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XYLAN & ARABINOXYLAN
(100 Assays per Kit) or (1000 Microplate Assays per Kit) or (1300 Auto-Analyser Assays per Kit) Megazyme International Ireland 2012 INTRODUCTION:
In nature, D-xylose occurs mainly in the polysaccharide form as xylan,
arabinoxylan, glucuronoarabinoxylan, xyloglucan and xylogalacturonan.
Mixed linkage D-xylans are also found in certain seaweed species and a
similar polysaccharide is thought to make up the backbone of psyllium
gum. Free D-xylose is found in guava, pears, blackberries, loganberries,
raspberries, aloe vera gel, kelp, echinacea, boswellia, broccoli, spinach,
eggplant, peas, green beans, okra, cabbage and corn. In humans, D-xylose
is used in an absorption test to help diagnose problems that prevent the
small intestine from absorbing nutrients, vitamins and minerals in food.
D-Xylose is normally easily absorbed by the intestine. When problems
with absorption occur, D-xylose is not absorbed and blood and urine
levels are low. A D-xylose test can help to determine the cause of a
child's failure to gain weight, especially when the child seems to be eating
enough food. If in a polysaccharide, the ratio of D-xylose to other sugars
etc. is known, then the amount of the polysaccharide can be quantitated
from this knowledge plus the determined concentration of D-xylose in
an acid hydrolysate. Xylans are a major portion of the polysaccharides
that could potentially be hydrolysed to fermentable sugar for biofuel
production.
PRINCIPLE:
Interconversion of the α- and β-anomeric forms of D-xylose is catalysed
by xylose mutarotase (XMR) (1).
The β-D-xylose is oxidised by NAD+ to D-xylonic acid in the presence of β-xylose dehydrogenase (β-XDH) at pH 7.5 (2).
(2) β-D-Xylose + NAD+ D-xylonic acid + NADH + H+ The amount of NADH formed in this reaction is stoichiometric with the amount of D-xylose. It is the NADH which is measured by the increase in absorbance at 340 nm.
SPECIFICITY, SENSITIVITY, LINEARITY AND PRECISION:
All reported forms of β-xylose dehydrogenase also act on D-glucose. At
concentrations of D-glucose similar to the levels of D-xylose measured
in this assay, the current enzyme acts slowly. However, at high
concentrations of D-glucose, the rate of reaction is very significant and
thus problematic. In many instances, there will be a need to measure
D-xylose in the presence of high concentrations of D-glucose (e.g. in mixtures of sugars obtained on acid hydrolysis of wheat flour), thus it is essential to remove the D-glucose. In the current assay protocol, this is achieved by a short pre-incubation of the sample extract with hexokinase in the presence of an excess of ATP (as per the procedure on page 4).
β-Xylose dehydrogenase has no action on L-arabinose. The smallest differentiating absorbance for the assay is 0.010 absorbance units, this corresponds to 0.350 mg/L of sample solution at the maximum sample volume of 2.00 mL (or to 7.01 mg/L with a sample volume of 0.1 mL). The detection limit is 0.701 which is derived from an absorbance difference of 0.020 and the maximum sample volume of 2.00 mL. The assay is linear over the range of 2 to 100 μg of D-xylose per assay. In duplicate determinations using one sample solution, an absorbance difference of 0.005 to 0.010 may occur. With a sample volume of 2.00 mL, this corresponds to a D-xylose concentration of approx. 0.175 to 0.350 mg/L of sample solution. If the sample is diluted during sample preparation, the result is multiplied by the dilution factor, F. If in sample preparation, the sample is weighed, e.g. 10 g/L, a difference of 0.02 to 0.05 g/100 g can be expected.
INTERFERENCE:
If the conversion of D-xylose has been completed within the time
specified in the assay (approx. 6 min), it can be generally concluded
that no interference has occurred. However, this can be further
checked by adding D-xylose (approx. 25 μg in 0.1 mL) to the
cuvette on completion of the reaction. A significant increase in the
absorbance should be observed.
