BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Pyruvic acid (PA), also known as 2-oxopropanoic acid, is a key intermediate in fundamental metabolic processes throughout organisms. It enables the interconversion of sugars, fats, and amino acids within the body via acetyl-CoA and the tricarboxylic acid (TCA) cycle, acting as a crucial metabolic hub. Pyruvate is the end product of anaerobic glucose metabolism (glycolysis). Researchers often study pyruvate and lactate together, using their ratio to assess the degree of circulatory failure. Pyruvate can be detected using the lactate dehydrogenase enzymatic method or the dinitrophenylhydrazine colorimetric method.
Assay Principle
Under acidic conditions, pyruvic acid reacts with 2,4-dinitrophenylhydrazine (DNPH) to form a pyruvic acid-dinitrophenylhydrazone complex. This complex develops a brownish-red color after oxidation. The absorbance at 520 nm is measured using a microplate reader. The PA concentration is calculated by comparing the absorbance to a standard curve. This kit can be used to detect endogenous pyruvic acid levels in samples such as plants, cells, tissues, and serum, and is particularly suitable for measuring pyruvic acid content in plant samples. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
| Component | 100T | Storage |
| Pyruvate Standard (6 mg/mL) | 1 mL | 2-8℃. Store in the dark. |
| Tissue Homogenization Buffer (3×) | 100 mL | RT. Store in the dark. |
| Phenylhydrazine Chromogenic Solution | 3 mL | 2-8℃. Store in the dark. |
| PA Assay Buffer (1.5×) | 10 mL | RT |
Required Materials Not Provided
Distilled Water, Microplate Reader, 96-well Plate, Centrifuge Tubes or Small Test Tubes
Experimental Procedure
1. Preparation of Tissue Homogenization Buffer (1×):
Add 20 mL of distilled water to 10 mL of Tissue Homogenization Buffer (3×) to prepare 30 mL of 1× buffer.
2. Preparation of PA Assay Buffer (1×):
Add 5 mL of distilled water to 10 mL of PA Assay Buffer (1.5×) to prepare 15 mL of 1× buffer.
3. Sample Preparation
3.1 Liquid Samples (e.g., Serum/Plasma)
Homogenize the sample on ice using a ratio of serum/plasma volume (mL) to 1× Tissue Homogenization Buffer (mL) between 1:5 and 1:10 (e.g., add 0.1 mL liquid sample to 1 mL Homogenization Buffer is recommended).
Let stand for 30 minutes.
Centrifuge at 8000 rpm, room temperature, for 10 minutes.
Collect the supernatant for assay.
3.2 Tissue Samples
Homogenize the tissue on ice using a ratio of tissue weight (g) to 1× Tissue Homogenization Buffer (mL) between 1:5 and 1:10 (e.g., add 1 mL Homogenization Buffer to approx. 0.1 g tissue is recommended).
Let stand for 30 minutes.
Centrifuge at 8000 rpm, room temperature, for 10 minutes.
Collect the supernatant for assay.
3.3 Bacteria or Cultured Cells
Collect bacteria or cells by centrifugation and discard the supernatant.
Homogenize using a ratio of bacteria/cell count (10⁴) to 1× Tissue Homogenization Buffer (mL) between 500~1000:1 (e.g., add 1 mL Homogenization Buffer to 5 million bacteria/cells is recommended). Sonicate on ice (200W, 3s pulse, 10s interval, repeat 30 times).
Let stand for 30 minutes.
Centrifuge at 8000 rpm, room temperature, for 10 minutes.
Collect the supernatant for assay.
3.4 High-Concentration Samples
If the sample contains high PA levels, dilute it with the original lysis buffer or PBS before assay (e.g., chicken serum can be diluted 5-10 times).
4. Preparation of Standard Working Solution
For serum, urine, etc.: Dilute Pyruvate Standard (6 mg/mL) with distilled water at a ratio of 1:99 to achieve a concentration of 60 µg/mL.
For tissue samples: Dilute Pyruvate Standard (6 mg/mL) with 1× Tissue Homogenization Buffer at a ratio of 1:99 to achieve a concentration of 60 µg/mL.
