Applications of Stable Isotope-Labeled Compounds in In Vivo Metabolic Studies
Applications of Stable Isotope-Labeled Compounds in In Vivo Metabolic Studies
Stable isotope-labeled compounds use ²H, ¹³C, ¹⁵N, or ¹⁸O to label metabolic substrates, allowing their transformation and fate in vivo to be identified by mass spectrometry or nuclear magnetic resonance. This technology can be used to determine substrate sources, metabolic pathways, interorgan exchange, and the synthesis and turnover of biological materials.
Keywords: stable isotopes; in vivo metabolic tracing; ¹³C; ¹⁵N; ²H; ¹⁸O; metabolic flux
1 Basic Principles of Stable Isotope Tracing
1.1 Research Value of Stable Isotope Tracing
Conventional metabolomics primarily measures metabolite abundance at specific time points. Although it can identify changes in metabolite levels, concentration alone cannot readily determine metabolite sources or metabolic direction. An increase in the concentration of a metabolite may result from enhanced synthesis, restricted degradation, or reduced export. By detecting the transfer of labeled atoms among different metabolites, stable isotope tracing extends metabolic research from static abundance comparisons to analyses of substrate origin, pathway connectivity, and metabolic flow.
1.2 Tracers, Tracees, and Isotopic Enrichment
(1) Tracers and tracees
A tracer is an exogenous molecule containing a stable heavy isotope, whereas a tracee is the corresponding unlabeled endogenous molecule with the same chemical structure. Labeled and unlabeled molecules have similar chemical properties and can enter the same metabolic networks. They can nevertheless be distinguished by differences in molecular mass, allowing the transfer of specific atoms into downstream metabolites to be recorded.
(2) Isotopic enrichment
Isotopic enrichment represents the proportion of labeled molecules within a target metabolite pool. After an exogenous tracer enters the body, it mixes with the endogenous unlabeled substrate and is progressively diluted or transferred during metabolism. Measuring the labeled fractions of different metabolites in blood and tissues allows the assessment of tissue substrate uptake, metabolic conversion, and clearance.
1.3 Isotopologues and Isotopomers
(1) Isotopologues
Isotopologues are distinguished by the number of heavy-isotope atoms within a molecule. For a six-carbon metabolite traced with ¹³C, M+0 indicates that no tracer-derived ¹³C is present, whereas M+1 to M+6 indicate the presence of one to six ¹³C atoms, respectively. The relative proportions of different M+n peaks constitute the mass isotopologue distribution (MID), which reflects the extent to which a labeled substrate enters downstream metabolites.
(2) Isotopomers
Isotopomers contain the same number of heavy-isotope atoms, but the labeled atoms occupy different positions. For example, two M+2 metabolites may carry ¹³C at different carbon positions, representing distinct atomic transfer pathways. Label positions generally require determination by tandem mass spectrometry, nuclear magnetic resonance, or carbon-skeleton derivatization analysis.
1.4 Common Stable Isotopes
Table 1 Stable Isotopes Commonly Used in In Vivo Metabolic Studies
Isotope | Commonly Labeled Substrates | Main Tracing Information | Application Characteristics |
²H | Heavy water, deuterated glucose, fatty acids, and drugs | Biosynthesis, molecular turnover, and substrate kinetics | Suitable for synthesis and turnover studies over relatively long timescales |
¹³C | Glucose, lactate, glutamine, acetate, and fatty acids | Carbon-skeleton transfer, pathway connectivity, and carbon-source contribution | Broadly applicable and suitable for central carbon metabolism studies |
¹⁵N | Amino acids, glutamine, ammonium salts, and urea | Transamination, nitrogen transfer, the urea cycle, and nucleotide synthesis | Suitable for amino acid and nitrogen-containing metabolism studies |
¹⁸O | Heavy-oxygen water, labeled oxygen, and oxygen-containing metabolites | Oxygen-atom incorporation, water exchange, and hydrolysis reactions | Suitable for studying specific reactions involving water or oxygen |
2 Labeling Strategies for Stable Isotope Tracers
2.1 Uniformly Labeled Tracers
(1) Labeling characteristics
Uniformly labeled tracers contain stable isotopes at all corresponding positions of the target element, such as [U-¹³C₆]glucose, [U-¹³C₅]glutamine, and [U-¹³C₁₆]palmitate. These tracers produce strong labeling signals and provide broad coverage of downstream pathways.
