1D-Xylose Assay Kit
Catalog # 6601

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to evaluate cellular permeability

FORMAT:

96-well ELISA Plate with non-removeable strips

ASSAY TYPE:

Colorimetric Assay

ASSAY TIME:

1 hour

STANDARD RANGE:

20 mM to 0.3 mM

NUMBER OF SAMPLES:

Up to 39 (duplicate) samples/plate

SAMPLE TYPES:

Serum and Plasma

RECOMMENDED SAMPLE DILUTIONS:

Undiluted

CHROMOGEN:

N/A (read at 554 nm)

STORAGE:

Room Temperature

VALIDATION DATA:

Intra-Assay (5.6-8.0%)/Inter-Assay (4.6-6.4%)/Spiking Test (95-104%)

NOTES:© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2

D-Xylose Assay Kit
Catalog # 6601
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
The transport of molecules from the gut into the body occurs either in between the cells or through the cells, paracellularly or transcellularly,
respectively. Fluorescent-labeled Dextrans can be used to evaluate paracellular permeability, while D-Xylose, a simple monosaccharide,can be used to evaluate transcellular uptake.
In fact, it may be necessary to use two different markers to evaluate permeability. For example, two groups of rats fed total parenteral nutrition
supplemented with two different amino acid cocktails showed an increase in fluorescein isothiocyanate (FITC)-labeled dextran permeability
in one group over the other, but no discernible difference in D-Xylose uptake between the two groups (1). This data shows that measuring
only one mode of transport may result in incorrect conclusions about permeability.
Chondrex, Inc. provides a D-Xylose Assay Kit (Cat # 6601) in a convenient 96-well plate format, as well as FITC-labeled Dextrans (Cat #4009:4 kDa and Cat # 4013:40 kDa) to evaluate cellular permeability in-vitro or in-vivo. Rhodamine-labeled Dextran (Cat # 4014: 70 kDa),
which fluoresces at a different wavelength than FITC, is also available, permitting the simultaneous use of two different sized Dextrans which
can be individually measured. Moreover, Chondrex, Inc. offers an array of mouse and human anti-bacteria and anti-lipopolysaccharides(LPS) antibody ELISA kits as an alternative method for evaluating gut barrier integrity and proper immune function against theseenvironmental factors. Please visit contact support@chondrex.com or www.chondrex.com for more information.

KIT COMPONENTS
Item

Quantity

AmountStorage

D-Xylose Standard Solution (66011)

1 vial

40 mM, 100 µl

Room Temperature

D-Xylose (66012)
Reagent 1 (66013)
Solution A (66014)
96-Well Plate

1 vial1 vial1 vial1 each500 mg
200 mg
1.5 ml8-well strips x 12

Room Temperature
Room Temperature
Room Temperature
Room Temperature

REAGENTS NOT PROVIDED
1.

Glacial Acetic Acid*

2.

Concentrated Hydrochloric Acid**Please use caution and wear the appropriate protective gear when working with strong acids

IN-VITRO PROTOCOL
Please refer to references 2-5 to establish a working protocol© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3IN-VIVO PROTOCOL
1.

Fast mice 4 hours before oral feeding and during the experiment.

2.

Feed each mouse 200 µl of the D-Xylose solution by oral gavage. The concentration of D-Xylose must be optimized for individual
experimental purposes. A 100 mg/ml D-Xylose solution (500 mg D-Xylose in 5 ml PBS) works for most purposes. For rats, feeding 2
ml of a 50 mg/ml solution works for most purposes (1).

3.

Maintain fasting conditions and wait 1 hour (time may vary depending on individual animals).

4.

Collect blood by retro-orbital bleeding, then spin, and collect serum (plasma). Proceed with D-Xylose Assay or store at -20°C until use.
NOTE: It is possible to simultaneously evaluate transcellular permeability with D-Xylose and paracellular permeability with fluorescentlabeled Dextrans (Catalog # 4009, 4013, and 4014). However, follow the appropriate protocols, as incubation times for paracellularpermeability and transcellular permeability differ. Also, consult the appropriate animal committee for multiple bleedings on the sameday.

ASSAY OUTLINE
Add 10 µl of diluted standards and samples to wells

Add 250 µl of color reagent solution to wells
Incubate at 60 degrees Celsius for 30 minutes
Cool plate at room temperature for 5 minutes

Read at 554 nm WITHIN 30 MINUTES OF COOLING

PLATE LAYOUT
123456789101112A

2020

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

1010

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C55

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

2.5

2.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

1.25

1.25

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F0.630.63

S6S6S14

S14

S22

S22

S30

S30

S38

S38

G0.310.31

S7S7S15S15

S23

S23

S31

S31S39S39

HBB

S8S8S16S16S24S24S32

S32

SBSBStandardsSamples© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.

ASSAY PROCEDURE
1.

Prepare D-Xylose Standard: The recommended standard range is 0.3-20 mM. To dilute the standard, take 50 µl of the D-Xylose
Standard (40 mM) and add to 50 µl of Solution A to make a 20 mM D-Xylose standard solution; then serially dilute it with Solution A.
For example, mix 50 µl of the standard (20 mM) with an equal volume of Solution A to make a 10 mM solution, and then repeat it five
more times to make 5, 2.5, 1.25, 0.63, and 0.31 mM standard solutions (Solution A will be the Blank). The remaining 40 mM Standard
Stock solution can be stored at room temperature for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions
for each assay.
50 ml

40 mM

2.

