Mass isotopomer pattern and precursor product relationship test

mass isotopomer pattern and precursor product relationship test

expansion; biological polymers; precursor-product relationship; probability analysis .. ternal pattern or relationships among mass isotopomer enrichments is uniquely have to test the effect of precursor enrichment on calcu- lated product. pattern of excess enrichment among mass isotopomers of de novo .. Statistical analyses. Data were tested for normality before using paired t-tests to compare . the application of the precursor-product relationship: def- inition of the precise. Jul 5, into urea and glutamine, predicts the pattern of isotopomers produced as a function of Three different nitrogen mass isotopomers of urea were found, Such methodology will enable us to test Meijer's hypothesis that there is therefore, there should be a precursor-product relationship between 15N.

One nitrogen is derived from ammonia and is incorporated into carbamoyl phosphate by carbamoyl-phosphate synthetase, which occurs in the mitochondrial matrix. This is then transferred to ornithine to give citrulline. The second nitrogen is derived from aspartate and is incorporated into argininosuccinate by argininosuccinate synthetase, which is found in the cytoplasm. The two nitrogens in urea are chemically and sterically identical so that it is not possible to identify the origin of a particular nitrogen atom in urea.

Urea synthesis occurs in the liver, where it is a major function of hepatocytes in the periportal and pericentral zones 4.

The glutamine produced also contains two atoms of nitrogen. However, they are distinguishable, the amide being derived from ammonia and the amino from glutamate. The present study examines the labeling of urea and glutamine when 15N-labeled ammonia is provided as substrate. We had two objectives in doing this. First, we wished to provide a rigorous theoretical, experimentally verified framework for understanding the determinants of urea isotopomer production.

This is particularly relevant, since a number of experimental studies have measured the incorporation of 15N-labeled substrates into urea 789. One set of studies, in particular, has used these data to suggest that urea synthesis in sheep is fundamentally different from other mammals 89.

mass isotopomer pattern and precursor product relationship test

The second reason for carrying out these studies was to introduce a methodology that would permit the determination of the isotopic enrichment of the two nitrogenous precursor pools involved in urea synthesis. Such methodology will enable us to test Meijer's hypothesis that there is metabolic channeling between glutaminase and carbamoyl-phosphate synthetase I such that the amide nitrogen of glutamine has preferential access to carbamoyl-phosphate synthetase without mixing with the mitochondrial pool of ammonia We have employed the single-pass isolated perfused liver as our experimental model, since this avoids problems due to recycling of substrate such as incorporation of ammonia into glutamine in perivenous hepatocytes and subsequent use of this glutamine nitrogen for urea synthesis in periportal hepatocytes, which could occur either in incubated hepatocytes or in a recirculating perfusionwhich could confound the interpretation.

When 15NH3 is provided as substrate the urea formed may have a mass of 60, 61, or 62, depending on whether zero, one, or two 15N atoms are incorporated. This, in turn, depends on the enrichment of 15N in the two relevant nitrogen pools, the mitochondrial ammonia pool and the cytoplasmic aspartate pool.

We present here a theoretical scheme that predicts the proportions of these three isotopomers of urea as a function of the 15N enrichment and an experimental means of determining the actual 15N enrichment of these pools.

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We have also considered the synthesis of glutamine isotopomers. When synthesized in the presence of 15NH3 four separate glutamine mass and positional isotopomers are produced, i. We present here a theoretical scheme that predicts the proportions of these four isotopomers of glutamine as a function of the 15N enrichment in the precursor pools, i.

Perfusion flow rate, pH, pCO2, and pO2 in influent and effluent media were monitored throughout, and oxygen consumption was calculated. After 15 min of perfusion, we changed to a medium containing 15NH4Cl final concentration, 0.

mass isotopomer pattern and precursor product relationship test

Separate perfusate reservoirs, each containing ammonia of different 15N enrichment, were employed to facilitate changes in perfusion media. Samples were taken from the influent and effluent media for chemical and GC-MS analyses. At the end of the perfusions livers were freeze-clamped with aluminum tongs precooled in liquid N2, the frozen livers were ground into a fine powder and extracted into perchloric acid, and the extracts were used for the analysis of adenine nucleotides by enzymatic techniques Urea and ammonia concentrations in the perfusion media were assayed by standard methods 13 Amino acid concentrations were determined by HPLC, utilizing precolumn derivatization with o-phthaldehyde A few perfusions were carried out to determine the rate of glutamine production due to proteolysis.

For these experiments the rats were pretreated with the glutamine synthetase inhibitor, methionine sulfoxamine, and this was included in the perfusate 6. The columns were washed with 3 ml of deionized water.

Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer mass spectrum. Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer.

Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically increases certainty that the analyte of interest is in the sample. The target analytes are extracted and mixed with water and introduced into an airtight chamber.

Mass isotopomer pattern and precursor-product relationship.