(2) Main uses
Uniformly labeled tracers can simultaneously transfer labeled atoms into multiple downstream metabolic branches. They are suitable for examining the overall distribution of substrates into metabolic pools associated with glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, amino acids, nucleotides, and lipids and are commonly used to construct comprehensive metabolic flow maps.
2.2 Position-Specific Labeled Tracers
(1) Labeling characteristics
Position-specific tracers contain a heavy isotope only at a predefined atomic position. When the labeled carbon is released during a decarboxylation step or enters a specific product after carbon-skeleton cleavage, downstream metabolites form pathway-characteristic labeling patterns.
(2) Main uses
Glucose labeled at different carbon positions can be used to analyze carbon-flow distribution among the oxidative pentose phosphate pathway, nonoxidative pentose phosphate pathway, and glycolysis. Separate administration of [1,2,3-¹³C₃]fructose and [4,5,6-¹³C₃]fructose can also reveal differences in how carbon atoms from the first and second halves of fructose enter glycerol, lactate, glucose, and other organic acids.
2.3 Dual-Element Labeling
(1) Carbon-skeleton tracing
The ¹³C component of ¹³C/¹⁵N dual-labeled amino acids is used to trace carbon-skeleton transformation. In glutamine, for example, the five ¹³C atoms can sequentially enter glutamate, α-ketoglutarate, and the tricarboxylic acid cycle, allowing the analysis of glutaminolysis, tricarboxylic acid cycle anaplerosis, and reductive carboxylation.
(2) Nitrogen-group tracing
The ¹⁵N component of dual-labeled amino acids can be used to analyze transamination, nucleotide synthesis, and nitrogen excretion. Glutamine contains amino and amide nitrogen atoms that participate in different reactions. Amide-position-specific ¹⁵N-labeled glutamine is more suitable for studying amide-nitrogen transfer during nucleotide synthesis.
2.4 Water Tracing
(1) ²H₂O tracing
After entering the body, ²H₂O rapidly mixes with the total body water pool. Through enzymatic reactions, ²H is incorporated into nonexchangeable positions in newly synthesized fatty acids, cholesterol, nucleotides, and proteins. It is suitable for studying lipid synthesis, cell proliferation, and protein turnover.
(2) H₂¹⁸O tracing
H₂¹⁸O can be used to investigate the incorporation of water-derived oxygen atoms, specific hydrolysis reactions, and phosphate-related reactions. Unlike ¹³C carbon-skeleton tracing, H₂¹⁸O primarily indicates whether oxygen atoms from water participate in a target reaction.
2.5 Principles of Tracer Selection
Table 2 Stable Isotope Tracing Strategies for Different Research Questions
Research Question | Preferred Tracing Strategy | Main Observations |
Whether a substrate enters multiple downstream metabolic pools | Uniformly ¹³C-labeled substrate | Overall labeling distribution in downstream metabolites |
Relative contribution of two pathways to the same product | Position-specific ¹³C tracing | Characteristic M+n peaks and label positions |
Coordinated carbon and nitrogen metabolism of amino acids | ¹³C/¹⁵N dual-labeled amino acids | Transfer of carbon skeletons and nitrogen-containing groups |
Lipid or protein synthesis | ²H₂O or labeled precursors | Isotope incorporation into newly synthesized products |
Appearance and clearance of circulating substrates | Continuous infusion of labeled substrates | Precursor enrichment and its dynamic changes |
Rapid tissue uptake of substrates | Bolus or short-term administration of labeled substrates | Rise and decay of labeling signals |
3 Major Applications of Stable Isotopes in In Vivo Metabolic Studies
3.1 Glucose and Central Carbon Metabolism
(1) Glycolysis and the lactate cycle
[U-¹³C₆]glucose produces M+3 pyruvate and M+3 lactate through glycolysis. Labeled lactate can enter the circulation and be taken up by the liver, heart, skeletal muscle, and other tissues for gluconeogenesis or oxidative metabolism. Comparing the isotopic distributions of glucose, pyruvate, lactate, and tricarboxylic acid cycle intermediates in plasma and tissues can reveal the distribution of glucose-derived carbon among glycolysis, the lactate cycle, and oxidative metabolism.