Interfering substances in the sample being analysed can be identifiedby including an internal standard. Quantitative recovery of thisstandard would be expected. Losses in sample handling andextraction are identified by performing recovery experiments, i.e. byadding D-xylose to the sample in the initial extraction steps. SAFETY:
The reagents used in the determination of D-xylose are not
hazardous materials in the sense of the Hazardous Substances
Regulations. However, the buffer concentrate contains sodium azide
(0.02% w/v) as a preservative. The general safety measures that apply
to all chemical substances should be adhered to.
KITS:
Kits suitable for performing 100 assays in manual format (or 1300
assays in auto-analyser format or 1000 assays in microplate format) are
available from Megazyme. The kits contain the full assay method plus:
Bottle 1:
Buffer (45 mL, pH 7.5) plus sodium azide (0.02% w/v) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2: (x2) NAD+ plus ATP.
Freeze dried powder. Stable for > 5 years at -20°C.
Bottle 3:
Hexokinase suspension (2.2 mL).
Stable for > 2 years at 4°C.
Bottle 4:
XDH/XMR solution (5.6 mL). Stable for > 2 years at -20°C.
Bottle 5:
D-Xylose standard solution (5 mL, 0.25 mg/mL). Stable for > 2 years at 4°C.
PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:
1.
Use the contents of bottle 1 as supplied.
Stable for > 2 years at 4°C.
Dissolve the contents of bottle 2 in 21 mL of distilled water. Divide into appropriately sized aliquots and store in polypropylene tubes at -20°C between use and keep cool during use if possible. Stable for > 2 years at -20°C.
Use the contents of bottle 3 as supplied. Before opening for the first time, shake the bottle to remove any protein that may have settled on the rubber stopper. Subsequently, store the bottle in an upright position. Stable for > 2 years at 4°C.
Use the contents of bottle 4 as supplied. Before opening for the first time, shake the bottle to remove any protein that may have settled on the rubber stopper. Subsequently, store the bottle in an upright position. Stable for > 2 years at -20°C.
Use the contents of bottle 5 as supplied.
Stable for > 2 years at 4°C.
NOTE: The D-xylose standard solution is only assayed where there
is some doubt about the accuracy of the spectrophotometer being used or where it is suspected that inhibition is being caused by substances in the sample. The concentration of D-xylose is determined directly from the extinction coefficient of NADH (page 5).
EQUIPMENT (RECOMMENDED):
1. Glass test tubes (round bottomed; 16 x 100 mm).
2. Disposable plastic cuvettes (1 cm light path, 3.0 mL).
3. Micro-pipettors, e.g. Gilson Pipetman® (20 μL and 100 μL).
4. Positive displacement pipettor, e.g. Eppendorf Multipette®
- with 5.0 mL Combitip® (to dispense 0.4 mL aliquots of buffer [bottle 1] and NAD+/ATP solution).
- with 25 mL Combitip® (to dispense 2.0 mL aliquots of distilled 5. Analytical balance.
6. Spectrophotometer set at 340 nm.
7. Vortex mixer (e.g. IKA® Yellowline Test Tube Shaker TTS2).
8. Stop clock.
9. Whatman No.1 (9 cm) filter papers.
10. Corning Culture Tubes. - screw cap, 16 x 125 mm [Fisher Scientific cat no. TKV-173-030B (tubes); TKV-178-020V (caps)]; Fisher Scientific, [email protected].
MANUAL ASSAY PROCEDURE:
Wavelength:
Cuvette: 1 cm light path (glass or plastic)
Final volume:
Sample solution:
2-100 μg of D-xylose per cuvette (in 0.1-2.0 mL sample volume) Read against air (without a cuvette in the light path) or against water
Pipette into cuvettes distilled water (at 25°C) solution 1 (buffer) solution 2 (NAD+/ATP) suspension 3 (Hexokinase) Mix*, read the absorbances of the solutions (A1) after at least 5 min and start the reaction by addition of: solution 4 (XDH/XMR) Mix*, read the absorbance of the solutions (A2) at the end of the reaction ( 6 min). * for example with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette cap or Parafilm®.
