Determination of leucine flux in vivo by gas chromatography—mass spectrometry utilizing stable isotopes for trace and internal standard

A simple and reliable method is described for the determination of leucine flux in vivo using two stable isotopes of leucine and gas chromatography—mass spectrometry (GC---MS). [6,6,6-²H₃]Leucine is administered as a primed-dose constant infusion in vivo and -[²H₇]leucine is added to plasma as an internal standard. Plasma leucine concentration and moles per cent enrichment of [²H₃]leucine can be determined simultaneously by GC---MS and selected ion monitoring. Leucine flux calculated from the [6,6,6-²H₃]leucine data was nearly identical to that obtained with -[U-¹⁴C]leucine in dogs. This method is readily applicable to the study of leucine metabolism in humans of all ages and laboratory animals.     

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein‐by‐protein basis. The ability to measure protein synthesis and degradation rates on a proteome‐wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well‐established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L‐leucine was replaced with a stable isotope labelled analogue ([²H₇]L‐leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.     

An approach to the differentiation of leucine and isoleucine residues in EI mass spectra of peptides

Residues of leucine and isoleucine cannot generally be distinguished in the electron impact (EI) generated mass spectra of N-acylated peptide esters. We have obtained the mass spectra of model peptide esters containing leucine or isoleucine in various positions and trifluoroacetyl perdeutero leucine as the N-terminal blocking group. The mass spectra of the peptide derivatives show a pair of peaks as a result of the elimination from the M⁺ ion of neutral fragment of perdeuterated isobutene (M⁺-64) from the leucine side chain of the N-terminal blocking group and isobutene or butene (M⁺-56) from leucine or isoleucine residues of the peptide. The ratios of the intensities of the peaks $\text{M}{}^{\mathrm{+}}\text{-56}\text{M}{}^{\mathrm{+}}\text{-64}$ show considerable variation with the position of leucine or isoleucine in the peptide chain and the length of the peptide, but for peptides which are identical except for the fact that one contains leucine and the other isoleucine in a given position the ratio is always smaller for the isoleucine containing peptide. The differences are sufficient to distinguish the isomeric residues if comparison spectra are available.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

Nutrient absorption,storage and remobilization in octopus vulgaris

Average absorption and conversion to ¹⁴CO₂for free leucine included in a meal were 96% and 30% after 24 h. The values for glucose were 98% and 48% and for palmitate 46% and 12.5%. Muscle was the major repository of leucine (38% of the total ingested) and glucose (44%), but the digestive gland contained most of the palmitate (20%).

During normal feeding ¹⁴CO₂ production from octopuses given leucine dropped to low stable levels after only 2 days, those given glucose required 4 to 5, but palmitate apparently did not enter a stable reserve. Fasting increased the release of ¹⁴CO₂ from octopuses given palmitate and leucine, but glucose animals showed little change. A 5‐fold increase in ¹⁴CO₂ production during forced exercise after fasting by octopuses given glucose may indicate increased carbohydrate catabolism. Reduced ¹⁴CO₂production in exercise after other substrates is consistent with this, suggesting muscle carbohydrate reserves may be exclusively for locomotion.

A comparison of nutrient uptake, catabolism and growth suggests that lipids are the limiting nutrient for octopuses on a crab diet.     

Simultaneous determination of the enantiomers of leucine and [2H7]leucine in plasma by capillary gas chromatography–mass spectrometry

A method for the stereoselective assay of d- and l-enantiomers of both leucine and [²H₇]leucine in rat plasma was developed using gas chromatography–mass spectrometry–selected-ion monitoring. dl-[²H₃]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.     

Labeling of Bifidobacterium longum Cells with 13C-Substituted Leucine for Quantitative Proteomic Analyses

Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [¹³C₆]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence.     

Relative Stability of Membrane Proteins in Escherichia coli

The relative stability of membrane proteins in Escherichia coli was investigated to determine whether these proteins are degraded at heterogeneous rates and, if so, whether the degradative rates are correlated with the sizes or charges of the proteins. Cells growing in a glucose-limited chemostat with a generation time of 15 h were labeled with [¹⁴C]leucine. After allowing 24 h for turnover of ¹⁴C-labeled proteins, the cells were labeled for 15 min with [³H]leucine. By this protocol, the rapidly degraded proteins have a high ratio of ³H to ¹⁴C, whereas the stable proteins have a lower ratio. The total cell envelope fraction was collected by differential centrifugation, and the proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The relative ratio for each protein was determined by dividing its ³H/¹⁴C ratio by the ³H/¹⁴C ratio of the total membrane fraction. Although most of the 125 membrane proteins had relative ratios close to the average for the total membrane fraction, 19 varied significantly from this value. These differences were also observed when the order of addition of [¹⁴C]leucine and [³H]leucine was reversed. In control cultures labeled simultaneously with both isotopes, the relative ratios of these 19 proteins were similar to that of the total membrane fraction. Thirteen of these proteins had low relative ratios, which suggested that they were more stable than the average protein. An experiment in which the normal labeling procedure was followed by a 60-min chase period in the presence of excess unlabeled leucine suggested that the low relative ratios of 3 of these 13 proteins may be due to a slow post-translational modification step. Six membrane proteins had high relative ratios, which indicated that they were degraded rapidly. In contrast to the relationships found for soluble proteins in mammalian cells, there were no strong correlations between the degradative rates and either the isoelectric points or the molecular weights of membrane proteins in E. coli.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins

