Blood metabolite analysis of spectrophotometric lactate assay

Introduction

To understand the metabolites, work in human, blood lactate measurement is one of the methods
to understand the neurotransmitter or substrate such as lactate, pyruvate, glutamine glutamate to
work in blood and brain. It is suggested that the concentration of them would have effect on other
substance balance level. (Li et al., 2011) The aim of this practical to understand the lactate
concentration in the deproteinated blood sample from healthy individuals as controls and compare
to the patients after they have myocardial infarction (MI). Lactate has related to acetylcholine,
pyruvate, glutamine as all of them are from glucose and citrate cycles. Li et al. (2011) also pointed
out the acetylcholine is the modulator of glutamatergic release. In this blood lactate analysis, the
blood centration on lactate would have different from normal healthy subject to patients after
myocardial infarction.

Myocardial infarction (MI) also known as heart attack, which can be defined death of myocardial cell
death due to the prolonged ischaemia when the cell death was classified as the coagulation or
contraction band necrosis, then usually it would change to oncosis which might formed a slight
degree from apoptosis it occurs when blood flow has increased or decreased to some part of heart
which causing direct or serious damage to heart muscle. (Thygesen, Alpert and White, 2007) Some
symptoms of having heart attack will be discomfort on chest or cheat pain, some of the pain may as
well travel to other body part. From Thygesen, Alpert and White (2007), Myocardial infraction (MI)
occurs as a minor event in a lifelong chronic disease would further lead to heart failure or cardiac
arrest. Some risk factors would also add risk of having MI, for example, high blood pressure,
diabetes, obesity, high blood cholesterol or lack of exercise. (Mehta, Wei and Wenger, 2015)

The Blood metabolite analysis on lactate concentration were the aim of this experiment as lactate
can be found in most tissues of body with the highest production in muscle. The lactate into the
pyruvate in cori cycle in liver and then pyruvate being converted in glucose. When glucose converted
into lactate again from the anaerobic metabolism, the cycle kept circulating the use of glucose and
lactate. Lactate level is important to be remain at certain level to keep other tissues functionally
normal. Some diseases or symptoms are related to the lactate level when it goes higher than the
required averaged value such as hypoperfusion, hypoxemia, dysfunction on liver or cellular
metabolism or external factors like having high intensity exercise or excessive muscle activity. To be
specific on high lactate levels associated is diabetes; when the lactate levels is gone higher the
insulin resistance was also gone higher.

Aims

To analyse blood metabolite in different patients and comparing with controls by measuring the
concentration of substrates in muscle (e.g. lactate, pyruvate) to determine importance of lactate for
having myocardial infarction (MI) disease with using interconversion of NAD/NADH or NADP/NADPH

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then use the spectrophotometric lactate assay to measure change of optical density to obtain lactate
level.

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Methods

8 deproteinated blood samples which sorted into two groups of control (C) and patient (P) on a 100
μl and waited the process within 30 minutes. In total, there were 16 samples which a duplicate
sample for the 8 samples to improve accuracy. The samples were marked as C1, C2, C3, C4, P1, P2,
P3, P4 and the alphabet were stand for first set and duplicate set. For each sample, pipettes (P100,
P200, P2000) were used. The pipette (P1000) were used for pipetting 1 ml of reaction mix into each
of the tubes, the reaction mix were mixture of 1.1mol/l hydrazine buffer and NAD. After that, all
samples were mixed well either by tapping the tubes or covering tubes with lip and put in the
spinner to help mixing. No fluid was allowed to fall back into the tube and the micro-cuvette were
used to be transferred and measured absorbency peak at 340 nm under spectrophotometer for
getting the E1 value as the absorbance peak of NADH is at 340 nm meanwhile the NAD+ would be
exhibited at this wavelength. After the absorbency value were obtained, all the fluids were being
transferred back to its tube and then a duration of 20 minutes time was waited the reaction after
added 5 μl LDH in each tube. In total, an 1105 μl of sample volume in each tube with the previous
added solution The final step was to measure all tubes under the spectrophotometer at 340 nm for
getting the E2 value.

Results

Final Lactate
Lactate Con.
Δ OD Mean Conc. (mM) [x10 for
Sample E1 E2 (E2-E1) Δ OD (mM) dilution factor]
C1A 0.220 0.241 0.021
C1B 0.218 0.239 0.021 0.021 0.03731 0.373
C2A 0.207 0.261 0.054
C2B 0.224 0.255 0.031 0.0425 0.07552 0.755
C3A 0.225 0.299 0.074
C3B 0.230 0.299 0.069 0.0715 0.12704 1.270
C4A 0.215 0.24 0.025
C4B 0.218 0.368 0.15 0.0875 0.15547 1.555
P1A 0.233 0.486 0.253
P1B 0.256 0.411 0.155 0.204 0.36248 3.625
P2A 0.241 0.596 0.355
P2B 0.233 0.504 0.271 0.313 0.55615 5.562
P3A 0.245 0.803 0.558
P3B 0.243 0.785 0.542 0.55 0.97726 9.773
P4A 0.276 1.018 0.742
P4B 0.276 0.685 0.409 0.5755 1.02257 10.226

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 Table 1. Results of the Blood Metabolite Analysis of control blood sample and patients’ blood sample
with the lactate concentration.