Determine the absorbance difference (A2-A1) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of the sample, thereby obtaining ΔAD-xylose. The value of ΔAD-xylose should as a rule be at least 0.100 absorbance units to achieve sufficiently accurate results. The concentration of D-xylose can be calculated as follows: = V x MW x ΔAD-xylose = final volume [mL] = molecular weight of D-xylose [g/mol] = extinction coefficient of NADH at 340 nm = 6300 [l x mol-1 x cm-1] = light path [cm] = sample volume [mL] It follows for D-xylose:
c
= 2.97 x 150.1 x ΔAD-xylose 6300 x 1.0 x 0.1 = 0.7076 x ΔAD-xylose If the sample has been diluted during preparation, the result must be
multiplied by the dilution factor, F.
When analysing solid and semi-solid samples which are weighed out
for sample preparation, the content (g/100 g) is calculated from the
amount weighed as follows:
Content of D-xylose
= cD-xylose [g/L sample solution] x 100
weightsample [g/L sample solution] Content of Arabinoxylan
= Content of D-xylose (g/100 g)
x 100/(D-xylose content of polymer) e.g. for wheat flour arabinoxylan with a D-xylose content of 62%, Arabinoxylan
= Content of D-xylose (g/100 g) x 100/62
NOTE: These calculations can be simplified by using the Megazyme
Mega-CalcTM, downloadable from where the product appears in
the Megazyme web site (www.megazyme.com).
AUTO-ANALYSER ASSAY PROCEDURE:
1. The Auto-Analyser Assay Procedure for D-xylose can be performed using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination of D-xylose either a single point standard or a calibration
curve must be performed concurrently using the same
batch of reagents.
Reagent preparation is performed as follows:
Preparation of R1:
solution 1 (buffer) solution 2 (NAD+/ATP) 8.4 mL (after adding 21 mL of H2O to bottle 2) suspension 3 (Hexokinase) 0.42 mLTotal volume Preparation of R2:
solution 4 (XDH/XMR) EXAMPLE METHOD:
R1:
Reaction time:
Wavelength:
Prepared reagent stability:
> 2 days when refrigerated Reaction direction: increase
Linearity:
up to 1 g/L of D-xylose using 0.01 mL sample volume MICROPLATE ASSAY PROCEDURE:
1. The Microplate Assay Procedure for D-xylose can be performed using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination of D-xylose either a single point standard or a calibration
curve must be performed concurrently using the same
batch of reagents.
Wavelength:
Microplate:
96-well (e.g. clear flat-bottomed, glass or plastic) Final volume:
Linearity:
0.1-10 μg of D-xylose per well (in 0.01-0.2 mL sample volume) Pipette into wells standard solution solution 1 (buffer) solution 2 (NAD+/ATP) suspension 3 (Hexokinase) Mix*, read the absorbances of the solutions (A1) after approx. 4 min and start the reactions by addition of:solution 4 (XDH/XMR) Mix*, read the absorbances of the solutions (A2) at the end of the reaction (approx. 6 min).
* for example using microplate shaker, shake function on a microplate reader, or repeated aspiration (e.g. using a pipettor set at 50 - 100 μL volume).
CALCULATION (Microplate Assay Procedure):
g/L
If the sample is diluted during preparation, the result must be multiplied by the dilution factor, F. SAMPLE PREPARATION:
1. Sample dilution.
The amount of D-xylose present in the cuvette (i.e. in the 0.1 mL
of sample being analysed) should range between 2 and 100 μg. The
sample solution must therefore be diluted sufficiently to yield a
D-xylose concentration between 0.002 and 1.00 g/L.