50 ml

50 ml

50 ml

50 ml

50 ml

50 ml

1234567850 ml

50 ml

50 ml

50 ml

50 ml

50 ml

50 ml

50 ml

20 mM

10 mM

5 mM

2.5 mM

1.3 mM

0.6 mM

0.3 mMBlank(Solution A)

Prepare Samples: Serum samples may be used undiluted. If D-Xylose levels in a sample are above 20 mM, then dilute the sample
2-3 times and re-assay.
NOTE: Chondrex, Inc. recommends preparing sample blanks using D-Xylose free serum (plasma) or D-Xylose free culture media
because serum or culture media components, such as glucose, may interfere with the assay. Refer to Plate Mapping.

3.

Prepare Color Reagent: 250 µl of color reagent is required for each well. For example, a 7-point standard, blank, sample blank, and15 samples, all in duplicate will be 48 wells. Therefore, 12 ml of color reagent is needed. Dissolve 60 mg of Reagent 1 in 12 ml ofconcentrated acetic acid (AcOH) and 1.2 ml of concentrated hydrochloric acid (HCl). Use caution when working with strong acids.
NOTE: Prepare the Color Reagent just before use. Do not store the prepared solution for reuse in a future assay.

4.

Add Standards, Samples and Sample Blank: Add 10 µl of solution A (blank), standards, sample blank, and samples into designatedwells.

5.

Add Color Reagent: Carefully add 250 µl of freshly prepared Color Reagent into each well. Seal the wells with a plate sealer toprevent evaporation during incubation. If only using half the plate, cut the plate sealer in half and save for a future assay. Incubate at60°C for 30 minutes.

6.

Read Plate: Remove the plate from the incubator and carefully remove the plate sealer. Allow the plate to cool at room temperaturefor 5 minutes. Next, read the OD values at 554 nm. Read the plate within 30 minutes of cooling. If the OD values of the samples are
greater than the OD value of the highest standard point, dilute the samples 2-3 times and re-assay.

© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

5

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank (and sample blank), standards, and test samples.

2.

Subtract the averaged blank OD value from the individual averaged standard, sample, and sample blank OD values. Subtract theaveraged sample blank OD value from the averaged sample OD values.

3.

Plot the subtracted OD values of the standards against the concentration (mM) of D-Xylose. Using a log/log plot will linearize the data.
Figure 1 shows representative OD values of the standard range from 0.3 - 20 mM.

4.

The D-Xylose levels in samples can be calculated using regression analysis. If necessary, multiply it by the sample dilution factor toobtain the D-Xylose concentration in the original sample.
NOTE 1: The D-Xylose concentration can be converted to mg/ml with the following equation.
150.13D-Xylose (mg/ml) =x D-Xylose (mM)
1000
NOTE 2: Gut bacteria may metabolize D-Xylose, resulting in a lower D-Xylose uptake in the case of gut bacteria overgrowth (6); furtheranalysis to distinguish bacteria overgrowth from impaired passive carrier-mediated uptake may be required.

Figure 1 – A Typical Standard Curve for the D-Xylose Assay Kit

ASSAY VALIDATION
Table 1 - Reproducibility Data for the D-Xylose Assay Kit
Test

0.6 mM

2.5 mM

10 mM

Intra-Assay CV (%)

5.6

6.7

8.0

Inter-Assay CV (%)

4.6

5.1

6.4

95%

103%

Spike Test* (%)
104%* Known amounts of D-Xylose were added to samples and then assayed.

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

6

COMPARING TRANSCELLULAR AND PARACELLULAR TESTS (7)Transcellular Test (D-Xylose)
This test measures the permeability of small sugarmolecules (342 Da or smaller)
Repair mechanisms can repair small openings in tight junctions within
hours.
The intestinal permeability to small sugar molecules does notnecessarily correlate with the uptake of much larger dietary antigens
and bacterial toxins, such as LPS.
Measuring permeability to small sugar molecules does
not correlate with gut dysbiosis, endotoxin release,microbial translocation, and activation of the mucosal
immune system.

Paracellular Test (Fluorescent-labeled Dextrans)
This test measures the permeability of large molecules(10,000 Da or larger).
Large openings in tight junctions (which are associated withstructural damage to tight-junction proteins) cannot be
repaired within hours.
The intestinal permeability to large molecules correlates withdigestion-resistant fragments of food antigens and bacterial
toxins, such as LPS.
Measuring permeability to large molecules such as LPS
does correlate with gut dysbiosis, microbial
translocation, and immune activation.

REFERENCES
1.

S. Haque, K. Chen, N. Usui, Y. Iiboshi, H. Okuyama, et al., Alanyl-glutamine dipeptide-supplemented parenteral nutrition improvesintestinal metabolism and prevents increased permeability in rats. Ann Surg 223, 334-41 (1996).

2.

N. Ohkohchi, M. Himukai, Y. Igarashi, M. Kasai, Mechanism of D-xylose transport in human small intestine. J Pediatr Gastroenterol
Nutr 5, 372-8 (1986).

3.

A. Polleri, P. Menozzi, D. Norman, O. Hechter, Effect of inhibitors on D-xylose permeability in rat diaphragm muscle. J Gen Physiol 44,479-86 (1961).

4.

H. Duthie, J. Hindmarsh, The effect of amino acids on the intestinal transport of L- and D-xylose in vitro. J Physiol 187, 195-200 (1966).

5.

N. Vinogradova, N. Nikol'skiĭ, E. Semenova, D-xylose transport in cultured mammalian cells. Tsitologiia 22, 303-9 (1980).

6.

K. Horváth, G. Horn, H. Bingadeem, N. Nemes, H. Bodánszky, The value of the d-xylose loading test in the diagnosis of malabsorption
syndromes. Orv Hetil 131, 1803-6, 1809 (1990).

7.

A. Vojdani, For the assessment of intestinal permeability, size matters. Altern Ther Health Med 19, 12-24 (2013).

© 2021 Chondrex, Inc. All Rights Reserved, 6601 3.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com