(2) Pentose phosphate pathway
Glucose C1 can be released during the decarboxylation step of the oxidative pentose phosphate pathway, whereas other carbon positions exhibit distinct retention and rearrangement patterns. Comparing lactate, ribose-5-phosphate, and other downstream products generated from [1-¹³C]glucose, [2-¹³C]glucose, or multiply labeled glucose can reveal the relative contributions of the oxidative and nonoxidative pentose phosphate pathways.
(3) Tricarboxylic acid cycle
When glucose-derived pyruvate is converted into acetyl-CoA by pyruvate dehydrogenase, labeled products such as M+2 citrate can be formed during the first turn of the tricarboxylic acid cycle. When pyruvate is converted into oxaloacetate by pyruvate carboxylase, isotopologues such as M+3 malate, aspartate, and citrate can be generated. These distinct labeling patterns can be used to analyze carbon-flow distribution between pyruvate oxidation and tricarboxylic acid cycle anaplerosis.
3.2 Glutamine, Amino Acid, and Nitrogen Metabolism
(1) Glutaminolysis
[U-¹³C₅]glutamine is converted by glutaminase into M+5 glutamate and subsequently into M+5 α-ketoglutarate through glutamate dehydrogenase or transamination reactions. After the labeled carbon is oxidized through the tricarboxylic acid cycle, M+4 isotopologues can be formed in succinate, fumarate, malate, and aspartate, allowing the contribution of glutamine-derived carbon to the tricarboxylic acid cycle to be evaluated.
(2) Reductive carboxylation
Under hypoxia, electron transport chain dysfunction, or restricted mitochondrial oxidative metabolism, α-ketoglutarate can undergo reductive carboxylation to generate isocitrate and citrate, providing carbon for lipid synthesis. [U-¹³C₅]glutamine can generate M+5 citrate and subsequently produce corresponding labeling in fatty acids, allowing glutamine-dependent reductive carboxylation to be analyzed.
(3) Transamination and nitrogen-containing metabolism
¹⁵N-labeled glutamine, glutamate, glycine, or arginine can be used to analyze nitrogen transfer into amino acids, purines, pyrimidines, and the urea cycle. The amide nitrogen of glutamine can enter purine and pyrimidine bases, the amino nitrogen of glutamate can enter aspartate and alanine through transamination, and labeled nitrogen from arginine can enter urea, nitric oxide, and ornithine-related metabolites.
3.3 Fatty Acid Oxidation and Lipid Synthesis
(1) Fatty acid uptake and β-oxidation
[U-¹³C₁₆]palmitate or [U-¹³C₁₈]oleate can be used to trace tissue fatty acid uptake, β-oxidation, re-esterification, and interorgan transport. Each round of β-oxidation releases M+2 acetyl-CoA, allowing the label to enter the tricarboxylic acid cycle and ketone bodies. The incorporation of intact labeled fatty acids into triglycerides, phospholipids, or cholesteryl esters reflects fatty acid re-esterification and lipid storage.
(2) De novo fatty acid synthesis
[U-¹³C₆]glucose and [¹³C₂]acetate can enter fatty acid synthesis through acetyl-CoA, generating newly synthesized fatty acids with M+2-based mass increments. These tracers are suitable for analyzing the carbon sources used for lipid synthesis. Through the total body water pool, ²H₂O supplies hydrogen to multiple positions during fatty acid synthesis and can be used to determine the proportion of newly synthesized fatty acids over a defined period.
