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ACCUCARETM
GLUCOSE-6-PHOSPHATASE DEHYDROGENASE (G-6PDH) QUANT |
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INTENDED USE Accucare GLUCOSE-6-Phosphate Dehydrogenase reagents are for the quantitative, ultraviolet, kinetic determination of g-6-PDH in blood at 340 nm. SUMMARY Glucose- 6 phosphate dehydrogenase, (G6PDH, D-glucose-6-phosphate : oxidoreductase ) catalyzes the first step in the pentose phosphate shunt, oxidizing glucose-6-phosphate (G-6-P) and reducing NADP to NADPH. The Accucare Procedure is a spectrophotometric method based on the following reaction G-6-P + NADP+ ------------------------------® 6-PG + NADPH + H+ Nicotinamide adenine dinucleotide phosphate (NADP) is reduced by G-6-PDH in the presence of G-6-P. The rate of formation of NADPH is proportional to the G-6-PDH activity and is measured spectrophotometrically as an increase in absorbance at 340 nm. Production of a second molar equivalent of NADPH by erythrocyte 6-phosphogluconate dehydrogenase (6-PGDH according to the reaction : G-6-P + NADP+ ------------------------------® Ribulose-5-Phosphate + NADPH + H+ + CO2 Is prevented by use of maleimide, an inhibtor of 6-PGDH. REAGENTS Reagent I : GLUCOSE-6-PHOSPHATE Assay Reagent Reagent II : G-6-PDH SUBSTRATE SOLUTION REAGENT PREPARATION Is prepared by reconstituting G-6-PDH assay vials with the volume of deionised water as stated on the vial. Swirl gently and invert several times to dissolve the contents. Wait 2-3 minutes and mix again. STORAGE AND STABILITY Store unopened G-6-PDH Reagent vials and the G-6-PDH Substrate Solution in refrigerator (2-8°C). Reagents are stable until expiration dates shown on the labels. Reconstitute G-6-PDH assay Solution is stable for a hrs at room temperature (18-26°C) or 5 days refrigerated (2-8°C). G-6-PDH SUBSTRATE SOLUTION Is supplied ready to use. SPECIMEN STORAGE AND COLLECTION Whole blood is collected in EDTA, Heparin or ACD is satisfactory. Red cell G-6-PDH is stable in whole blood for one week refrigerated (2-8°C), but is unable in red cell hemolysates. Freezing of Blood is not recommended. Since activity is reported in terms of number of red cells or grams of hemoglobin. The red cell count or hemoglobin concentration should be determined prior to performing the G-6-PDH assay. The integrity of erythrocytes collected in ACD id preserved even after prolonged storage so that obtaining accurate red cells count usually poses no problem. However, red cell counts on specimens collected in heparin become unreliable after about 2 days. Thus, for heparinized samples, results are best reported in terms of hemoglobin concentration. CALCULATIONS ( frm catalogue) pA per min = FINAL A - INITIAL A 5 G-6-PDH activity is expressed as U/1012 erythrocytes (RBC) or U/g hemoglobin (Hb). G-6-PDH (U/1012 RBC) = pA per min x 3.01 x 10 x TCF 0.01 x 6.22 x (N x 1012) x 1000 Where : 3.01 = Total reaction volume (mL) 1012 = Factor for expressing activity in 1012 cells 0.01 = Sample volume (mL) 6.22 = Millimolar absorptivity of NADPH at 340 nm N x 1012 = Red cell count (red cells/mm3 ) determined for each specimen 1000 = Convesion of red cell count from mm3 to mL TCf = Temperature correction factor (1 at 30°C) This equation reduces to : G-6-PDH (U/1012RBC) = pA per min x 48,390 x TCF N Where : N = Red cell count divided by 106 TCF = Temperature correction factor (1 at 30°C) G-6-PDH (U/g Hb) = pA per min x 100 x 3.01 x TCF 0.01 x 6.22 x Hb(g/dL) = pA per min x 4839 x TCF Hb(g/dL) Where : 100 = Factor to convert activity to 100 mL 3.01 = Total reaction volume (mL) 0.01 = Sample volume (mL) 6.22 = Millimolar absorptivity of NADPH at 340 nm Hb(g/dL) = Hemoglobin concentration determined for each specimen TCF = Temperature correction factor (1 at 30°C) |
