Photometric Method for Detecting
Fructose in Seminal Plasma
FOR RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC PROCEDURES
FructoScreen® measures the amount of fructose in seminal plasma.
In the first step, all proteins as well as particulates are separated from the seminal plasma which contains fructose.
In the second step, the deproteinized seminal plasma is incubated with indole. The indole changes to a yellow-orange color in the presence of fructose. The intensity of this color can be measured in a microplate reader and is directly related to the amount of fructose in the seminal plasma. Using a formula, both the concentration of fructose and the total amount of fructose present can be calculated.
Solution A: 12 ml zinc solution. Ready to use.
Solution B: 8 ml sodium hydroxide solution. Ready to use. Warning: Avoid contact with skin.
Indole: 4 ml indole solution. Ready to use. Warning: Avoid contact with skin.
Fructose Standard: 1.5 ml 6.5 mM fructose solution with sodium azide. Ready to use. Warning: Dispose of with care.
Empty Dropper Bottle: Capacity for 15 ml concentrated hydrochloric acid with label reading Concentrated Hydrochloric Acid. Ready to be filled.
Plate and Lid: 96 wells.
Materials Required But Not Provided:
1. Concentrated hydrochloric acid.
2. Test tubes and rack.
3. Pipettors and tips.
4. Distilled or deionized water.
5. Collecting cups.
6. Centrifuge, capable of 1500g.
7. Air incubator.
8. Microplate reader, capable of reading at 470 - 490 nm.
Special Instructions for Filling Empty Dropper Bottle with Concentrated Hydrochloric Acid:
1. Wear gloves and safety goggles.
2. Place your bottle of concentrated hydrochloric acid and the test dropper bottle labeled Concentrated Hydrochloric Acid in a venting hood.
3. Remove cap and dropper tip from dropper bottle.
4. Uncap your concentrated hydrochloric acid bottle and carefully remove plastic cork (if present).
5. Pour acid into test dropper bottle. Avoid breathing acid fumes. Avoid spilling acid.
6. Replace plastic cork in your acid bottle and then screw on cap.
7. Holding dropper bottle firmly, force dropper tip into neck of dropper bottle. Screw on cap.
8. In case of contact, flush skin and eyes with copious amounts of water for at least 15 minutes.
Storage and Stability:
Store the Indole below 0oC and thaw fully before using. Store the other components at room temperature. They can be used until the expiration date shown on each reagent label.
Warning and Precautions:
All semen samples should be considered potentially infectious. Handle all specimens as if capable of transmitting HIV and hepatitis. Specimens should be disposed of in accordance with OSHA guidelines.
Avoid inhaling the Concentrated Hydrochloric Acid while performing the test. Avoid skin contact with the Concentrated Hydrochloric Acid. In the event that the acid gets on your skin or in your eyes, flush with copious amounts of water for at least 15 minutes.
Specimen Collection and Batching:
Semen should be collected in a clean cup. The semen sample can be stored at room temperature until using. Or, semen can be stored frozen by placing specimen in an ordinary freezer until assaying at a later time. No special procedures are needed for freezing and thawing.
1. Allow semen sample to liquify and measure total volume of semen.
2. Pipette 100 ul semen into a test tube.
3. Add 3 drops of Solution A to the test tube.
4. Add 2 drops of Solution B to the test tube and mix.
5. Centrifuge 5 minutes at 1500g.
6. Pipette 50 ul supernatant into an empty well of the Plate.
7. Pipette 50 ul Fructose Standard into an empty well.
8. Pipette 50 ul water, as a negative control, into an empty well.
9. Add exactly 1 drop Indole to each well.
10. Add exactly 4 drops Concentrated Hydrochloric Acid to each well.
11. Briefly agitate Plate (horizontally) to mix contents. Cover Plate with Lid and incubate 30 minutes at 37oC.
12. Read optical density (OD) of wells at 470 (or 490) nm using a microplate reader. First adjust the microplate reader to zero while reading an empty well.
Calculation of Fructose Concentration:
[Fructose] = ODseminal plasma x 14
where 14 is a constant determined by the concentration of fructose in the standard and the dilution of the specimen.
Calculation of Total Amount of Fructose:
Fructose = [Fructose] x Volume
Example: At 490 nm the following were obtained for a semen specimen:
Semen Volume = 2 ml
Mean OD of Specimen = 0.255
Mean OD of Fructose Standard = 0.168
OD of Water (negative control) = 0.020
Applying the formula for fructose concentration:
0.255 X 14 = 21.2 mmol/L [fructose]
Applying the formula for the total amount of fructose:
21.2 X 2 = 42.5 umol fructose/ejaculate
A semen specimen was assayed 10 times for fructose:
No. Mean, umol S.D. C.V.
10 35.8 +1.60 4%
World Health Organization. 1999. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press. Fourth Edition.