The overall goal of the following experiment is to non-invasively monitor real-time metabolic alterations at the whole animal level within glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle that are caused by individual metabolic pathway, genetic alterations, and or pharmacologic treatments. This is achieved by feeding stable isotopes to living nematodes to provide sufficient isotopic enrichment in more metabolites to permit quantitation of genetic and or pharmacologic alterations in intermediary metabolic pathways. As a second step, isotopic incorporation in atmospheric and dissolved carbon dioxide are measured by gas ratio mass spectrometry, which provides more direct assessment of intermediary metabolic flux over short periods of time.
Next samples are processed for whole worm free amino acid analysis using HPLC as well as by isotopic enrichment in amino acids and organic acids by GCMS in order to quantify metabolic flux alteration in nematodes. Following stable isotope exposure, results are obtained that show effects of variable durations of isotope exposure, bacterial clearing, and alternative worm disruption in wild type worms to interrogate isotopic abundance in intermediary metabolites. War with method can provide insight into sail against intermediary metabolism.
It can also be applied to our systems such as mammalian cell lines and tissues. We first had the idea for this method when we observed consistent abnormalities in free worm amino acid analysis by HPLC that were suggestive of impaired intermediary metabolic flux in primary respiratory changes Function. To begin this Procedure.
Worms that have been exposed to a stable isotope labeled metabolic precursor either during development or in adulthood, are collected in a 1.5 milliliter plastic centrifuge tube. We usually dilute the worms to a concentration of 1000 worms per one milliliter. Add 60%per chloric acid containing epsilon aminocaproic acid as an internal standard to the worms to a final concentration of 4%per chloric acid and 20 nanomolar of internal standard.
Let the worm settle by gravity for five minutes, then remove and save the supernatant in another tube. The following step is one of the most critical aspects of this procedure. The worms have to be completely disrupted by screening prior to free metabolite analysis Using a plastic homogenizer and motorized drill, grind the worm samples for 15 seconds.
Visually confirm worm disruption by light microscopy and repeat the grinding if necessary. In this image, panels A and B show worms prior to grinding. In contrast to panel C and D that show worms after grinding transfer the previously saved supernatant to the 1.5 milliliter plastic centrifuge tubes with the worm Homogenate centrifuge.
The samples at 2, 250 RPM 1300 Gs for five minutes. After centrifugation, transfer the supernatant to a fresh seven milliliter glass tube. Save the pellet for later use in a protein concentration assay.
Now add four normal potassium hydroxide to the SUPERNAT until the pH range of the samples is seven to eight centrifuge. The neutralized samples in the seven milliliter glass tubes at 2, 250 RPM 1300 Gs for five minutes To remove salts, transfer the supernatant from each sample to a fresh seven milliliter glass tube. The neutralized samples can now be prepared for metabolite quantitation by high performance liquid chromatography or HPLC and gas chromatography, mass spectrometry or GCMS for HPLC.
Separate 50 microliters of each neutralized sample for direct injection into the HPLC. Free amino acid quantitation is performed using precal derivitization with al aldehyde and fluorescent detection. As previously described, the remaining neutralized samples will undergo extraction using an ion exchange resin for GCMS to determine isotopic enrichment in amino acids and organic acids as shown in the next Section.
To prepare The beads, add one normal hydrochloric acid to a G one and a G 50 beads separately. In one liter beakers, stir each Flask for 30 minutes with a magnetic stir. After that, wash the beads with deionized water 10 Times until the pH of the washings is equal to the pH of the water.
Next, we will Show our method for preparing custom made low-cost columns using glass pipettes. Pre-made columns are also commercially available. To prepare a column, use forceps to insert a cotton plug just above the narrowest part of a pasture pipette tip, use a G one beads for organic acid extraction and a G 50 beads.
For amino acid extraction, fill each column to approximately one third of the column height. If a sample is already at neutral pH, nothing is added to the sample before applying it to the AG one column. To extract organic acids to extract amino acids, add one milliliter of 0.1 normal hydrochloric acid to the sample before applying it to the AG 50 column.
Now apply the previously prepared neutralized samples to each column. Wash each column 10 times with water until washings are neutral. After the washing is complete, elute each column to fresh labeled four milliliter glass sample vials For organic acid extraction, add three milliliters of three normal hydrochloric acid to the AG one column.
