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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 interesting phenomenon in which glycolysis is used predominantly for energy production and aerobic mitochondrial respiration, which involves the TCA cycle and electron transport chain, is downregulated in mammalian cancer cells^1,2. We showed recently that the lactate levels and the consequent lactate/pyruvate (L/P) ratio decreased during aging in the model organism Caenorhabditis elegans (C. elegans). Likewise, we found that the mammalian tumor suppressor p53 ortholog CEP-1 in C. elegans has an important role in the age-related alterations of energy metabolism through the activation of its transcriptional targets³.

In biological assays, such as the measurement of lactate and pyruvate concentrations in cells, the sensitivity, accuracy, sample size, and incubation time of colorimetric/fluorimetric assay kits have been improved dramatically. Owing to technological innovations, we are now able to analyze various metabolites and intermediate metabolites without the large-scale culture of C. elegans, which is difficult given its small size. In general, the sensitivity of a colorimetric assay is an order of magnitude smaller than that of a fluorimetric assay; however, the colorimetric approach is more suitable in the setting of common laboratories. Furthermore, an extraction technique containing homogenization and protein precipitation is crucial for the quantitative determination of lactate and pyruvate concentrations in C. elegans cells because this nematode is enclosed in an exoskeleton called the cuticle, unlike mammalian cultured cell lines^4,5. Here, we describe a protocol to analyze lactate and pyruvate concentrations using commercial colorimetric assay kits including tips for sample extraction from C. elegans.

Protokół

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 peptone, 17 g of agar, and 975 mL of deionized water. Autoclave. Cool to 55 °C, then sterilely add, in order, 1 mL of 1 M MgSO₄, 1 mL of 1 M CaCl₂, 1 mL of 5 mg/mL cholesterol in EtOH, and 25 mL of 1 M potassium phosphate pH 6.0 in 90-mm Petri dishes.
   1. For 1 M potassium phosphate pH 6.0, use 108.3 g of KH₂PO₄ and 46.6 g of K₂HPO₄, and add deionized water to 1 L. Autoclave⁶.
3. Spread 1-2 mL of the cultured OP50 on the NGM agar plates. To make a thin layer of OP50, incubate the plates overnight at room temperature before adding any nematodes.
   NOTE: The NGM agar plates inoculated OP50 can be stored at room temperature for 2-3 weeks.
4. Add at least 100 worms onto an NGM agar plate with OP50, and culture at 20 °C until the adult stage. At least three plates are required.
5. To collect eggs in utero, transfer gravid hermaphrodites from the three NGM agar plates each with 5 mL of S buffer in a 15-mL conical tube, and wash the worms 3 times with 15 mL of S buffer using centrifugation at 300 x g for 30 s at room temperature.
   1. To make S buffer, use 5.9 g of NaCl and 50 mL of 1 M potassium phosphate pH 6.0, add to 1 L with deionized water. Autoclave⁶.
6. Dissolve the worms in an alkaline hypochlorite solution for axenization and bulk egg isolation (0.5 mL of fresh bleach or equivalent: 5-6% sodium hypochlorite, 0.1 mL of 10 M NaOH, approximately 4.5 mL of S buffer)⁶, and stand the solution for 10-15 min at room temperature with mixing by inverting.
   NOTE: Within 15 min, adult worms should dissolve, leaving a hazy solution of eggs liberated from their carcasses (the liberated eggs should be confirmed using a stereoscopic microscope). Instead of 0.1 mL of 10 N NaOH, 0.2 mL of 5 N NaOH can also be used.
7. After hypochlorite treatment, wash the egg pellet 3 times with 15 mL of S buffer, and resuspend in 5-6 mL of S buffer. Hatch the released eggs during an overnight incubation at 20 °C in S buffer without E. coli for an age-synchronous culture of L1 stage larvae.
8. To determine the approximate number of L1 stage larvae, count the worms using a stereoscopic microscope in 10 µL of S buffer after resuspending the larvae at least 3 times, and calculate the average. Then, transfer the L1 stage larvae to five NGM agar plates with OP50 (1,500-3,000 worms per plate using a 90-mm Petri dish), and culture at 20 °C until they have grown to the young adult stage, when the self-fertilization begins and a few eggs are laid (ordinarily after 3 days).

