Blood Metabolite Analysis of Spectrophotometric Lactate Assay.

BLOOD METABOLITE ANALYSIS OF SPECTROPHOTOMETRIC LACTATE ASSAY.

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 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.2

Introduction
Laboratory tests, which involve measurement of blood lactate levels are important in

clinical settings especially in the diagnosis of Myocardial infarction. This is because an increase

in lactate concentration in blood is an evidence of decreased systemic blood flow, which is a

characteristic of myocardial infarction. In a study which was done recently by USA Department

of Public Health, that measured lactic acid in human veins showed that the most prevalent and

reliable clinical sign by patients with myocardial infarction is a rapid rise in blood lactate levels

(Shimojo et al., 2011). This transpired a recommendation by (Li et al., 2011) on the use of blood

lactate levels as a molecular marker in the diagnosis of cardiogenic shock. Therefore lactate, as a

blood metabolite will be found elevated in patients with clinical signs of myocardial infarction.

The main objective of this lab analysis is modeling and understanding the lactate concentration

profile in blood samples from patients with myocardial infarction in comparison to a

deproteinated blood sample from normal healthy persons. Lactate in the body is formed through

fermentation, as cells breakdown glucose in the absence of molecular oxygen to yield energy.

Glucose is converted to pyruvate, which is then reduced to lactate with the action of enzyme

lactate dehydrogenase. The lactate formed is utilized by other body tissues including heart

muscle as a source of fuel when it's further oxidized (Li et al. (2011).

Myocardial infarction (MI) is the clinical name for heart attack. Heart attack which

brought up due to reduced blood flow to the heart as a consequence of prolonged ischemia and

myocardial cell death. The stoppage or decreased blood flow to heart muscles can cause heart

muscle and tissue damage in server cases (Thygesen, Alpert and White, 2007). Myocardial

infarction is associated with the following clinical signs: chest pains and congestions

(discomfort). Myocardial (MI) infarction has been thought to be a lifetime chronic ailment that

could lead to heart failure and death (Thygesen, Alpert and White, 2007). Apart from reduced
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.3

blood flow to the heart muscles, other factors which increase the risk of one being diagnosed

with Myocardial Infraction (MI) include obesity, high blood sugar (diabetes), increased dietary

cholesterol levels in blood, the lack of exercise and high blood pressure (Andersen et al., 2013).

Derangements in lactate levels (an increase or decrease from normal ranges) result in

certain diseases which include liver failure, hypoperfusion, disturbed cellular metabolism, and

hypoxemia. This may be caused by intense exercise or overworked muscles. During diagnosis

factors, such as high blood pressure diabetes which could result in elevated lactate concentrations

in the blood due to insulin resistance should be eliminated.

This experiment is based on the reaction of lactate in the presence of NAD+ to give

pyruvate, NADH, and H+. The amount of lactate in the sample solution is measured by

determining the concentration of NADH produced the test reaction. NADH produced above is

measured spectrophotometrically by use of the optical density. NADH absorbs at a maximum

wavelength of 340 nm. In the experiment hydrazine is added to remove the pyruvate which is

formed ensuring the reaction proceeds to the right since the equilibrium of the reaction lies to the

left (Junghans et al. (2019).

Aims.

1. To compare lactate concentration in deproteinated blood samples from 4 healthy

individuals and in 4 patients after a myocardial infarction spectrophotometrically.

2. To determine the importance of lactate as a marker in the diagnosis of patients having

myocardial infarction (MI) disease.

3. To study the correlation between Myocardial Infraction and blood lactate concentration.

Methodology.
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.4

100ul of 8 deproteinated samples grouped into 2 groups: control (C) and patient (P) were

provided in duplicate. The samples were marked as C1, C2, C3, C4, P1, P2, P3, P4, and the

alphabet were stood for the first set and duplicate set. To each of the 16 tubes, 1 ml of the

reaction mix (hydrazine buffer and NAD) was added. The tubes were then tapped to mix the

content well and vortexed thereafter at a low speed. No fluid was allowed to fall back into the

tube. Using a micro cuvette the samples were transferred and measured absorbance at a

wavelength of 340 nm using a spectrophotometer to get E1 values. ((NADH has an absorbance

peak at 340 nm, whilst NAD+ exhibits little absorbance at this wavelength). The solutions were

transferred back into their test tube and 5 μl of LDH added in each tube. The tubes were left to

stand for 20 minutes then the solution was transferred into the cuvettes and absorbance of each

tube measured (E2). Lactate concentrations were then calculated for each tube.