Dilution Table
Estimated concentration of
factor (F) with water No dilution required If the value of ΔAD-xylose is too low (e.g. < 0.100), weigh out more sample or dilute less strongly. Alternatively, the sample volume to be pipetted into the cuvette can be increased up to 2.00 mL, making sure that the sum of the sample and distilled water components in the reaction is 2.10 mL and using the new sample volume in the equation.
2. Sample clarification.
a. Solutions:
Carrez I solution. Dissolve 3.60 g of potassium hexacyanoferrate
(II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P-9387) in 100 mL of distilled water. Store at room temperature.
Carrez II solution. Dissolve 7.20 g of zinc sulphate (ZnSO4.7H2O)
(Sigma cat. no. Z-4750) in 100 mL of distilled water. Store at room
temperature.
Sodium hydroxide (NaOH, 100 mM). Dissolve 4 g of NaOH in
1 L of distilled water. Store at room temperature.
b. Procedure:
Pipette the liquid sample into a 100 mL volumetric flask which
contains approx. 60 mL of distilled water, or weigh sufficient quantity
of the sample into a 100 mL volumetric flask and add 60 mL of
distilled water. Carefully add 5 mL of Carrez I solution, 5 mL of
Carrez II solution and 10 mL of NaOH solution (100 mM). Mix after
each addition. Fill the volumetric flask to the mark, mix and filter.
3. General considerations.
(a) Liquid samples: clear, slightly coloured and approximately
neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used
undiluted (such as wine or fruit juice), the pH of the solution should
be increased to approx. 7.5 using 2 M NaOH, and the solution
incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing a significant amount of
carbon dioxide should be degassed by increasing the pH to approx.
7.5 with 2 M NaOH and gentle stirring, or by stirring with a glass
rod.
(d) Coloured samples: an additional sample blank, i.e. sample with
no β-XDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly
coloured samples should be treated by the addition of 0.2 g of
polyvinylpyrrolidone (PVPP)/10 mL of sample. Shake the tube
vigorously for 5 min and then filter through Whatman No. 1 filter
paper.
(f) Solid samples: homogenise or crush solid samples in distilled
water and filter if necessary.
(g) Samples containing fat: extract such samples with hot
water at a temperature above the melting point of the fat, e.g. in a
100 mL volumetric flask. Adjust to room temperature and fill the
volumetric flask to the mark with distilled water. Store on ice or
in a refrigerator for 15-30 min and then filter. Discard the first few
mL of filtrate, and use the clear supernatant (which may be slightly
opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing
protein with Carrez reagents.
SAMPLE PREPARATION EXAMPLES:
(a) Determination of D-xylose in plant samples.
Mill plant materials to pass a 0.5 mm screen. Weigh out 1.0 g of sample and extract with 90 mL of water (heated to 80°C). Quantitatively transfer to a volumetric flask and dilute to the mark with distilled water. Mix, filter and use the appropriately diluted, clear solution for the assay.
(b) Determination of D-xylose in fermentation samples and
cell culture medium.
Incubate an aliquot (approx. 10 mL) of the solution at approx. 90-95°C for 10 min to inactivate enzyme activity. Centrifuge or filter and use the supernatant or clear filtrate (diluted according to the dilution table, if necessary) for the assay. Alternatively, deproteinisation can be performed with Carrez reagents. Homogenise gelatinous agar media with water and treat further as described above.
(c) Determination of D-xylose in polysaccharides and fibrous
plant material.
