Biochemical Basis of the Reaction
The enzyme lactate dehydrogenase mediates a reversible oxidation-reduction reaction. Depending on the prevailing metabolic pathway (glycolysis or gluconeogenesis), the process can proceed in either the forward or reverse direction.
The substrates for this reaction are pyruvate or lactate. An essential participant is the coenzyme NAD (in the form of NAD$^+$ or NADH). The biosynthesis of this coenzyme requires its precursor vitamin — vitamin PP (also known as vitamin B$_3$, niacin, nicotinic acid, or nicotinamide).
The overall reaction equation is: Pyruvate + NADH + H$^+$ ↔ Lactate + NAD$^+$
The source of the enzyme for laboratory diagnostics is the patient's blood serum, where the enzyme is released following normal cell turnover or cell lysis.
Conditions for the Laboratory Assay
To accurately determine enzyme activity in vitro, conditions must be optimized so that lactate dehydrogenase operates at peak efficiency. These parameters are strictly controlled during the assay:
- Temperature: Optimal physiological temperature for human enzymes is usually maintained at 37 °C. In some protocols, standard laboratory temperatures (25 °C or 30 °C) are used.
- pH: The reaction requires an optimal buffer system to maintain the pH between 7.4 and 7.8.
- Duration: Incubation time is precisely regulated. In standard educational and laboratory problems, it is usually exactly 10 minutes.
Reaction Rate Assessment and Activity Calculation
The primary parameter used to assess enzyme activity is the reaction velocity. This rate is evaluated by measuring the change in reactant quantities per unit time — either by substrate depletion or product accumulation.
Spectrophotometry is widely used for this purpose in modern clinical laboratories. Changes in the optical density of the solution are measured at a wavelength of 340 nm, which directly reflects the changing concentration of the NADH coenzyme.
When calculating activity based on substrate depletion, the enzymatic activity ($A$) is expressed in International Units per liter (IU/L or U/L) using the 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 initial amount $S_{\text{initial}}$ and remaining amount $S_{\text{residual}}$);
- $t$ is the incubation time in minutes;
- $V$ is the volume of the serum sample in milliliters;
- $1000$ is the conversion factor to translate milliliters to liters.
Clinical Example: Interpretation of Results
Let us examine a practical application of the formula by comparing two patients. In both cases, the incubation time was 10 minutes, the serum volume was 5 mL, and the initial substrate amount was 40 µmol.
Patient 1 Analysis:
- Residual substrate was 31 µmol. Substrate depletion ($\Delta S$): 40 - 31 = 9 µmol.
- Activity calculation: $A_1 = \frac{9}{10 \cdot 5} \cdot 1000 = \frac{0.9}{5} \cdot 1000 = 180$ IU/L.
Patient 2 Analysis:
- Residual substrate was 4 µmol. Substrate depletion ($\Delta S$): 40 - 4 = 36 µmol.
- 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 LDH activity in the second patient (720 IU/L) is several times higher than in the first patient (180 IU/L). Such a marked elevation in serum LDH activity points to a pathological process accompanied by cellular destruction, such as hemolysis, hepatocellular injury, or myocardial infarction.