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®.

 

Product Page (K-LACTUL)