Novel method for the specific, convenient, cost effective and rapid assay of L-asparagine, L-glutamine and ammonia as acrylamide precursors in the food industry, or as cell culture media / supernatant components, or in other materials.
INTRODUCTION:
The simple and rapid measurement of L-asparagine and ammonia together has recently become of great significance with respect to two major applications:
1. Cell Culture: L-Asparagine is an essential component of certain cell culture media. However, the incorporation of this amino acid into growth media presents two major problems; firstly, L-asparagine is labile, and spontaneously breaks down to L-aspartate and free ammonium ions. Secondly, the released ammonium ions are very toxic to the cells. To overcome these issues L-asparagine is added just prior to use, and its concentration along with that of ammonia is frequently monitored during culturing.
This kit (K-ASNAM), based on the use of advanced recombinant enzymes, is very rapid (~ 20 min), and also measures both
L-asparagine and ammonia in a very simple format. A value for
L-glutamine is also produced as part of the method, that may also be of significance in cell culture applications. Both manual (see page 6, “A”) and microplate (see page 9, “B”) formats are described. Other rapid tissue culture test kits are also available from Megazyme for ammonia (K-AMIAR), L-glutamine / ammonia (K-GLNAM), D-glucose (K-GLUHKR or K-GLUC), and L-lactic acid (K-LATE).
2. Acrylamide Precursors: It is now well known that when
L-asparagine, ammonium ions, D-fructose and / or D-glucose are heated above approximately 160°C, significant levels of the carcinogenic compound acrylamide are formed, with obvious concerns for human health. Food products affected include potato crisps and chips, roasted potatoes and other fried, toasted or roasted foods, such as bakery goods, breakfast cereals and coffee. Asparaginase, an enzyme that converts L-asparagine into L-aspartic acid, thus has potential applications in this area for reducing acrylamide levels. However, if asparaginase treatment is adopted, it will be necessary to confirm that the free L-asparagine has been successfully converted to L-aspartic acid. This kit (K-ASNAM) is ideal for this application, and along with the Megazyme D-fructose / D-glucose kit (K-FRUGL) enables the concentrations of all four key acrylamide precursors to be determined both simply, and cost effectively.
This kit is also suitable for the analysis of L-asparagine, L-glutamine and ammonia in a wide range of other samples. Most notably, this method first converts / quantifies L-glutamine, an amino acid that would otherwise lead to interference due to the low but significant action of asparaginase on this compound.
KITS:
Kits suitable for performing 50 assays each of L-asparagine, L-glutamine and ammonia are available from Megazyme. The kits contain the full assay method plus:
Bottle 1: Buffer (11 mL, pH 4.9) plus sodium azide
(0.02% w/v) as
a preservative. Stable for > 2 years at 4°C.
Bottle 2: (x2) Buffer (25.5 mL, pH 8.0) plus 2-oxoglutarate and sodium azide (0.02% w/v) as a preservative.
Stable for
> 2 years at 4°C.
4
Bottle 3: (x2) NADPH. Lyophilised powder.
Stable for > 5 years at -20°C.
Bottle 4: Glutaminase suspension (1.1 mL).
Stable for
> 2 years at 4°C.
Bottle 5: Glutamate dehydrogenase suspension (2.2 mL). Stable for > 2 years at 4°C.
Bottle 6: Asparaginase suspension (1.1 mL).
Stable for
> 2 years at 4°C.
Bottle 7: Ammonia standard solution (5 mL, 0.04 mg/mL) in 0.02% sodium azide.
Stable for
> 2 years at 4°C.
Bottle 8: L-Asparagine control powder (~ 2 g).
Stable for
> 2 years at 4°C.