0.2ml enzyme (Reactions 1 & 2). 1.2 Reversibility of the reaction: Reduction of NAD+ , effect of hydrazine on the reverse reaction, and pyruvate effect on the reaction rate Table 2. –Showing Absorbance (Au) readings obtained for Reactions 3, 3a and 4 at 15 second intervals until equilibrium has been reached at 340nm Time (in seconds) Reaction 3 (with lactate as substrate, glycine buffer, pH 9) Reaction 3a (with lactate as substrate and added pyruvate, glycine buffer, pH 9) Reaction 4 (carried out with lactate as substrate and glycine- hydrazine buffer, pH 9) 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 600 900 1200 1500 1500 1515 1530 1545 1560 1575 1590 1605 0.004 0.093 0.115 0.135 0.15 0.162 0.174 0.182 0.19 0.197 0.202 0.208 0.212 0.216 0.22 0.223 0.225 0.228 0.23 0.232 0.234 0.249 0.255 0.259 0.259 0.069 0.07 0.069 0.069 0.069 0.069 0.069 0.069 0.003 0.113 0.158 0.204 0.242 0.282 0.305 0.335 0.369 0.399 0.41 0.42 0.433 0.441 0.449 0.458 0.463 0.471 0.477 0.482 0.485 0.485 0.485 0.485 0.485 As Reactions 3 & 4 are the opposite of Reactions 1 & 2, a gradual increase in the Absorbance value is observed, concomittant with increasing formation of NADH. This is clearly seen in Fig. 2 which depicts plots of absorbance value versus time under different conditions such as the presence and absence of hydrazine, and when pyruvate is added to reverse the ongoing reaction. Fig. 2. Changes in Absorbance at 340nm observed in Reactions 3, 3a & 4 using lactate as substrate in the presence and absence of hydrazine, and after Pyruvate (0.1M) addition. Reaction 3 conducted in glycine buffer at pH 9.0 reached the end point around 3 min with the absorbance remaining steady at a value around 0.22 until about 270 secs (i.e., 4 and a half min) after which the absorbance started to increase very gradually. Absorbance was still rising until about 20 min when it reached a plateau and 0.1M pyruvate was added (Reaction 3a). The absorbance showed a sudden and steep decrease (from 0.259 to 0.069) and leveled off. When the reverse LDH reaction was performed with glycine-hydrazine buffer at pH 9.0, the rate of reaction was much higher than that done without hydrazine as seen in Fig. 2. A comparison of the increase in absorbance values between 30 sec and 90 sec showed that in the presence of hydrazine, the increase was 0.147 as compared to 0.059 in the absence of hydrazine. This means that the addition of hydrazine enhanced the reaction rate by nearly 2.5 times (i.e., 0.147/0.059 = 2.49) Discussion and Conclusion Experiment 1.1 : Effect of enzyme concentration in LDH activity assay The results of Reactions 1 and 2 conducted with two different amounts of the enzyme protein showed that the rate of NADH consumption as seen from the drop in absorbance was much higher when the quantity of enzyme used was higher. Theoretically it is known that the rate of an enzyme-catalysed reaction depends on the concentrations of enzyme and, as the concentration of enzyme increases the rate of reaction also increases provided an excess of substrate is present. This is to be expected since the presence of more enzyme molecules presents more active sites for the formation of the enzyme-substrate complex, leading to higher rates of substrate consumption and product synthesis. If the enzyme present is insufficient, then again the reaction will not proceed as fast as it otherwise would because all of the active sites would be occupied with the substrate. That the reaction rate increases as enzyme concentration increases but levels off earlier due to substrate limitation is evident from Fig. 1 which shows the reaction in the case of Reaction 2 levelling off around 3 min (180 sec) whereas in Reaction 1 in which less enzyme (0.1ml) was used, continued much longer that is, until around 5 min. Experiment 1.2: Reversibility of the reaction: Reduction of NAD+ , effect of hydrazine on the reverse reaction, and pyruvate effect on the reaction rate That the LDH assay is reversible was clear from the increase in absorbance noted at 340nm (Fig. 2) which indicated the formation of NADH from NAD when lactate was the substrate used. The low equilibrium constant of the reaction (Keq = 2 x 10-2 M) favours reduction of pyruvate to lactate. Therefore, in order to promote Lactate  Pyruvate reaction, pyruvate needs to be removed as it is being formed. This was also evident from our assay (Reaction 4) done in presence of hydrazine in which ~2.5 times higher reaction rate was observed as compared to the reaction performed without hydrazine (Reaction 3) (Fig. 2). Hydrazine binds and removes pyruvate, thereby preventing the reaction from going in the direction of Pyruvate  Lactate. As opposed to the Reaction 4 result, the addition of pyruvate (Reaction 3a) caused an almost instantaneous drop in the absorbance from 0.259 to 0.069 (Table 2, Fig. 2) indicating that the reaction had reversed and had used up the NADH that had been produced in the system earlier by the oxidation of lactate. As seen from Fig. 2, until about 2 min the reaction is zero order; hence, the enzyme activity is directly proportional to substrate (i.e.,lactate) concentration. Also, an alkaline pH shifts the equilibrium in the direction of pyruvate. In conclusion, including hydrazine in the assay mixture and using an alkaline pH, will render the absorbance of NADH measured at 340nm at 300 seconds (5 min), directly proportional to the lactate concentration when hydrazine and NADH are present in excess in the reaction medium. Experimental protocol for lactate estimation in plasma Clinically, measuring lactate in whole blood or plasma is important in cases of hypoxemic shock, lactate acidosis etc. The aim of the experiment was to design a pilot study that could be further developed into an assay protocol for the evaluation of plasma lactate levels using the catalytic reaction of lactate dehydrogenase. An experimental methodology based on the results of the Pilot study described above is proposed as follows: A. 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) B. Experimental Procedure 1. Preparation of working solutions of the L-lactate standard Normal values2 of lactate in plasma have been reported to be in the range of 0.5-2.2 mM .Therefore, suitable working standards of 0.25, 0.50, 0.75, 1.00, 1.25, 1.5, 2.00, and 2.20 mM L-lactate are prepared by diluting the given stock DL- lactate solution (4.4 mM) with distilled water for setting up the calibration curve. The dilution process is described in Table 3. Table 3. 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 Tube # 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 = 7.50 ; y = 2.50 1.50 4 2.0 x = 6.25 ; y = 3.75 1.25 4 2.0 x = 5.00 ; y = 5.00 1.00 5 1.0 x = 7.50 ; y = 2.50 0.75 6 1.0 x = 5.00 ; y = 5.00 0.50 7 1.0 x = 2.50 ; y = 7.50 0.25 * Refers to the concentration of L-Lactate present in the DL-Lactate solution 2. 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 20 min. The tubes are next placed in the spectrophotometer previously adjusted to 340 nm and the absorbance values at zero time are recorded. The tubes are 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 solution mixed well and absorbance readings are noted at the end of 5 min. The reaction with each working standard is performed in duplicate. If the values differ by >5%, the reaction is repeated. For values showing Read More