(3) Cholesterol synthesis and turnover
¹³C-labeled acetate or glucose can be used to analyze the contribution of acetyl-CoA-derived carbon to the sterol skeleton. ²H₂O can be used to determine the proportion of newly synthesized cholesterol, whereas labeled cholesterol can be used to investigate plasma lipoprotein transport, tissue uptake, esterification, and clearance.
3.4 Interorgan Metabolism
Orally ingested nutrients may first be metabolized in the gastrointestinal tract and liver, producing lactate, glucose, amino acids, and other circulating metabolites that are subsequently used by distal tissues. Comparing isotopic distributions in the systemic circulation, organ-draining veins, and different tissues can reveal substrate exchange among the liver, small intestine, skeletal muscle, adipose tissue, and heart and identify the indirect entry of an original tracer into target tissues through circulating metabolites.
3.5 In Vivo Tumor Metabolism
Tumor tissues can use glucose, lactate, glutamine, acetate, and fatty acids to support energy production and biosynthesis. Administration of the corresponding ¹³C-labeled substrates to tumor-bearing animals, followed by comparison of isotopic distributions among tumors, adjacent normal tissues, and circulating metabolites, can reveal tumor substrate selection, tricarboxylic acid cycle anaplerosis, reductive carboxylation, nucleotide synthesis, and lipid synthesis.
3.6 Biological Macromolecule Synthesis and Turnover
Incorporation of labeled amino acids into proteins can be used to analyze protein synthesis and degradation. Incorporation of ²H₂O into nonexchangeable positions in deoxyribose, fatty acids, or proteins can be used to investigate cell proliferation, lipogenesis, and protein turnover. Labeled glucose can also be used to analyze glycogen and ribose synthesis, whereas labeled fatty acids and glycerol can be used to investigate triglyceride and phospholipid renewal.
3.7 Metabolic Diseases and Drug Effects
In metabolic diseases such as diabetes, obesity, and fatty liver disease, stable isotope tracing can be used to investigate hepatic glucose output, peripheral glucose utilization, fatty acid oxidation, lipid synthesis, and triglyceride turnover. Comparing substrate contributions, labeling patterns, and turnover rates before and after intervention can determine whether a drug changes the utilization or pathway allocation of a specific substrate and distinguish whether changes in metabolite abundance arise from synthesis, degradation, uptake, or clearance.
4 Detection and Interpretation of Stable Isotope Signals
4.1 Detection of Stable Isotope Signals
Stable isotopes are nonradioactive, and their tracer signals are primarily detected by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Mass spectrometry distinguishes unlabeled metabolites from metabolites with different degrees of labeling according to changes in molecular mass and provides high sensitivity and broad detection coverage. NMR provides positional information about labeled atoms and is suitable for analyzing carbon-skeleton rearrangements and specific metabolic reactions.
4.2 Isotopologue Labeling Patterns
The relative proportions of different M+n peaks collectively constitute the mass isotopologue distribution and can reflect the extent to which a labeled substrate enters downstream metabolites and how carbon or nitrogen skeletons are transferred. Raw results generally require correction for natural isotope abundance and should be analyzed together with tracer purity, precursor enrichment, metabolite abundance, and sampling time.
4.3 Basic Interpretation of In Vivo Tracing Results
The presence of labeling in a downstream metabolite indicates that atoms from the tracer substrate have entered that metabolite, but the labeled fraction cannot be directly equated with metabolic flux. Interorgan exchange and circulating metabolism occur in vivo; for example, labeled glucose may first be converted into labeled lactate and subsequently taken up by other tissues. Label origin, pathway contribution, and metabolic flux can only be evaluated further by integrating precursor enrichment, metabolite abundance, dynamic sampling, and appropriate metabolic models.
