Low Lactose ERP - Review Book

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-05 14:27:52

Name

Brian Lang

E-mail

brian.lang@nist.gov

Organization

NIST

Title of Method

K-LOLAC Enzymatic Low Lactose Assay

AOAC Candidate Method Number (e.g. ALN-01)

LAC-006

Applicable SMPR

2018.009

I. Summary of the Method

The Megazyme Lactose Assay (LOLAC) uses a series of enzymes to convert lactose to ribulose 6 phosphate and NADPH. Quantification of lactose is determined spectrophotometrically though the absorbance of NADPH. After extraction a portion of the test sample (dilution in water for liquids and solubilization in 50 °C water for solids), proteins are precipitated using Carrez solution, and the supernatant is removed and filtered. Samples are then pretreated with Glucose Oxidase/Catalase at room temperature to convert glucose to D-gluconic acid. Samples are then heated to 100°C for five minutes to denature the Glucose oxidase and Catalase since these will interfere in the subsequent analysis. Next, a 0.1 ml portion of the samples is added to a 1 cm cuvette with 0.9 ml water and a solution containing NADP+ and ATP. After initial blank measurements on a spectrophotometer at 340 nm , a solution of hexokinase , glucose-6-phosphate dehydrogenase, and 6-Phosphogluconate dehydrogenase is added to remove any remaining glucose in the solution. Hexokinase first converts D-glucose to glucose 6 phosphate in the presence of ATP. Then glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to gluconate 6 phosphate with the simultaneous reduction of NADP+ to NADPH. Finally, the gluconate 6 phosphate is converted by 6-Phosphogluconate dehydrogenase to ribulose 5 phosphate with a reduction of NADP+ to NADPH. The formation of NADPH is then measured at 340 nm as a determination the residual glucose in the solution. A final solution of ß galactosidase is added to the analytical sample to hydrolyze lactose into D galactose and D glucose. The enzymes present in the analytical solution convert D glucose into ribulose 5 phosphate with the simultaneous production of NADPH from NADP+. After incubation, the absorbance of the NADPH is measured at 340 nm . The absorbance of NADPH is then used to calculate the concentration of lactose.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

Yes