If it is suspected that the measurements of K-LACTUL are not correct and there is doubt regarding the performance of the kit then the following steps should be checked.
1. Check that the cuvettes are
1.5 mL microcuvettes and that the volume of the liquid in the cuvettes is high
enough for the spectrophotometer.
2. Check the temperature of
the reactions is correct.
Using the standard lactulose/fructose solution (bottle 8) that is supplied with the kit will help determine where issues are occurring with the measurement of lactulose samples. The obvious steps where issues may occur are: A. Sample Preparation (page 7 K-LACTUL booklet) and B. Enzymatic
Determination Reaction (page 8 K-LACTUL booklet).
3. The performance of
K-LACTUL can be tested as follows:
(A. Sample Preparation
(page 7 K-LACTUL booklet) Use 0.5 mL of the standard lactulose /fructose solution (Bottle 8) which contains 0.1 mg/mL lactulose and 0.05 mg/mL fructose. The typical individual absorbance values are: A1 = 0.2, A2 = 0.2, A3 = 1.0. This should generate a final absorbance difference of (A3-A2) of approximately 0.8 (Note: this measurement
includes the lactulose and fructose measurement and is not just lactulose
content only).
Note: If the correct values
are obtained for the performance of K-LACTUL then there is no need to check the
performance of the Enzymatic Determination Reaction step separately.
4. The performance of the Enzymatic
Determination Reaction step can be tested separately as follows:
B. Enzymatic Determination
Reaction (page 8 K-LACTUL booklet) This test uses 0.1 mL of the standard lactulose
/fructose solution (Bottle 8) which contains 0.05 mg/mL fructose. This is
equivalent to 5 μg of fructose added to the cuvette and should generate an
absorbance difference (A3-A2) of approximately 0.3. If this absorbance
difference is obtained then it can be concluded that the step is performing
correctly.
B. ENZYMATIC DETERMINATION
REACTION:
Wavelength: 340 nm
Cuvette: 1 cm light path (glass or
plastic; 1.5 mL semi-micro)
Final volume: 1.16 mL
Sample solution: 0.65-65 μg of lactulose per
cuvette (in 0.1-1.0 mL sample volume)
Read against air (without cuvette in the light
path) or against water Pipette
Pipette into cuvettes | Sample | Blank |
standard 8 (lactulose/fructose solution) distilled water solution 3 (imidazole buffer) solution 4 (NADP+/ATP) | 0.10 mL 0.90 mL 0.05 mL 0.05 mL | - 1.00 mL 0.05 mL 0.05 mL |
Mix*, read absorbance of the solutions (A1) after approx. 3 min and start the reactions by addition of: | ||
suspension 5 (HK/G-6-PDH) suspension 6 (6-PGDH) | 0.02 mL 0.02 mL | 0.02 mL 0.02 mL |
Mix*, read absorbance of the solutions (A2) at the end of the reaction (approx. 10 min). Then add: | ||
suspension 7 (PGI) | 0.02 mL | 0.02 mL |
Mix*, read absorbance of the solutions (A3) at the end of the reaction (approx. 15 min). | ||
* for example with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette cap or Parafilm®.