5 Products Related to Stable Isotope Tracing
5.1 Products Related to Tracing Glucose Metabolism, the Tricarboxylic Acid Cycle, and Short-Chain Carbon Sources
Catalog # | Product Name | Grade & Purity | Labeling Form and Tracing Position | Research Application |
D-Glucose-¹³C₆ | ≥99 atom% 13C,≥98% | All six carbon positions are labeled with ¹³C; glycolysis generates M+3 pyruvate and lactate, and entry of pyruvate into the tricarboxylic acid cycle through acetyl-CoA produces the corresponding M+n isotopologues | Glucose carbon-source contribution and central carbon metabolism | |
D-Glucose-¹³C₆,1,2,3,4,5,6,6-d₇ | ≥99 atom% 13C,≥97 atom% D,≥98% | All six carbon positions are labeled with ¹³C, while hydrogen atoms at positions 1, 2, 3, 4, 5, 6, and 6 are labeled with ²H; carbon-skeleton and hydrogen-isotope signals can be detected separately, although some ²H may undergo exchange during metabolism | Dual carbon–hydrogen isotope methodology | |
D-Glucose-1-¹³C | ≥99 atom% 13C | The C1 position of glucose is labeled with ¹³C; when this carbon enters the oxidative pentose phosphate pathway, it can be released as ¹³CO₂ during decarboxylation | Contribution of the oxidative pentose phosphate pathway | |
D-Glucose-2-¹³C | ≥99 atom% 13C,≥98% | The C2 position of glucose is labeled with ¹³C; this carbon exhibits distinct retention and rearrangement patterns in glycolysis and the pentose phosphate pathway | Discrimination of glucose pathway branches | |
Sodium pyruvate-¹³C₃ | Moligand™, ≥99 atom% 13C,≥98% | All three carbon positions of pyruvate are labeled with ¹³C; lactate and alanine appear as M+3, acetyl-CoA as M+2, and oxaloacetate and its derivatives may appear as M+3 | Pyruvate oxidation and anaplerosis | |
Sodium pyruvate-1-¹³C | ≥99 atom%,≥98% | The C1 carboxyl carbon of pyruvate is labeled with ¹³C; after decarboxylation by pyruvate dehydrogenase, the label enters the ¹³CO₂ and bicarbonate pools | Pyruvate decarboxylation and oxidative metabolism | |
Sodium acetate-¹³C₂ | ≥99 atom% 13C | Both carbon positions of acetate are labeled with ¹³C; conversion produces M+2 acetyl-CoA, which transfers the two-carbon label to citrate, fatty acids, and cholesterol | Acetate-derived carbon and acetyl-CoA utilization | |
Sodium acetate-1-(¹³C) | ≥99 atom% 13C | The C1 carboxyl carbon of acetate is labeled with ¹³C; after entry into acetyl-CoA, it corresponds to the carbonyl carbon of the acetyl group and provides positional complementarity with C2-labeled acetate | Acetate oxidation and decarboxylation | |
Sodium acetate-2-¹³C | ≥99 atom% 13C,≥98% | The C2 methyl carbon of acetate is labeled with ¹³C; after entry into acetyl-CoA, it corresponds to the methyl carbon of the acetyl group and provides positional complementarity with C1-labeled acetate | Acetate-supported biosynthesis | |
Glycerol-1,3-¹³C₂ | ≥99 atom% 13C | The C1 and C3 positions of glycerol are labeled with ¹³C; after conversion through glycerol-3-phosphate, the label enters dihydroxyacetone phosphate or is retained in the glycerolipid backbone | Gluconeogenesis and glycerolipid synthesis | |
Glycerol-¹³C₃, d₈ | ≥98 atom% D,≥99 atom% 13C | All three carbon positions of glycerol are labeled with ¹³C and the molecule contains eight ²H atoms; ¹³C is retained in the glycerol carbon skeleton, whereas some ²H is transferred or lost through redox and exchange reactions | Glycerol turnover and hydrogen-exchange studies | |
L-Lactic acid-3-13C, 99 atom % 13C, 98% (CP) | ≥99 atom% | The C3 methyl carbon of lactate is labeled with ¹³C; after conversion into pyruvate by lactate dehydrogenase, the label is retained at pyruvate C3 and in its downstream carbon skeletons | Lactate cycling and gluconeogenesis |
