TITLE: Determination of microglucosuria
AUTHORS: El Hadji Malick Ndour, Khuthala Mnika, Fatou Gueye Tall, Moussa Seck, Indou Deme Ly, Victoria Nembaware, Gaston Kuzamunu Mazandu, Helene Ange Therese Sagna-Bassene, Rokhaya Dione, Aliou Abdoulaye Ndongo, Jean Pascal Demba Diop, Nene Oumou Kesso Barry, Moustapha Djite, Rokhaya Ndiaye Diallo, Papa Madieye Gueye, Saliou Diop, Ibrahima Diagne, Aynina Cisse, Ambroise Wonkam, Philomene Lopez Sall

[DESCRIPTION]
This protocol describes how to determine microglucosuria in patients living with sickle cell disease in order to diagnose a nephropathy as a result of proximal convoluted tubule damaged by heme catabolites.

[STEPS]
SECTION: DETERMINATION OF MICROGLUCOSURIA
1. . OBJECTIVE
The aim is to describe how microglucosuria is determined.
SECTION: DETERMINATION OF MICROGLUCOSURIA
2. SAMPLING

Urine 
A meadstream voiding urine sample at random  at any time between 8 a.m. and 2 p.m. is collected. The urine sample is centrifuged before performing the test on the same day of collection with the undiluted supernatant.
SECTION: DETERMINATION OF MICROGLUCOSURIA
3. PRINCIPLE 
Several methods can be used, including hexokinase, glucose oxidase or glucose dehydrogenase. 
The method used in this work is that of glucose oxidase / peroxidase whose principle is as follows: 
Glucose, under the action of glucose-oxidase, is oxidized to gluconic acid with formation of hydrogen peroxide. 
In the presence of peroxidase, the hydrogen peroxide produced transforms a colorless reduced chromogen into an oxidized chromogen colored pink whose intensity of coloration measurable by spectrophotometry at 505 nm is proportional to the glucose concentration of the sample.
                                      Glucose oxydase
Glucose + O2 + H2O --------------------------→ Acide gluconique + H2O2
                                                                    Peroxydase
H2O2 + phénol + amino 4 phenazone ----------------------→ Quinone imine + 2 H2O

SECTION: DETERMINATION OF MICROGLUCOSURIA
4. PROCEDURE
-Place the reagents at room temperature for 5 minutes.
-Pipette in test tubes according to the following table:
 
 
 
 
            Blanc      Standard      Control      Sample        Blanc      -      -      -      -        Standard      -      10 µl      -      -        Control      -      -      10 µl      -        Sample      -      -      -      10 µl        Reagent       1 ml      1 ml      1 ml      1 ml   

-Shake well and incubate the tubes for 10 minutes at room temperature or for 5 minutes 37°C. A pink coloration is obtained (stable at least 1 hour).
   -Read the concentrations by spectrophotometer at 505 nm.
 
 
 
 
SECTION: DETERMINATION OF MICROGLUCOSURIA
5. RESULTS
 
The urine of a healthy subject does not contain glucose. 
SECTION: DETERMINATION OF MICROGLUCOSURIA
6. INTERPRETATION
To interpret glucosuria, the RGCU (urinary glucose/creatinine ratio) which is the ratio of glucosuria (mg/l) to creatininuria (g/l) is first calculated.
If RGCU ˂ 20 mg/g, it is called physiological glucosuria.

If the RGCU ≥ 20 mg/g, with test strip results indicating an absence of glucose in the urine, it is called microglucosuria.

If RGCU ≥ 20 mg/g with test strip results indicating the presence of glucose in the urine, this is called glucosuria.