Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Summary

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Abstract

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Introduction

Lactate and pyruvate concentrations are widely regarded as intermediates of energy metabolism, and are related to the states of glycolysis, tricarboxylic acid (TCA) cycle, and electron transport chain in the cells of aerobic organisms. A series of reactions in glycolysis oxidize glucose to pyruvate, which lies at a metabolic crossroads and can be converted to carbohydrates through gluconeogenesis, to fatty acids and energy metabolism through acetyl-CoA, and to the amino acid alanine. The TCA cycle occurs under the presence of sufficient dissolved oxygen and is fundamental for the conversion of glucose to energy. Especially, the alteration of secondary metabolism is an....

Protocol

1. Synchronized Culture of C. elegans

1. Before seeding, culture the Escherichia coli (E. coli) strain OP50 overnight at 37 °C in 300 mL of Luria-Bertani (LB) broth liquid medium. Store the cultured OP50 at -4 °C.
   1. To make LB broth liquid medium, use 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1.5 mL of 1 N NaOH, and add to 1 L with deionized water. Autoclave.
      NOTE: OP50 and C. elegans strains are available from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN, USA).
2. To make nematode growth medium (NGM) agar, use 3 g of NaCl, 2.5 g of....

Results

Using the colorimetric assays for the quantitative determination of lactate and pyruvate concentrations, we showed the accuracy of these assays compared with previous reports in C. elegans^7,8. Here, the process of protein precipitation during sample extraction was the most crucial step to generate accurate values. For protein precipitation, common precipitants (e.g., TCA, PCA, or metaphosphoric acid) can be used .......

Discussion

When utilizing these colorimetric assay kits, the most critical step in sample extraction to detect cellular lactate and pyruvate accurately in C. elegans is the process of protein precipitation during homogenization (Figure 1). It is not strictly necessary to use a Teflon homogenizer, as other homogenizers (e.g., Dounce and tapered tissue grinders, or bead mills) are also suited to the small-scale extraction of worms. We did not detect cellular lactate and pyruvate in test.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lactate Colorimetric/Fluorimetric Assay kit	BioVision	#K607-100	colorimetric/fluorimetric 100 assays; Store at -20oC
EnzyChrom Pyruvate Assay Kit	BioAssay Systems	#EPYR-100	colorimetric/ fluorometric 100 assays; Store at -20oC
BCA Protein Assay Kit	Thermo Scientific	#23225	colorimetric assay; store at  room temperature
Trichloroacetic Acid	Wako Pure Chemical	#207-04955	store at room temperature
Teflon homogenizer	Iwaki/Pyrex	#358034 (Wheaton)	Instead of Iwaki/Pyrex, available by Wheaton