Results.

Final Lactate
Con.
Δ OD Lactate (mM) [x10 for
Sampl (E2- Mean Conc. dilution
e E1 E2 E1) Δ OD (mM) factor]
C1A 0.220 0.241 0.021 0.0373
C1B 0.218 0.239 0.021 0.021 1 0.373
C2A 0.207 0.261 0.054 0.0755
C2B 0.224 0.255 0.031 0.0425 2 0.755
C3A 0.225 0.299 0.074 0.1270
C3B 0.230 0.299 0.069 0.0715 4 1.270
C4A 0.215 0.24 0.025 0.1554
C4B 0.218 0.368 0.15 0.0875 7 1.555
P1A 0.233 0.486 0.253 0.3624
P1B 0.256 0.411 0.155 0.204 8 3.625
P2A 0.241 0.596 0.355 0.5561
P2B 0.233 0.504 0.271 0.313 5 5.562
P3A 0.245 0.803 0.558 0.9772
P3B 0.243 0.785 0.542 0.55 6 9.773
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.5

P4A 0.276 1.018 0.742 1.0225

P4B 0.276 0.685 0.409 0.5755 7 10.226

Table 1. Results of the Blood Metabolite analysis of control blood sample and patients’ blood
sample showing absorbance values and the final calculated lactate concentration.

14
y = 1.5358x - 2.7687
R² = 0.8875
12

10.226
Concentration (mM) [x10 for dilution factor]

9.773
10

6 5.562

4 3.625

2 1.555
1.270
0.755
0.373
0
C1 C2 C3 C4 P1 P2 P3 P4

-2
Sample

Lactate concentration Linear (Lactate concentration)

Figure 1. Graph showing concentration of blood samples (controls and patients) against their
final lactate calculated concentration in (10 times dilution factor).

Standard deviation of Control Samples 0.456488

Standard deviation of Patient Samples 2.793001
Table 2. The standard deviation values of both control samples and patient samples.
Absorbance readings from the spectrometer at a wavelength of 340 nm were read and recorded

twice. That is E1 (that is absorbance for hydrazine buffer and the NAD mixture only) and E2

after which the LDH solution was added. The concentration of lactate in the solution was
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.6

calculated using the change in optical density in each tube (that is E2-E1 then multiplied by the

total volume of solution in each tube e.g. for tube C1 it will be 0.1608 x 11.05). Besides the

average values were also obtained from the original and duplicate samples and used in

calculating the lactate concentration. Finally, a dilution factor of X10 was used in constructing a

graph sample concentration against the calculated lactate concentration to observe the variation

between the metabolite concentrations across the different sample types

It is evident from the results obtained for the control tubes C1, C2, C3, and C4 from the

graph above a slow gradual increment in the concentration of lactate for C1 sample followed

then by a slight increase for C3 sample and similar increase also reported for C3 to C4. The

results for the patient also showed an increase however in a double stretched curve. One can pick

out the P1 and P2 values, which have almost doubled from the C1 and C2. The P3 and P4 double

increase are more observable compared to that of P1 and P2. The standard deviation for control

and patient that s 2.108 and 4.505 respectively doubling confirmed the theory above of doubling

of values from control to patient values.

Discussion.

This experiment aimed to evaluate and determine variations in blood lactate

concentrations profile between individuals with and without Myocardial Infraction condition.

From the results presented above from the experiment, it shows that healthy individuals have

lower levels of blood lactate compared to individuals having myocardial infraction ailment, who

have higher values. The increased levels of lactate in the blood may due to damage of heart

muscles as a result of decreased blood flow to the heart (Mehta, Wei, and Wenger, 2015).