EXAMPLE : Assay of a specimen which had a red cell count of 4.6 x / mm3 and a hemoglobin concentration 15.2 g/dL resulted in a pA per min at 30°C of 0.026. G-6-PDH (U/1012 RBC) = 0.026 x 48,390 = 295 4.6 G-6-PDH(U/g Hb) = 0.026 x 4839 = 8.9 15.2 NOTE : If pA per min is greater than 0.060, repeat determination using 5mL blood ad multiply the result by 2 Both copper, which completely inhibits the enzyme at a concentration of 100mmol/L, and sulfate ions (0.005 mol/L) will decrease observed values of G-6-PDH activity, Certain drugs and other substances are known to influence circulating levels of G-6-PDH Reticulocytes have higher G-6-PDH levels than mature red cells. Therefore it is not recommended that assays be performed after a severe hemolytic crisis, since G-6-PDH levels appears falsely elevated. Under those conditions, detection of deficiency may require family studies. Testing may be more helpful after the level of mature red cells has returned to normal. Under normal circumstances, activity contributed by leucocytes, platelets and serum is relatively small. However, in cases of extreme anaemia, grossly elevated white counts or very low levels of red cells G-6-PDH activity, the contribution to the total made under these conditions may be significant. See "Use of Buffy-Coat-Free Samples" section. PROCEDURE The temperature of the reaction mixture should be maintained at 30°C or some other constant temperatures (see "Temperature Correction " section). 1 Prepare reaction mixture a) Add 0.01 ml blood to 1 ml of G-6-PDH Assay solution and mix thoroughly to completely suspended erythrocytes. Let stand at room temperature (18-26°C) for 5-10 minutes. b) Add 2.0ml G-6-PDH Substrate solution to the above hemolysate and mix gently by inverting several times. c) Transfer contents to Cuvette labeled Test & proceed with Step2. 2. Place Cuvet in constant temperature cuvet compartment or water bath and incubate for approximately 5 minutes to obtain thermal equilibrium. 3. Read and record absorbance (A) of Test at 340nm vs water or Pottasium Dichromate solution. This is INITIAL A. (if using the water bath or incubator, return the cuvete to it) 4. Exactly 5 minutes later, again read and record absorbance. 5. To determine G-6-PDH activity, refer to "calculations" section. CALIBRATION The procedure is standardized on the basis of the milimolar absorpivity of NADPH, which is 6.22 at 340nm. The oxidative conservation of G-6-P by G-6-PDH leads to reduction of NADP to NADPH on a molar equivalent basis. Measurement of the rate of increase in absorbance (pA) at 340 nm serves to quantitate enzymatic activity. The maximum G-6-PDH activity which may be measured by this procedure is approximately 650 U/1012 RBC or 19.5 U/g Hb. QUALITY CONTROL The reliability of test results should be mentioned with each run by routine use of control sera with known G-6-PDH activity. Under normal circumstances G-6-PDH activity contributed by leucocytes, platelets and serum is relatively small. However, as reported by Echler and others, more accurate measurement of G-6-PDH activity specially in the presence of anaemia and / or leucocytosis, can be achieved by using buffy-coat-free blood samples for assay. Thus in case of a borderline value obtained with whole blood, it may be warranted to repeat the assay on a buffy coat-free sample. TEMPERATURE CORRECTION When temperature of 30°C, no temperature correction factor (TCF) is required in the calculations. If the assay Is performed at a room temperature other than 30°C , a TCF must be used. UNIT DEFINATION One international unit (U) is that amount G-6-PDH activity that will convert 1 micromole of substrate per minute under the conditions specified in this insert. Activity may be expressed in terms of either a standard number of cells or amount of hemoglobin. Since it is preferred, despite the fact that is believed by some that red cell counts are subject to considerable uncertainty. Hemoglobin concentration may be determined with greater accuracy, but the amount of hemoglobin contained in a cell is under separate genetic control and may vary independently of G-6-PDH activity. EXPECTED VALUES The following range of G-6-PDH values measured at 30°C was obtained in or laboratory for 100 clinically healthy males and females : G-6-PDH Activity 146-376 (U/1012RBC) 4.6-13.5 (U/g Hb) Values for newborns may range somewhat higher. It is recommended that each laboratory establish its own normal range. It has been determined that G-6-PDH deficiency in re cells is the basis for certain drug induced hemolytic anemia's. This type of suspectibility to drug-induced Haemolysis is often called "primaquine sensitivity" because studies which led to its characterization were made during investigations of the hemolytic properties of this antimalarial compound. |
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Lab
Care Diagnostics (India) Pvt. Ltd. 16-A, 'A' Wing , 1st Floor, Sita Estate Mahul Road, Aziz Baug, Chembur, Mumbai 400 074. India Phone: 00 91 22 2554 2109/ 00 91 22 2554 1558 Fax: 00 91 22 2554 3541 E-mail : accucare@labcarediagnostics.com |