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Monitoring Lactate Concentration in Culture Medium, Serum, and Urine Samples

Background- The concentration lactate, a byproduct of glycolysis, is a reliable biochemical
indicator of anaerobic metabolism. Lactate is formed from pyruvate in the cytosol in a
reversible reaction catalyzed by the enzyme lactate dehydrogenase, which is often termed
reaction-11 of glycolysis or anaerobic glycolysis. If the amount of oxygen is limiting, as
occurs in muscle during intense activity, much of the pyruvate is reduced to lactate. This
reaction catalyzed by lactate dehydrogenase regenerates NAD+ thereby allowing continued
glycolysis with the continued production of ATP. This also occurs in a variety of
microorganisms which ferment glucose. For example yogurt is prepared by growing
lactate-producing bacteria in skim milk; as the concentration of acid increases, casein is
denatured and coagulates.

Cultured cells produce increased levels of lactate in response to hypoxic conditions.
Lactate synthesis occurs in the presence of a rapid increase in metabolic rate or when
oxygen delivery to the mitochondria declines. When the blood flow is decreased in patients,
oxygen delivery is no longer adequate to maintain aerobic glucose metabolism, thus
creating an oxygen debt, which may lead to hyperlactemia (2-5 mM blood lactate) or lactic
acidosis (>5 mM blood lactate). The normal blood lactate concentration in healthy,
unstressed individuals is approximately 1 mM. Patients who develop severe septic shock
usually exhibit hyperlactemia and lactic acidosis.

Lactate producers are skeletal muscle (white fiber), the gut, the brain, and mature red
blood cells whereas lactate metabolizers are the liver, the kidneys, and the heart. The
amount of lactate produced is generally thought to correlate with the body oxygen debt, the
degree of tissue hypoperfusion, and the severity of shock (clinical syndrome resulting from
an imbalance between tissue oxygen demand and supply). At the molecular and cellular
levels, increased lactate production may be caused by activation of glycolysis, inhibition of
pyruvate dehydrogenase activity, inborn errors of metabolism, or defects in the electron
transport chain.

In the past, lactate assays were difficult and tedious to perform. Although newer lactate
autoanalyzers using electrochemical methods can rapidly measure lactate concentration
within minutes, the machines are not designed for research purpose and are not readily
available. The Lactate Assay Kit developed by us is based on the reduction of the
tetrazolium salt INT in a NADH-coupled enzymatic reaction to formazan, which is
water-soluble and exhibits an absorption maximum at 492 nm. Since the intensity of the red
color formed is proportional to the lactate concentration, the assay, using a set of lactate
standards, can measure the concentration of lactate released to the culture medium or
circulation in a semi-quantitative manner. The kit can be directly used with culture medium,
serum, plasma, and urine samples (no TCA or PCA treatment is required). The assay time
required is 30 min.

Biomedical Research Service