An inert gas such as Nitrogen N2 is bubbled through the water; this is known as purging or sparging. The volatile compounds move into the headspace above the water and are drawn along a pressure gradient caused by the introduction of the purge gas out of the chamber. The volatile compounds are drawn along a heated line onto a 'trap'.

The trap is a column of adsorbent material at ambient temperature that holds the compounds by returning them to the liquid phase. The trap is then heated and the sample compounds are introduced to the GC-MS column via a volatiles interface, which is a split inlet system. In this system the inert gas is bubbled through the water until the concentrations of organic compounds in the vapor phase are at equilibrium with concentrations in the aqueous phase.

The gas phase is then analysed directly. Another relatively common detector is the ion trap mass spectrometer. Additionally one may find a magnetic sector mass spectrometer, however these particular instruments are expensive and bulky and not typically found in high-throughput service laboratories. Other detectors may be encountered such as time of flight TOFtandem quadrupoles MS-MS see belowor in the case of an ion trap MSn where n indicates the number mass spectrometry stages.

The first quadrupole Q1 is connected with a collision cell Q2 and another quadrupole Q3. Types of analysis include product ion scan, precursor ion scan, selected reaction monitoring SRM sometimes referred to as multiple reaction monitoring MRM and neutral loss scan. When Q1 is in static mode looking at one mass only as in SIMand Q3 is in scanning mode, one obtains a so-called product ion spectrum also called "daughter spectrum".

From this spectrum, one can select a prominent product ion which can be the product ion for the chosen precursor ion. The pair is called a "transition" and forms the basis for SRM. SRM is highly specific and virtually eliminates matrix background.

mass isotopomer pattern and precursor product relationship test

Ionization[ edit ] After the molecules travel the length of the column, pass through the transfer line and enter into the mass spectrometer they are ionized by various methods with typically only one method being used at any given time. Once the sample is fragmented it will then be detected, usually by an electron multiplierwhich essentially turns the ionized mass fragment into an electrical signal that is then detected.

The ionization technique chosen is independent of using full scan or SIM. Block diagram for gas chromatography using electron ionization for collecting mass spectrum. Electron ionization[ edit ] By far the most common and perhaps standard form of ionization is electron ionization EI. The molecules enter into the MS the source is a quadrupole or the ion trap itself in an ion trap MS where they are bombarded with free electrons emitted from a filament, not unlike the filament one would find in a standard light bulb.

The electrons bombard the molecules, causing the molecule to fragment in a characteristic and reproducible way. Hard ionization is considered by mass spectrometrists as the employ of molecular electron bombardment, whereas "soft ionization" is charge by molecular collision with an introduced gas. The molecular fragmentation pattern is dependent upon the electron energy applied to the system, typically 70 eV electron Volts.

Spectral library searches employ matching algorithms such as Probability Based Matching [10] and dot-product [11] matching that are used with methods of analysis written by many method standardization agencies. Cold electron ionization[ edit ] The "hard ionization" process of electron ionization can be softened by the cooling of the molecules before their ionization, resulting in mass spectra that are richer in information.

Collisions with the make up gas at the expanding supersonic jet reduce the internal vibrational and rotational energy of the analyte molecules, hence reducing the degree of fragmentation caused by the electrons during the ionization process.

The enhanced molecular ions increase the identification probabilities of both known and unknown compounds, amplify isomer mass spectral effects and enable the use of isotope abundance analysis for the elucidation of elemental formulae. Chemical ionization In chemical ionization a reagent gas, typically methane or ammonia is introduced into the mass spectrometer. Depending on the technique positive CI or negative CI chosen, this reagent gas will interact with the electrons and analyte and cause a 'soft' ionization of the molecule of interest.

A softer ionization fragments the molecule to a lower degree than the hard ionization of EI. One of the main benefits of using chemical ionization is that a mass fragment closely corresponding to the molecular weight of the analyte of interest is produced.

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In positive chemical ionization PCI the reagent gas interacts with the target molecule, most often with a proton exchange. This produces the species in relatively high amounts.

In negative chemical ionization NCI the reagent gas decreases the impact of the free electrons on the target analyte. This decreased energy typically leaves the fragment in great supply. Analysis[ edit ] A mass spectrometer is typically utilized in one of two ways: The typical GC-MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup of the particular instrument.

The primary goal of instrument analysis is to quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum.

mass isotopomer pattern and precursor product relationship test

Two kinds of analysis are possible, comparative and original. Comparative analysis essentially compares the given spectrum to a spectrum library to see if its characteristics are present for some sample in the library. This is best performed by a computer because there are a myriad of visual distortions that can take place due to variations in scale.

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Computers can also simultaneously correlate more data such as the retention times identified by GCto more accurately relate certain data. Deep learning was shown to lead to promising results in the identification of VOCs from raw GC-MS data [18] Another method of analysis measures the peaks in relation to one another.

The total mass of the unknown compound is normally indicated by the parent peak.