Question

The measurement of alanine aminotransferase activity (reaction rate) usually includes an excess of pure lactate dehydrogenase and NADH in the reaction system. The rate of alanine disappearance is equal to the rate of NADH disappearance measured spectrophotometrically. Explain how this assay works.

Short answer

ALT activity is measured by coupling it to the conversion of pyruvate into lactate using LDH and NADH, with NADH disappearance detected spectrophotometrically at 340 nm.

Step-by-step solution

Understand the Role of Alanine Aminotransferase

Alanine aminotransferase (ALT) is an enzyme that catalyzes the transfer of an amino group from alanine to α-ketoglutarate, forming pyruvate and glutamate. The measurement of its activity is important for assessing liver function.

Role of the Coupling Reaction

In the assay, ALT activity is indirectly measured by linking it to a second reaction. The pyruvate produced by ALT is used as a substrate by lactate dehydrogenase (LDH). LDH catalyzes the conversion of pyruvate to lactate, using NADH as a coenzyme. This coupling increases the sensitivity of the assay because NADH is a strong absorbance marker at 340 nm.

Spectrophotometric Measurement of NADH

During the coupled reaction, NADH is oxidized to NAD+. This oxidation can be monitored spectrophotometrically because NADH absorbs light at 340 nm, while NAD+ does not. The decrease in absorbance at 340 nm is proportional to the conversion of NADH to NAD+, which in turn is proportional to the ALT activity.

Assay Interpretation

The change in absorbance over time is recorded, and the rate of change is used to calculate ALT activity. A higher rate of NADH disappearance indicates higher ALT activity, as more pyruvate is being processed into lactate, utilizing NADH.

Key concepts

Alanine Aminotransferase

Alanine aminotransferase (ALT) is a crucial enzyme primarily found in the liver. It plays a key role in amino acid metabolism by transferring the amino group from alanine to α-ketoglutarate. This transformation forms two important compounds - pyruvate and glutamate. The liver's health can be assessed by measuring ALT levels, as damage or disease often causes ALT to leak into the bloodstream.
Doctors commonly use ALT as an indicator of liver injury. Elevated ALT levels suggest liver cell injury or inflammation, pointing to conditions such as hepatitis or cirrhosis. Keeping an eye on ALT levels provides valuable insights into liver functionality and possible underlying health issues.

Spectrophotometry

Spectrophotometry refers to a method used to measure how much light a chemical substance absorbs by passing a light beam through a solution. It helps identify and quantify substances based on their specific light absorption characteristics. In our context, it is essential for monitoring enzymatic reactions like the assay of ALT.

• Light Source: A light source generates beams covering broad or specific wavelengths.
• Sample Cuvette: Holds the solution where the enzymatic reaction occurs.
• Detector: Captures transmitted light, noting changes in light intensity, corresponding to absorbance.

During the ALT assay, spectrophotometry is applied to track the disappearance of NADH, hence indirectly measuring ALT activity. Light absorbed by NADH at 340 nm is particularly critical, as its reduction in absorbance indicates NADH's conversion along with LDH activity.

Enzyme Activity

Enzyme activity refers to the rate at which an enzyme catalyzes a reaction. It is measured by tracking the transformation of substrates into products over time, providing insights into how enzymes function under specific conditions.
ALT plays a pivotal role in enzyme activity studies. Its function is monitored based on how rapidly it converts alanine into pyruvate. In this context, enzyme activity gives a direct indication of physiological processes and potential disturbances.
When assessing enzyme activity, particularly ALT, coupling the reaction to a NADH-utilizing reaction enhances the sensitivity and accuracy. This ensures more effective monitoring of liver enzyme function, which is essential for diagnosing various health conditions.

Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is a significant enzyme found in almost all body cells. It facilitates the conversion of pyruvate, derived from glucose via glycolysis, into lactate when oxygen is scarce.
In biochemical assays like the one measuring ALT, LDH has an additional role, serving in a coupling reaction. Pyruvate, produced by ALT's activity, acts as a substrate for LDH, which then converts it into lactate, in the process utilizing NADH. This dependency allows for the indirect measurement of ALT activity through changes in NADH levels.

• Working in tandem with NADH, LDH ensures accurate tracking of ALT activity.
• It allows the usage of spectrophotometry to monitor the reaction, as changes in NADH absorbance directly correlate with ALT function.

Thus, LDH not only supports cellular energy production but also enhances the effectiveness of ALT assays.