5.2 Products Related to Tracing Amino Acids, Nitrogen Metabolism, and Protein Turnover
Catalog # | Product Name | Grade & Purity | Labeling Form and Tracing Position | Research Application |
L-Glutamine-¹³C₅ | Moligand™, ≥98 atom% 13C,≥95% | All five carbon positions are labeled with ¹³C; glutamate and α-ketoglutarate appear as M+5, oxidative four-carbon intermediates as M+4, and citrate generated through reductive carboxylation as M+5 | Glutaminolysis and reductive carboxylation | |
L-Glutamine-¹³C₅,¹⁵N₂ | ≥98% | All five carbon positions are labeled with ¹³C, and both the amino and amide nitrogen atoms are labeled with ¹⁵N; downstream products may separately retain the carbon skeleton, amino nitrogen, or amide nitrogen signals | Coupled glutamine carbon and nitrogen metabolism | |
L-Glutamine-(amide-¹⁵N) | ≥98 atom% 15N,≥98% | Only the side-chain amide nitrogen of glutamine is labeled with ¹⁵N; glutaminase transfers it into the ammonia pool, whereas amidotransferase reactions transfer it into nucleotides and other products | Analysis of amide-nitrogen sources in nucleotide synthesis | |
L-Glutamic acid-¹³C₅ | ≥98 atom% 13C,≥95% | All five carbon positions are labeled with ¹³C; α-ketoglutarate appears as M+5, four-carbon tricarboxylic acid cycle intermediates appear as M+4 after oxidation, and GABA can retain four labeled carbons | Glutamate catabolism and GABA metabolism | |
L-Glutamic acid-1-¹³C | ≥99 atom% 13C,≥98% | The C1 carboxyl carbon of glutamate is labeled with ¹³C; during glutamate decarboxylation or oxidative decarboxylation of α-ketoglutarate, this carbon is released as ¹³CO₂ | Glutamate decarboxylation studies | |
L-Glutamic acid-¹³C₅,¹⁵N | ≥98 atom% 13C,≥98 atom% 15N,≥95% | All five carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; after transamination, ¹⁵N enters the acceptor amino acid, whereas the ¹³C carbon skeleton enters α-ketoglutarate and its downstream products | Transamination and carbon–nitrogen allocation | |
L-Serine-¹³C₃,¹⁵N | ≥99 atom% 13C,≥99 atom% 15N,≥98% | All three carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; serine conversion can generate M+2 glycine, ¹³C-labeled one-carbon units, and corresponding ¹⁵N-containing products | Serine–glycine–one-carbon metabolism | |
L-Serine-3-¹³C | ≥99 atom% 13C | The C3 hydroxymethyl carbon of serine is labeled with ¹³C; after the serine hydroxymethyltransferase reaction, this carbon enters tetrahydrofolate-bound one-carbon units | One-carbon unit generation and utilization | |
Glycine-15N | ≥99 atom%,≥98.5% | The amino nitrogen of glycine is labeled with ¹⁵N; this nitrogen enters purines, porphyrins, creatine, and other nitrogen-containing products through the corresponding biosynthetic reactions | Contribution of glycine-derived nitrogen | |
Glycine-¹³C₂,¹⁵N | ≥99 atom% 13C,≥98 atom% 15N | Both carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; intact glycine incorporation and separate transfer of carbon and nitrogen atoms produce distinct isotope combinations | Coordinated glycine carbon and nitrogen metabolism | |
L-Proline-¹³C₅,¹⁵N | ≥98 atom% 13C,≥98 atom% 15N,≥95% | All five carbon positions are labeled with ¹³C, and the ring nitrogen is labeled with ¹⁵N; after oxidation, the labels enter pyrroline-5-carboxylate, glutamate, and α-ketoglutarate, while the intact molecule can also be incorporated into proteins | Proline oxidation and collagen turnover | |
