Mar 13, 2023
Determination of microglucosuria
- El Hadji Malick Ndour1,2,
- Khuthala Mnika3,
- Fatou Gueye Tall1,2,
- Moussa Seck4,
- Indou Deme Ly2,
- Victoria Nembaware3,
- Gaston Kuzamunu Mazandu3,
- Helene Ange Therese Sagna-Bassene2,
- Rokhaya Dione2,
- Aliou Abdoulaye Ndongo5,
- Jean Pascal Demba Diop6,
- Nene Oumou Kesso Barry1,
- Moustapha Djite1,
- Rokhaya Ndiaye Diallo6,
- Papa Madieye Gueye1,
- Saliou Diop4,
- Ibrahima Diagne7,
- Aynina Cisse1,
- Ambroise Wonkam3,
- Philomene Lopez Sall1,2,2
- 1Department of Pharmaceutical Biochemistry, Faculty of Medicine, Pharmacy and Dentistry, Cheikh Anta Diop University, Dakar, Senegal;
- 2Albert Royer National Children's Hospital Center, Dakar, Senegal;
- 3Division of Human Genetics, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa;
- 4National Blood Transfusion Center, Dakar, Senegal;
- 5Pediatrics Department of Aristide Le Dantec Hospital, Dakar, Senegal;
- 6Department of Human Genetics, Faculty of Medicine, Pharmacy and Dentistry, Cheikh Anta Diop University, Dakar, Senegal;
- 7Health Sciences Training and Research Unit, Gaston Berger University, Saint-Louis, Senegal
Protocol Citation: 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 2023. Determination of microglucosuria. protocols.io https://dx.doi.org/10.17504/protocols.io.b52rq8d6
Manuscript citation:
Ndour EHM, Mnika K, Tall FG, Seck M, Ly ID, Nembaware V, et al. (2022) Biomarkers of sickle cell nephropathy in Senegal. PLoS ONE
17(11): e0273745. https://doi.org/10.1371/journal.pone.0273745
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 07, 2022
Last Modified: March 13, 2023
Protocol Integer ID: 59185
Keywords: Microglucosuria, determination of microglucosuria, microglucosuria, microglucosuria in patient, sickle cell disease, sickle cell disease in order, nephropathy, heme catabolite, proximal convoluted tubule
Abstract
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.
Materials
Spectrophotometer
Troubleshooting
DETERMINATION OF MICROGLUCOSURIA
. OBJECTIVE
The aim is to describe how microglucosuria is determined.
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.
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
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.
RESULTS
The urine of a healthy subject does not contain glucose.
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.