A bioassay to measure the incorporation of [¹⁴C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [¹⁴C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [¹⁴C]leucine uptake. The effectiveness of the wall fragments in inhibiting [¹⁴C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

Effect of Jasmonic, Salicylic, and Abscisic Acids on [14C]Leucine Incorporation into Proteins of Pea Leaves

All investigated exogenous phytohormones (jasmonic, salicylic, and abscisic acids) induced the appearance of ¹⁴C-label in a polypeptide with molecular mass 29 kD that was not found in the control; these acids also increased [¹⁴C]leucine incorporation into a 25-kD polypeptide and decreased such incorporation into a 45-kD polypeptide. This can be considered as a nonspecific response of the plants to the action of these hormones. Salicylic and abscisic (but not jasmonic) acids induced the synthesis of a 19-kD polypeptide, and jasmonate induced the synthesis of a 96-kD polypeptide.     

Leucine enkephalin-tyrosinase reaction products — Identification and biological activity

Leucine enkephalin (1 mM) was reacted with mushroom tyrosinase under reductive conditions (ascorbic acid, 50 mM). Reaction products were isolated by high-performance liquid chromatography and identified using electrospray ionization mass spectrometry. The products of the reaction were found to be hydroxylated at the Tyr¹ moiety of the peptide. The major product was a monohydroxylated derivative of leucine enkephalin ([HO-Tyr¹]LE) and the minor product of the reaction was a dihydroxylated derivative ([(HO)₂-Tyr¹]LE). The affinity of [HO-Tyr¹]LE to receptors in rat brain homogenate was compared to that of leucine enkephalin itself. Hydroxylation of LE was found to decrease receptor affinity to both μ and δ opioid receptor sites by a factor of about 20.     

Biochemical differentiation of lone star tick,Amblyomma americanum (L), salivary glands: Effects of attachment,feeding and mating

Salivary glands of female Amblyomma americanum (L.) are stimulated to differentiate by attachment to a host, subsequent feeding and mating. Incorporation of [³H]uridine into ribosomal and transfer RNAs as well as the synthesis of poly(A⁺)mRNA and protein parallel the pattern of increasing enzymatic activity and secretory ability of the glands. Unfed ticks contained 3.5 ± 0.47 ng poly(A⁺)mRNA/gland pr. By the second day of feeding this had increased more than 5-fold. The greatest amount of poly(A⁺)mRNA found in rapid-feeding phase females (body wt > 100 mg) was 370 ± 80 ng/gland pr. Poly(A⁺)mRNA mass doubles on the final day of feeding, just as the ticks exceeded 100 mg in wt. Ticks attached 1 to 10 days had increasingly greater amounts of salivary monosomes, 60 and 40S ribosomal subunits, and polysomes. Polysomal mass/gland pr also attained its maximum above 100 mg tick wt at the slow/rapid-feeding phase boundary; exceeding by 20 times that of unfed ticks. Degenerating glands from replete ticks continued to synthesize protein. In vitro incorporation of [³H]leucine was greatest within 24 h of attachment. Fluorographs of [³H]leucine labeled protein showed that mating caused a drop in incorporation after the 4th day of feeding. Glands from unmated females attached the same number of days continued to incorporate [³H]leucine at higher levels than those from mated females.     

LEUCINE OXIDATION IN BRAIN SLICES AND NERVE ENDINGS1

—It is generally believed that leucine serves primarily as a precursor for protein synthesis in the central nervous system. However, leucine is also oxidized to CO₂ in brain. The present investigation compares leucine oxidation and incorporation into protein in brain slices and synaptosomes. In brain slices from adult rats, these processes were linear for 90min and ¹⁴CO₂ production from 0·1 mm -l -[l-¹⁴C]leucine was 23 times more rapid than incorporation into protein. The rate of oxidation increased further with greater leucine concentrations. Experiments with l -[U-¹⁴C]leucine suggested that all of the carbons from leucine were oxidized to CO₂ with very little incorporation into lipid. Oxidation of leucine also occurred in synaptosomes. In slices, leucine oxidation and incorporation into protein were inhibited by removal of glucose or Na⁺, or addition of ouabain. In synaptosomes, replacement of Na⁺ by choline also reduced leucine oxidation; and this effect did not appear to be due to inhibition of leucine transport. The rate of leucine oxidation did not change in brain slices prepared from fasted animals. Fasting, however, reduced the incorporation of leucine into protein in brain slices prepared from young but not from adult rats. These findings indicate that oxidation is the major metabolic fate of leucine in brain of fed and fasted animals.     

Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP⁺-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean

To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. `Wells'), ¹⁴C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with ¹⁴C-sucrose and ¹⁴CO₂ assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.