Biochemical essence of the reaction
The enzyme lactate dehydrogenase is responsible for a reversible redox reaction. Depending on the active metabolic pathway (glycolysis or gluconeogenesis), the process can proceed in either the forward or reverse direction.
Pyruvate or lactate serves as the substrate in this reaction. An essential participant in the process is the coenzyme NAD (in the form of NAD$^+$ or NADH). For the body to synthesize this coenzyme, the precursor vitamin PP (also known as vitamin B$_3$, niacin, nicotinic acid, or nicotinamide) is required.
The overall reaction equation is as follows: Pyruvate + NADH + H$^+$ ↔ Lactate + NAD$^+$
The source of the enzyme for laboratory diagnostics is the patient's blood serum, where the enzyme appears following normal cell turnover or cell destruction.
Laboratory test conditions
To accurately determine enzyme activity in vitro, conditions must be established under which lactate dehydrogenase operates with maximum efficiency. These parameters are strictly controlled during the experiment:
- Temperature regime: usually maintained at the optimal temperature for human enzymes, which is 37 °C. In some cases, standard laboratory values (25 °C or 30 °C) are used.
- Medium acidity (pH): the reaction requires an optimal buffer solution that maintains pH in the range from 7.4 to 7.8.
- Duration: incubation time is fixed with high precision. In standard educational and laboratory problems, it is most often exactly 10 minutes.
Reaction rate assessment and activity calculation
The primary parameter used to evaluate enzyme activity is the reaction rate. This rate is assessed by the change in the amount of reactants per unit of time—either by substrate depletion or product accumulation.
In modern laboratory practice, spectrophotometry is widely used for this purpose. Measurement of the change in solution optical density is performed at a wavelength of 340 nm—this parameter directly reflects the dynamics of NADH coenzyme concentration changes.
If the assessment is based on the amount of depleted substrate, enzyme activity (A) is calculated in international units per liter (IU/L, U/L) using a special formula:
$$ A = \frac{\Delta S}{t \cdot V} \cdot 1000 $$
Where:
- $\Delta S$ is the amount of consumed substrate in micromoles (calculated as the difference between the initial amount $S_{\text{initial}}$ and the remaining amount $S_{\text{residual}}$);
- $t$ is the incubation time in minutes;
- $V$ is the volume of serum taken in milliliters;
- $1000$ is the standardization coefficient required to convert volume from milliliters to liters.
Clinical example: result interpretation
Let us examine the practical application of the formula by comparing two patients. In both cases, the incubation time was 10 minutes, and the volume of the tested serum was 5 ml. The initial amount of substrate was 40 µmol.
Patient 1 analysis:
- The remaining substrate was 31 µmol. We find the depletion ($\Delta S$): 40 - 31 = 9 µmol.
- We perform the activity calculation: $A_1 = \frac{9}{10 \cdot 5} \cdot 1000 = \frac{0.9}{5} \cdot 1000 = 180$ IU/L.
Patient 2 analysis:
- The remaining substrate was only 4 µmol. Depletion ($\Delta S$): 40 - 4 = 36 µmol.
- We perform the activity calculation: $A_2 = \frac{36}{10 \cdot 5} \cdot 1000 = \frac{3.6}{5} \cdot 1000 = 720$ IU/L.
Clinical conclusion: The results show that lactate dehydrogenase activity in the second patient (720 IU/L) is several times higher than that of the first patient (180 IU/L). Such a marked increase in serum LDH activity may indicate a pathological process accompanied by cell destruction, such as hemolysis, liver tissue damage, or myocardial infarction.
Briefly: answers to topical questions
What lactate dehydrogenase isoenzymes exist in the human body?
There are five isoenzymes (isoforms) of lactate dehydrogenase in the human body. The LDH molecule consists of four subunits of two types: H (heart) and M (muscle), combinations of which form these isoenzymes:
- LDH₁ (HHHH)
- LDH₂ (HHHM)
- LDH₃ (HHMM)
- LDH₄ (HMMM)
- LDH₅ (MMMM)
In which organs and tissues are LDH-1 and LDH-2 isoenzymes predominantly localized?
LDH-1 and LDH-2 isoenzymes are most active and predominantly localized in tissues with aerobic metabolism, specifically in the heart muscle (myocardium), brain, and renal cortex.
How does the isoenzyme spectrum of lactate dehydrogenase change in acute viral hepatitis?
In hepatitis, there is an elevation in the LDH₅ isoenzyme level, which appears on the plasma protein electrophoresis as a rightward shift of the spectrum. This isoform predominates in the liver, and its appearance in the blood indicates damage to this organ.
To remember the variables in the formula, use the mnemonic: Substrate divided by Time and Volume, multiplied by Thousand.
Class and exam questions
Which coenzyme is required for lactate dehydrogenase function?
In the reaction, NAD (nicotinamide adenine dinucleotide) acts as a coenzyme that reversibly transitions from its oxidized form to its reduced form.
Why measure optical density at 340 nm?
Measurement at a wavelength of 340 nm using spectrophotometry allows precise tracking of changes in NADH coenzyme concentration during the reaction, which is proportional to the enzyme's reaction rate.
What does high serum LDH activity indicate?
A manifold increase in LDH activity indicates massive leakage of the enzyme from cells into the bloodstream, which is characteristic of pathologies such as myocardial infarction, erythrocyte hemolysis, or liver damage.
What else to study on this topic
- Features of the spectrophotometric method for determining enzyme activity
- Principles of buffer systems and their effect on the pH of enzymatic reactions
- Glycolysis and gluconeogenesis: the role of LDH in the regulation of metabolic pathways
- B-complex vitamins as precursors of critical coenzymes
- Mechanisms of intracellular enzyme release into blood plasma during tissue necrosis
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