- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
NOTES:
1. The Auto-Analyser Assay Procedure for pyruvic acid can be performed using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination of pyruvic acid either a single point standard or a calibration curve must be performed concurrently using the same batch of reagents.
- C. MICROPLATE ASSAY PROCEDURE:
NOTES:
1. The Microplate Assay Procedure for pyruvic acid can be performed using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination of pyruvic acid either a single point standard or a calibration curve must be performed concurrently using the same batch of reagents.
- SAMPLE PREPARATION:
1. Sample dilution.
The amount of pyruvic acid present in the cuvette (i.e. in the 0.1 mL of sample being analysed) should range between 0.3 and 40 μg. The sample solution must therefore be diluted sufficiently to yield a pyruvic acid concentration between 0.003 and 0.40 g/L.
2. Sample clarification.
a. Solutions:
Carrez I solution. Dissolve 3.60 g of potassium hexacyanoferrate (II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P9387) in 100 mL of distilled water. Store at room temperature.
Carrez II solution. Dissolve 7.20 g of zinc sulphate (ZnSO4.7H2O) (Sigma cat. no. Z4750) in 100 mL of distilled water. Store at room temperature.
Sodium hydroxide (NaOH, 100 mM). Dissolve 4 g of NaOH in 1 L of distilled water. Store at room temperature.
b. Procedure:
Pipette the liquid sample into a 100 mL volumetric flask which contains approx. 60 mL of distilled water, or weigh sufficient quantity of the sample into a 100 mL volumetric flask and add 60 mL of distilled water. Carefully add 5 mL of Carrez I solution, 5 mL of Carrez II solution and 10 mL of NaOH solution (100 mM). Mix after each addition. Fill the volumetric flask to the mark, mix and filter.
3. General considerations.
(a) Liquid samples: for clear, slightly coloured liquid samples, adjust the pH to approx. 7.4 and use directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 7.4 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing a significant amount of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 7.4 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no D-LDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of polyvinylpolypyrrolidone (PVPP)/10 mL of sample. Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask. Adjust to room temperature and fill the volumetric flask to the mark with distilled water. Store on ice or in a refrigerator for 15-30 min to allow the fat to separate and then filter. Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay.
(h) Samples containing protein: deproteinise samples containing protein with Carrez reagents.
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100 assays (manual) / 1000 assays (microplate) / 1000 assays (auto-analyser)
Bottle 1:
Buffer (22 mL, pH 7.4) plus sodium azide (0.02% w/v) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2:
NADH.
Stable for > 2 years below -10°C.
Bottle 3:
D-Lactate dehydrogenase suspension (2.2 mL).
Stable for > 2 years at 4°C.
Bottle 4:
Pyruvic acid standard solution (5 mL, 0.20 mg pyruvate/mL).
Stable for > 2 years; store sealed at 4°C.