This permits analysis of isotope enrichment in total number of labeled carbons among three carbon species, four carbon species, five carbon species, and six carbon species. For amino acid extraction, add three milliliters of four normal ammonium hydroxide to the a G 50 column. This permits analysis of isotope enrichment in total number of labeled carbons among three carbon species and five carbon species.
At the completion of both the organic acid and amino acid extractions, place the sample vials in a reactive app three evaporator overnight until they are dry. The samples are then dert and analyzed by GCMS as detailed in the Protocol text. Begin This procedure by transferring a thousand young adult worms to an experimental 35 millimeter NGM agri plate containing universally labeled carbon 13 glucose and e coli.
Next place the experimental NGM plate without its cover. In a custom glass chamber fitted with a well sealed three-way stop cock to allow for precise atmosphere sampling and replacement. Cover the seal where the optically transparent glass disc sits on the plate with high vacuum grease.
Seal the chamber with the glass disc. Record the time. As time zero, 30 minutes later, use a 20 milliliter syringe to sample 10 milliliters of the atmosphere from the glass chamber by the three-way stop cock.
Close the stop cock to the chamber After sampling, transfer the sample to a red tub vacuumed glass tube containing one milliliter of sodium bicarbonate in 0.1 molar sodium hydroxide. Next, inject 10 milliliters of air into the glass chamber through the three-way stop cock. Using a 20 milliliter syringe, close the stop cock to the chamber After re-injecting atmosphere and before resuming incubation, repeat sampling of the atmosphere from the glass chamber at 60 90 and 120 minutes.
Analyze these atmospheric carbon dioxide samples for labeled carbon enrichment using a gas ratio mass spectrometer as described in the Protocol text, stable Isotopes are well incorporated after two hours into young adult worms as evidenced here by labeled carbon measured in atmospheric carbon dioxide and dissolved worm carbon dioxide in worms fed either living or UV irradiated killed OP 50 e coli. The ratio of carbon 13 carbon dioxide to carbon 12 carbon dioxide was greater in the dissolved worm carbon dioxide fraction relative to the released atmospheric carbon dioxide. It was observed that prolonged exposure to a stable isotope from the larval period and subsequently clearing worms on NGM Aate without bacteria increased isotopic enrichment of free glutamate in young adult worms.
In this graph, L one represents worms fed with universally labeled carbon 13 glucose and OP 50 bacteria from the L one larval stage through the 959 cell young adult stage YA represents worms fed with carbon 13 glucose and OP 50 bacteria for 48 hours beginning when they reached the first day of egg laying young adult stage. The x axis indicates the total number of labeled carbon atoms in each glutamate species, and the Y axis indicates percent enrichment. All animals were cleared of excess isotopic label prior to downstream sample preparation.
For GCMS analyses, the green bars indicate worms cleared following isotopic exposure by feeding with OP 50 E coli on NGM plates for two hours prior to PCA extraction. The blue and gray bars indicate worms cleared following isotopic exposure on NGM plates without bacteria for two or six hours respectively prior to PCA extraction regardless of the isotopic exposure time course. No significant difference in isotopic enrichment in intermediary metabolites was observed when animals were cleared on plates for either two or six hours.
In both isotopic exposure courses, less isotopic enrichment was observed in intermediary metabolites when animals were cleared by feeding unlabeled bacteria for two hours. Therefore, the optimal clearing protocol for worms grown on bacterial spread N GM plates with isotope was determined to be clearing for two hours on unspread NGM plates to optimize percent enrichment in intermediary metabolites. Following treatment for 24 hours in liquid culture with one six carbon 13 glucose without bacteria.
A comparison was made of worm samples disrupted by sonication alone or sonication plus grinding as shown in this figure. Worms disrupted by Sonication plus grinding had greater isotopic enrichment than samples disrupted only by Sonication finally feeding living worms with a stable isotopic precursor either during development from L one stage on plates or beginning on the first day of egg, laying as young adults for 24 hours in liquid culture. Permit sensitive analysis of isotopic enrichment among metabolites indicative of flux through glycolysis, pyruvate metabolism in the tricarboxylic acid Cycle after its development.
This technique paved the way for mitochondrial disease, researches to explore intermediate metabolic flux in sea elegance. After watching this video, you should have a good understanding of how to perform stable isotopic, profiling of intermediary metabolism and metabolic flux in the nematodes, The elegance.