Mill plant material or polysaccharide to pass a 0.5 mm screen using a Retsch centrifugal mill, or similar. Accurately weigh approx. 100 mg of material into a Corning screw-cap culture tube (16 x 125 mm). Add 5 mL of 1.3 M HCl to each tube and cap the tubes. Incubate the tubes at 100oC for 1 h. Stir the tubes intermittently during the incubation. Cool the tubes to room temperature, carefully loosen the caps and add 5 mL of 1.3 M NaOH. Quantitatively transfer the contents of the tube to a 100 mL volumetric flask using distilled water and adjust the volume to 100 mL with distilled water. Mix thoroughly by inversion and filter an aliquot of the solution through Whatman No. 1 filter paper or centrifuge at 1,500 g for 10 min. Typically, no further dilution is required and a sample volume of 0.1 mL is (d) Determination of D-xylose in whole blood samples.
a. Solutions:
Concentrated Carrez I solution. Dissolve 30 g of potassium
hexacyanoferrate (II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P-9387) in 200 mL of distilled water. Store at room temperature.
Concentrated Carrez II solution. Dissolve 60 g of zinc sulphate
{ZnSO4.7H2O} (Sigma cat. no. Z-4750) in 200 mL of distilled water. Store at room temperature.
b. Procedure:
Heat 1 mL of whole blood sample at approx. 80°C for 20 min in a microfuge tube then centrifuge at 13,000 x g for 10 min and recover
the supernatant. Add 20 μL Carrez Reagent II and mix thoroughly,
then add 20 μL Carrez Reagent I and mix thoroughly. Centrifuge
the sample again at 13,000 x g for 10 min and recover the clarified
supernatant for use in the assay. If required, dilute the sample
appropriately in distilled water for the assay.
Note: The final volume of the clarified supernatant will be
approximately one quarter of the starting volume of the original
sample. Therefore adjust the volume of the starting material as
required to obtain sufficient volume of clarified sample for the test.
(e) Determination of D-xylose in biological tissue samples.
Accurately weigh approx. 5 g of representative biological tissue into a 100 mL Duran® bottle. Add 20 mL of 1 M perchloric acid and homogenise for 2 min using a Ultraturrax® or Polytron® homogeniser (or equivalent). Quantitatively transfer to a 40 mL glass beaker and adjust the pH to approx. 8.0 using 2 M KOH. Quantitatively transfer to a 100 mL volumetric flask and adjust to the mark with distilled water (ensuring the fat containing layer is "above" the mark, and the aqueous layer is "at" the mark). Store on ice for 20 min to precipitate potassium perchlorate and allow separation of the fat (if
present). Centrifuge an appropriate volume of the sample at 13,000 x
g for 10 min and recover the clarified supernatant for use in the assay,
alternatively filter through Whatman No. 1 filter paper, discarding the
first 3-5 mL, and use the clear filtrate for the assay. If required, dilute
the sample appropriately in distilled water for the assay.
Note: The amount of starting material and volumes used can be
adjusted accordingly depending on the amount of analyte present in
the sample.
(f) Determination of D-xylose in biological fluid samples (e.g.
urine and serum).
For some biological fluid samples it may be sufficient to test them directly without any sample preparation other than appropriate dilution in distilled water. If this is not adequate then deproteinisation with either perchloric acid or trichloracetic acid may be required.
Deproteinise biological samples by adding an equal volume of ice-cold 1 M perchloric acid with mixing. Centrifuge an appropriate volume of the sample at 1,500 x g for 10 min and recover the supernatant for use in the assay, alternatively filter through Whatman No. 1 filter paper, discarding the first 3-5 mL, and use the filtrate for the assay. If required, dilute the sample appropriately in distilled water for the assay. Alternatively, use 50% (w/v) trichloroacetic acid instead of perchloric acid.



      
Figure 1. Increase in absorbance at 340 nm on incubation of
0-125 μg of D-xylose with β-xylose dehydrogenase plus xylose Megazyme International Ireland,
Bray Business Park, Bray,
Co. Wicklow,
Telephone: (353.1) 286 1220
Facsimile: (353.1) 286 1264
Internet: www.megazyme.com
The information contained in this booklet is, to the best of our knowledge, true and accurate, but since the conditions of use are beyond our control, no warranty is given or is implied in respect of any recommendation or suggestions which may be made or that any use will not infringe any patents.

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