2. Extraction of Cellular Fraction from C. elegans

1. Collect the young adult stage (5-day-old animals) worms from the five NGM agar plates with S buffer (Figure 1A).
   1. To select only living worms using the sucrose method⁶ for flotation on 30% (w/v) sucrose, mix the worms suspended in 3-4 mL of S buffer with an equal volume of ice-cold 60% (w/v) sucrose in a 15-mL conical tube. Spin the tube at 1,500 x g for 15 s at 4 °C and remove the floating worms into a fresh tube by moving them off the wall of the tube with a Pasteur pipette.
2. Wash the worms 3 times with S buffer by centrifugation at 1,500 x g for 30 s at 4 °C. Check the wet volume of washed worms after centrifugation using a 1,000 µL micropipette tip (Figure 1B).
3. Add the washed worms to an equal volume of ice-cold 10% (w/v) trichloroacetic acid (TCA; final concentration of 5%) for protein precipitation (Figure 1C). Instead of TCA, perchloric acid (PCA) or metaphosphoric acid can be used.
4. Homogenize the worms with the precipitant using 40 strokes of a pestle in a Teflon homogenizer (Potter-Elvehjem tissue grinder) with rotation at up to 1,300 rpm on ice.
5. Transfer the homogenate into a fresh 1.5-mL microtube with a Pasteur pipette, and sonicate using an ultrasonic homogenizer for 3 min (3 times of 1 min) with a 20% duty cycle on ice.
6. Clarify the homogenate by centrifugation at 8,000 x g for 10 min at 4 °C. Neutralize the supernatants with 4 M KOH (0.25 volume to 10% TCA) for 20 min on ice, and centrifuge at 8,000 x g for 10 min at 4 °C. The supernatant (as a test sample) can be stored at -80 °C until the following assays.

3. Lactate Assay Using a Colorimetric Assay Kit

1. Measure the concentration of lactate in the test samples using a colorimetric assay kit (Table of Materials). Carry out duplex examinations for the test samples. Add 5 or 10 µL of the test samples to a 96-well plate and adjust the volume to 50 µL per well with the Lactate Assay Buffer provided with the kit.
2. For the lactate standard curve, dilute 100 mM L(+)-Lactate Standard to 1 mM with Lactate Assay Buffer. Add 0, 2, 4, 6, 8, and 10 µL of the 1 mM L(+)-Lactate Standard, which is provided with the kit, into a series of wells.
3. Add 50 µL of Reaction Mix (containing 46:2:2 of Lactate Assay Buffer, Lactate Enzyme Mix and Lactate Probe in DMSO, anhydrous; all reagents are provided with the kit) or Background Control Mix (containing 48:2 of Lactate Assay Buffer and Lactate Probe) into each well and incubate at room temperature for 30-60 min while the samples are protected from light.
4. Measure the absorbance of each well at 570 nm using a microplate reader and subtract the absorbance of the Background Control Mix from the absorbance of the Reaction Mix.
5. Plot the lactate standard curve. Calculate the lactate concentrations of the test samples from the lactate standard curve.

4. Pyruvate Assay Using a Colorimetric Assay Kit

1. Measure the concentration of pyruvate in the supernatants (test samples) using a colorimetric assay kit (Table of Materials). Carry out duplex examinations for the test samples. Add 10 µL of the test samples and 90 µL of Working Reagent (containing 94:1 of Enzyme Mix and Dye Reagent, which are provided with the kit) into a 96-well plate and incubate at room temperature for 30 min while the samples are protected from light.
2. Measure the absorbance of each well at 570 nm using a microplate reader.
3. Plot the pyruvate standard curve. Calculate the pyruvate concentrations of the test samples from the pyruvate standard curve.

5. Protein Assay for Normalization with Protein Content

1. Measure the protein concentration in the supernatants (test samples) using a colorimetric assay kit (Table of Materials). However, this step is not limited to an assay kit, and other approaches can be utilized to measure the protein concentration.
2. Normalize the values of lactate and pyruvate concentrations among the test samples using each total protein concentration.
   Note: Protein concentration in the test samples is sufficiently detected even after protein precipitation and is utilized for the normalization step among samples.

Wyniki

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 ...

Dyskusje

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...

Materiały

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