It is seen from the graph that the rate of reaction is faster with the higher concentration of the enzyme with the oxidation of NADH reaching a plateau around 3 min (180 sec) while the reaction goes beyond 5min (300 sec) when less enzyme is used. Fig. 1. Graph showing NADH consumption or lactate formation from pyruvate with time catalysed by lactate dehydrogenase. A: 0.1ml enzyme, B: 0.2ml enzyme (Reactions 1 & 2). 1.2 Reversibility of the reaction: Reduction of NAD+ , effect of hydrazine on the reverse reaction, and pyruvate effect on the reaction rate Table 2.

–Showing Absorbance (Au) readings obtained for Reactions 3, 3a and 4 at 15 second intervals until equilibrium has been reached at 340nm Time (in seconds) Reaction 3 (with lactate as substrate, glycine buffer, pH 9) Reaction 3a (with lactate as substrate and added pyruvate, glycine buffer, pH 9) Reaction 4 (carried out with lactate as substrate and glycine- hydrazine buffer, pH 9) 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 600 900 1200 1500 1500 1515 1530 1545 1560 1575 1590 1605 0.004 0.093 0.115 0.135 0.15 0.162 0.174 0.182 0.19 0.197 0.202 0.208 0.212 0.216 0.22 0.223 0.225 0.228 0.23 0.232 0.234 0.249 0.255 0.259 0.259 0.069 0.07 0.069 0.069 0.069 0.069 0.069 0.069 0.003 0.113 0.158 0.204 0.242 0.282 0.305 0.335 0.369 0.399 0.41 0.42 0.433 0.441 0.449 0.458 0.463 0.471 0.477 0.482 0.485 0.485 0.485 0.485 0.485 As Reactions 3 & 4 are the opposite of Reactions 1 & 2, a gradual increase in the Absorbance value is observed, concomittant with increasing formation of NADH.

This is clearly seen in Fig. 2 which depicts plots of absorbance value versus time under different conditions such as the presence and absence of hydrazine, and when pyruvate is added to reverse the ongoing reaction. Fig. 2. Changes in Absorbance at 340nm observed in Reactions 3, 3a & 4 using lactate as substrate in the presence and absence of hydrazine, and after Pyruvate (0.1M) addition. Reaction 3 conducted in glycine buffer at pH 9.

0 reached the end point around 3 min with the absorbance remaining steady at a value around 0.22 until about 270 secs (i.e., 4 and a half min) after which the absorbance started to increase very gradually. Absorbance was still rising until about 20 min when it reached a plateau and 0.1M pyruvate was added (Reaction 3a). The absorbance showed a sudden and steep decrease (from 0.259 to 0.069) and leveled off. When the reverse LDH reaction was performed with glycine-hydrazine buffer at pH 9.0, the rate of reaction was much higher than that done without hydrazine as seen in Fig. 2. A comparison of the increase in absorbance values between 30 sec and 90 sec showed that in the presence of hydrazine, the increase was 0.

147 as compared to 0.059 in the absence of hydrazine. This means that the addition of hydrazine enhanced the reaction rate by nearly 2.5 times (i.e., 0.147/0.059 = 2.49) Discussion and Conclusion Experiment 1.1 : Effect of enzyme concentration in LDH activity assay The results of Reactions 1 and 2 conducted with two different amounts of the enzyme protein showed that the rate of NADH consumption as seen from the drop in absorbance was much higher when the quantity of enzyme used was higher. Theoretically it is known that the rate of an enzyme-catalysed reaction depends on the concentrations of enzyme and, as the concentration of enzyme increases the rate of reaction also increases provided an excess of substrate is present.

This is to be expected since the presence of more enzyme molecules presents more active sites for the formation of the enzyme-substrate complex, leading to higher rates of substrate consumption and product synthesis. If the enzyme present is insufficient, then again the reaction will not proceed as fast as it otherwise would because all of the active sites would be occupied with the substrate.

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