4.1 Standard Dilution for Serum/Urine Samples (using 96-well plate):
| Tube | 1 | 2 | 3 | 4 | 5 | 6 |
| Pyruvate Standard (60 µg/mL, in H₂O) (µL) | 2.5 | 5 | 7.5 | 15 | 30 | 45 |
| Distilled Water (µL) | 72.5 | 70 | 67.5 | 60 | 45 | 30 |
| Final PA Concentration (µg/mL) | 2 | 4 | 6 | 12 | 24 | 36 |
4.2 Standard Dilution for Tissue Samples (using 96-well plate):
| Tube | 1 | 2 | 3 | 4 | 5 | 6 |
| Pyruvate Standard (60 µg/mL, in Homog. Buffer) (µL) | 2.5 | 5 | 7.5 | 15 | 30 | 45 |
| 1× Tissue Homogenization Buffer (µL) | 72.5 | 70 | 67.5 | 60 | 45 | 30 |
| Final PA Concentration (µg/mL) | 2 | 4 | 6 | 12 | 24 | 36 |
5. PA Assay Setup
Set up Blank, Standard, and Test wells as below. Add reagents in the specified order, avoiding bubbles. For high PA concentrations, reduce sample volume or dilute appropriately. Duplicate wells are recommended.
5.1 For Serum/Urine Samples (using 96-well plate):
| Reagent (µL) | Blank Well | Standard Well | Test Well |
| Distilled Water | 75 | — | — |
| Diluted Pyruvate Standard (in H₂O) | — | 75 | — |
| Sample Supernatant | — | — | 75 |
| Phenylhydrazine Chromogenic Solution | 25 | 25 | 25 |
Mix thoroughly;
| PA Assay Buffer (1×) | 125 | 125 | 125 |
5.2 For Tissue Samples (using 96-well plate):
| Reagent (µL) | Blank Well | Standard Well | Test Well |
| 1× Tissue Homogenization Buffer | 75 | — | — |
| Diluted Pyruvate Standard (in Homog. Buffer) | — | 75 | — |
| Sample Supernatant | — | — | 75 |
| Phenylhydrazine Chromogenic Solution | 25 | 25 | 25 |
Mix thoroughly;
| PA Assay Buffer (1×) | 125 | 125 | 125 |
6. PA Measurement
Mix and incubate at room temperature for 10 minutes. The Blank well should be colorless or light yellow, while the Standard wells show varying shades of brownish-red.
Zero the microplate reader using the Blank well.
Measure the absorbance of Standard wells and Test wells at 520 nm (recorded as Astandard and Atest).
7. Calculation of Results
Plot the standard curve with the pyruvate standard concentrations (Tubes 1-6) on the X-axis and the corresponding Astandard values on the Y-axis. The pyruvate content for serum/urine is directly obtained from the standard curve.
7.1 Based on Serum/Plasma Volume:
Pyruvate (µg/mL) = A × (Vextract + Vsample) ÷ Vliquid × D
7.2 Based on Sample Protein Concentration:
Pyruvate (µg/mg prot) = A × Vextract ÷ (Vliquid × Cpr) × D = A ÷ Cpr × D
7.3 Based on Sample Weight:
Pyruvate (µg/g weight) = A × Vextract ÷ W
7.4 Based on Bacteria/Cell Count:
Pyruvate (µg/10⁴ cells) = A × Vextract ÷ N
Parameter Definitions:
A: Pyruvate concentration of the sample calculated from the standard curve (µg/mL)
Vextract: Volume of Homogenization Buffer added (mL)
Vsample: Volume of liquid sample added (mL)
Vliquid: Volume of liquid sample used (assumed equal to Vsample for calculation if not specified otherwise; typically 0.1 mL as per protocol)
Cpr: Sample protein concentration (mg/mL)
D: Dilution factor
W: Sample weight (g)
N: Bacteria/Cell number (in units of 10⁴)
Notes
1. The order of reagent addition must not be changed: first add pyruvate standard or test sample, then add Phenylhydrazine Chromogenic Solution, finally add PA Assay Buffer (1×).
2. Prepared Pyruvate Standard (60 µg/mL) should be stored at 4°C protected from light and is stable for 24 hours.
3. Tissue Homogenization Buffer is corrosive. Handle with care.
4. The reference range for pyruvic acid in fasting venous whole blood of healthy adults is 3–9 µg/mL.
5. If a microplate reader is unavailable, a spectrophotometer can be used, requiring adjustments to sample volumes.
6. Standard curve points should be evenly distributed and cover an appropriate range.
7. For your safety and health, wear lab coats and disposable gloves during operation.
8. Use reagents promptly after opening to avoid affecting subsequent experimental results.
Standard Curve Reference
When measured at 520 nm using a microplate reader at room temperature, the absorbance of pyruvate standards in the range of 1~60 µg/mL typically falls between 0.10 and 2.40. Based on the absorbance of pyruvate standards at 1, 2, 4, 6, 12, 24, 36, 48, and 60 µg/mL, the standard curve is as follows:
Y = 0.037X + 0.0746, R² = 0.9994
Note: Reference ranges may vary due to differences in instruments and operational techniques. For precise pyruvate quantification, use a multi-point standard curve with replicate measurements. Empirical data suggests deviations in the standard curve at standard concentrations below 1 µg/mL and above 60 µg/mL.
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