An automated plasma D-lactate assay with a new sample preparation method to prevent interference from L-lactate and L-lactate dehydrogenase.

OBJECTIVE

To establish an automated plasma D-lactate assay without interference from L-lactate and L-lactate dehydrogenase (L-LDH).

METHODS AND MATERIALS

The D-lactate assay was programmed as a 2-point endpoint assay on the Roche Modular P using the D-lactic acid kit from Biocontrol Systems, USA. In the chemical reaction, D-lactate was oxidized to pyruvate by NAD(+) in the presence of D-lactate dehydrogenase. The resultant pyruvate was converted to alanine in the presence of alanine aminotransferase. The amount of NADH formed in the coupled reaction, measured by the change in the absorbance at 340 nm, was proportional to the concentration of D-lactate in the sample. Human serum albumin (HSA) solutions and plasma from pigs with experimentally-induced gut ischemia were used in this study. Blood samples were collected into Venosafe® tubes.

RESULTS

The D-lactate assay was linear up to 1.000 mmol/L in HSA solutions and plasma. The detection limit was 0.003 mmol/L. Within-run CVs ≤ 2.0% and total CVs ≤ 3.2% were obtained in the control material. Recovery was 87.1 ± 5.2 % (Mean ± SD). The L-LDH activity was completely inactivated in plasma samples by the addition of 20 µL of a 5 mol/L NaOH solution to 500 µL of plasma (pH 11.5). No interference could be detected from concentrations of bilirubin < 450 µmol/L, haemoglobin < 0.2 mmol/L or Intralipid® < 2.5 g/L.

CONCLUSIONS

The performance of the established D-lactate assay meets the requirements to be implemented into hospital laboratories. The sample preparation method is simple, cheap and requires minimal labour.