Q: I have used the Megazyme K-LACGAR and K-LOLAC kits for determination of lactose in “lactose-free” milk and I have obtained values of 1% with K-LACGAR and 0.2% with K-LOLAC. Why is there such a difference in values and which value should I believe?”
A: It is not unusual to see a difference in lactose concentration as measured in K-LOLAC versus K-LACGAR for low-lactose and lactose-free products. Below is a typical low lactose milk sample with the various galacto-oligosaccharide (GOS) peaks identified. Note that glucose and galactose are off-scale but the remaining peaks are all on scale. While HPAEC-PAD is not strictly quantitative, the peak area responses do give an excellent indication of relative concentration, particularly when the compounds of interest are structurally related as is the case here. Below is a table that converts the “area” peak value into expected response in the LOLAC or LACGAR enzymatic assays, based on our knowledge of the selectivity of the β-galactosidase employed in the assay and the component monosaccharides that are released by each compound in the event of hydrolysis.
Peak | Compound | Peak Area | LOLAC | LACGAR | Comment |
3 | 1,6-Galactobiose | 15.794 | 0.000 | 31.588 | Contains two galactosyl residues (response increased by 100% in LACGAR) |
6 | 1,6-Galactosyl-glucose | 16.250 | 0.000 | 16.250 | Slowly hydrolysed in LOLAC but accounted for by creep calculator |
7 | Lactose | 9.756 | 9.756 | 9.756 | |
8 | 1,3-Galactosyl-galactose | 5.000 | 0.000 | 10.000 | Contains two galactosyl residues (response increased by 100% in LACGAR) |
9 | 1,6-Galactosyl-lactose | 6.758 | 0.000 | 10.137 | Contains two galactosyl residues (response increased by 150% in LACGAR) |
10 | 1,4-Galactosyl-galactose | 1.561 | 0.000 | 3.122 | Contains two galactosyl residues (response increased by 100% in LACGAR) |
11 | 1,3-Galactosyl-glucose | 1.546 | 1.546 | 1.546 | Hydrolysed in both assays |
Total | 11.302 | 82.399 |
The selectivity in LOLAC is not perfect, with full hydrolysis of the minor compound, 1,3-galactosyl-glucose, but the selectivity provided by the β-galactosidase used in LOLAC, coupled with the fact that detection in LOLAC is based on glucose as opposed to galactose results in a significantly more accurate value for lactose. The expected response for this sample analysed by LACGAR can be even ~7 times higher than that recorded in LOLAC. Note also that the lactose value obtained in the LOLAC assay may be higher than the “true” lactose value in the sample although the overestimation will be minor.