Patients experience inhibited systemic perfusion which is subject to cardiac ischemia. Infection

of the myocardial cells causes the heart muscle walls to rapture resulting in dilation of arteries
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.7

and arterioles of the heart. Transport of Lactate from other body parts is inhibited as a result of a

reduction in blood flow hence an accumulation or increase in concentration in blood.

Accumulation of lactic result in a condition known as lactic (Goodwin et al., 2007)

To show how the other secondary factors (e.g. diabetes and high blood pressure) that are

associated with myocardial infarction could also cause an increase in lactate levels in the blood,

Lactate Dehydrogenase (LDH) was added to the test mixture. The graph shown in the figure

shows an increase in lactate levels in patients with Myocardial Infarction. The concentration of

lactate is seen to double when lactate dehydrogenase is added into the test solutions. Lactate

dehydrogenase levels indicate higher levels of glucose and blood pressure. Based on this one can

conclude that higher levels of glucose in the blood and increased blood pressure result in

increased blood lactate concentration (Jaiswar et al., 2011)

 Results obtained above, support the hypothesis that individuals with myocardial

infarction have a higher concentration of blood lactate to its entirety. This evident and is

confirmed by the data in this experiment which is prepared in duplicate. Both the experimental

and duplicate sets of results showed great variability in lactate levels between the two groups.

This confirmation by the duplicate data validates our results, making them reliable. This also

ensured that data collected in the experiment is maintained in a linear plot rather than having

presumptive conclusions. 

Using a blood sample to measure the lactate level is efficient and reliable. This procedure

provided data to compare with and blood samples are taken and recorded within a time frame of

12 hours. Other methods like using test stipe are recommended in determining blood lactate

concentration. One can also use the lactase oxidase method which also determines lactate levels
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.8

in comparison to lactate dehydrogenase. This method relatively cheaper and faster compared to

the other methods.

Conclusion.

In conclusion, the experiment was a success, as absorbance readings of individual blood samples

were obtained and used to plot a curve. Results obtained support the hypothesis that individuals

with Myocardial Infraction (MI) have higher lactate levels than those who are normally healthy.
 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.9

References

Andersen, L., Mackenhauer, J., Roberts, J., Berg, K., Cocchi, M. and Donnino, M. (2013).

Etiology and Therapeutic Approach to Elevated Lactate Levels. Mayo Clinic Proceedings,

88(10), pp.1127-1140.

Goodwin, M., Harris, J., Hernández, A. and Gladden, L. (2007). Blood Lactate Measurements

and Analysis during Exercise: A Guide for Clinicians. Journal of Diabetes Science and

Technology, 1(4), pp.558-569.

Jaiswar, S., Gupta, A., Sachan, R., Natu, S. and Shaili, M. (2011). Lactic Dehydrogenase: A

Biochemical Marker for Preeclampsia–Eclampsia. The Journal of Obstetrics and Gynecology of

India, 61(6), pp.645-648.

Junghans, L., Teleki, A., Wijaya, A., Becker, M., Schweikert, M. and Takors, R. (2019). From

nutritional wealth to autophagy: In vivo metabolic dynamics in the cytosol, mitochondrion and

shuttles of IgG producing CHO cells. Metabolic Engineering, 54, pp.145-159.

Li, J., von Pföstl, V., Zaldivar, D., Zhang, X., Logothetis, N. and Rauch, A. (2011). Measuring

multiple neurochemicals and related metabolites in blood and brain of the rhesus monkey by

using dual microdialysis sampling and capillary hydrophilic interaction chromatography–mass

spectrometry. Analytical and Bioanalytical Chemistry, 402(8), pp.2545-2554.

Mehta, P., Wei, J. and Wenger, N. (2015). Ischemic heart disease in women: A focus on risk

factors. Trends in Cardiovascular Medicine, 25(2), pp.140-151.

 Blood Metabolite Analysis of Spectrophotometric Lactate Assay.10

Shimojo, N., Naka, K., Nakajima, C., Yoshikawa, C., Okuda, K. and Okada, K. (1989). Test-

strip method for measuring lactate in whole blood. Clinical Chemistry, 35(9), pp.1992-1994.