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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. Plotting of blood metabolite analysis with each sample (controls and patients) against
theirfinal lactate 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.

After getting all the values from the spectrophotometer at 340 nm twice; once the E1 value only
added for hydrazine buffer and the NAD; E2 for having the previous added solution and an extra LDH
were added. Thus, the lactate concentration was calculated used the change in optical density (OD)
which was E2 minus E1 and then times the given value of absorbency of NADH with the total volume
of each tube contained, (0.1608 × 11.05) or × 1.785. The mean values were obtained from each
original and duplicate sample and then used to calculate the lactate concentration. An ×10 dilation
factor were used to constructed a plotted curve to observe the relationship for blood metabolite
analysis

For the control tube results from C1, C2 , C3 and C4, a slowly gradually increased from C1 samples
can be observed, then a slightly high increase wad happened in C3 and a similar result was close to

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C3 is C4. The results of patient showed a similar increased trend but in a double stretched curve. The
P1 and P2 value observed to have a nearly doubled increase from C1 and C2. The P3 and P4 samples
had a expeditiously double increase as more cleared from the C1, C2 and P1, P2 comparison.
Therefore, to calculate the standard deviation values for control and patient samples are 2.1084622
in control and 4.5056585 in patient samples which showed a doubly increase on the values.

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Discussion

Firstly, the purpose of the study to outline the difference of the lactate level with normal individuals
and patients after having MI. From the results, the normal individuals have a lower value of the
blood lactate level which patients have a higher value. It may be caused by the damage part of
muscle of heart. (Mehta, Wei and Wenger, 2015) As infection were cause from the myocardial cell,
the muscle wall might have damaged and thus the diameter of the arterioles or arteries. Since then
the lactate level required from other tissues might have remained and the blood vessels might not
be able to fulfil the required levels which decreased blood flow and with that blood flow lactate level
has to be increased to travel to other tissue. This further was lead to more lactate to be in one
travel.

Secondly, from the previous risk factor which had discussed, such as diabetes and high blood
pressure. Since higher level of lactate and higher level of glucose or insulin resistance, so the result
curve from figure 1 was given out the conclusion on how the level of lactate to affect the patients
after having MI. The values of patient samples were doubled as the lactate dehydrogenase (LDH)
was added in these samples. The LDH levels were significant related to blood pressure as higher LDH
levels also indicated high blood pressure. From this, the risk was increased by increased lactate level
and blood pressure. (Jaiswar et al., 2011)

Thirdly, the results matched the presumption on normal individuals have a healthier blood lactate
level when the patient samples were much higher. The variability in the data was high as every value
was obtained from a duplicated sample and the included different level of blood lactate. The present
data was kept a in a quite linear plot, instead of having some data were out of the presumption by
not matching the trendlines.

Fourthly, the methods of using blood sample that testing for lactate level were reliable to have the
reliable as the blood sample were reliable and provided a result to compare the and hence, the most
accurate of the subject on the day that the sample was taken usually recorded around 12-hour
movement of the blood. (Shimojo et al., 1989) Moreover, the test stripe was other recommendation
on testing blood lactate concentration. The other alternative method to test blood lactate
concentration is testing lactate oxidase method which specifically tested the lactate response with
the LHD and it is also reliable and valid when compare to other methods, it cost less expensive than
other methods also the a quickly speed on testing than others. (White, Yaeger and Stavrianeas,
2009)

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Conclusion

In conclusion, the result of the practical was obtained and well plotted on the curve to measure or
make presumption on how the levels of lactate to have impact on normal healthy individuals and the
patients after having MI. The data of patient’s sample were higher than the normal samples which
supported the damage after MI caused in blood vessel or heart muscle would bring up the lactate
level. The problems which due to have a high lactate level will have diabetes and high blood
pressure and by adding LDH, the values also suggested there is correlation on them.

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Reference
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(20), pp.1127-40.

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.

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.

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.

Thygesen, K., Alpert, J. and White, H. (2007). Universal Definition of Myocardial Infarction. Journal of
the American College of Cardiology, 50(22), pp.2173-2195.

White, R., Yaeger, D. and Stavrianeas, S. (2009). Determination of blood lactate concentration:
reliability and validity of a lactate oxidase-based method. International Journal of Exercise Science,
2(2), p.2.