L-Arginine-¹⁵N₄ hydrochloride | ≥98 atom% 15N,≥97% | All four nitrogen positions of arginine are labeled with ¹⁵N; arginase and nitric oxide synthase reactions transfer the labels into urea, ornithine, citrulline, and nitric oxide-related products | Urea cycle and nitric oxide production | |
L-Leucine-¹³C₆,¹⁵N | ≥98 atom% 13C,≥98 atom% 15N,≥95% | All six carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; the carbon skeleton enters α-ketoisocaproate, acetyl-CoA, and acetoacetate, while the intact molecule can be incorporated into proteins | Leucine oxidation and protein turnover | |
L-Isoleucine-¹³C₆,¹⁵N | ≥98 atom% 13C,≥98 atom% 15N,≥95% | All six carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; after catabolism, the carbon labels are distributed into acetyl-CoA and propionyl-CoA, while the nitrogen label enters transamination products | Isoleucine catabolism and tissue utilization | |
L-Phenylalanine-¹³C₉,¹⁵N | ≥98 atom% 13C,≥98 atom% 15N,≥95%,≥97%(Chiral Purity) | All nine carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; the intact molecule retains all labels, and the carbon and nitrogen labels remain in tyrosine after hydroxylation | Whole-body protein synthesis and turnover | |
L-Valine-¹³C5,¹⁵N | ≥98 atom% 15N,≥98 atom% 13C,≥95% | All five carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; the carbon skeleton enters succinyl-CoA through propionyl-CoA and methylmalonyl-CoA, while the intact molecule can be incorporated into proteins | Valine catabolism and protein turnover | |
L-Aspartic acid-¹³C₄,¹⁵N | ≥99 atom% 13C,≥99 atom% 15N,≥98% | All four carbon positions are labeled with ¹³C, and the amino nitrogen is labeled with ¹⁵N; the carbon labels enter oxaloacetate and nucleotide skeletons, while the nitrogen label enters the urea cycle and nitrogen-containing products | Aspartate utilization and nitrogen-containing biosynthesis |
5.3 Products Related to Tracing Lipid Synthesis, Body Water, and Other Metabolic Processes
Catalog # | Product Name | Grade & Purity | Labeling Form and Tracing Position | Research Application |
Palmitic acid-¹³C₁₆ | ≥98 atom% 13C,≥98% | All 16 carbon positions are labeled with ¹³C; the intact acyl chain appears as M+16 in complex lipids, whereas β-oxidation successively generates M+2 acetyl-CoA | Fatty acid uptake, oxidation, and re-esterification | |
Palmitic acid-1-(¹³C) | ≥99 atom% 13C | The C1 carboxyl carbon of palmitate is labeled with ¹³C; after the first round of β-oxidation, this carbon becomes the carbonyl carbon of the first acetyl-CoA molecule | Fate of the palmitate carboxyl carbon | |
Oleic acid-¹³C₁₈ | ≥98 atom% 13C,≥95% | All 18 carbon positions are labeled with ¹³C; the intact oleoyl chain appears as M+18 in complex lipids, whereas β-oxidation generates successive M+2 acetyl units | Oleate utilization and lipid remodeling | |
Cholesterol-2,3,4-¹³C₃ | ≥99 atom% 13C,≥98% | The C2, C3, and C4 positions of the cholesterol sterol skeleton are labeled with ¹³C; the three labels are concentrated in ring A and retained in cholesteryl esters and corresponding sterol-conversion products | Cholesterol transport, esterification, and clearance | |
Deuterium oxide | ≥99.9 atom% D | The hydrogen atoms in water are labeled with ²H; after entering the total body water pool, ²H is incorporated into nonexchangeable positions in fatty acids, cholesterol, nucleotides, and proteins during biosynthesis | Whole-body synthesis and renewal | |
Water-¹⁸O | ≥97 atom% 18O | The oxygen atom in water is labeled with ¹⁸O; hydrolysis, oxygen exchange, and related synthetic reactions incorporate ¹⁸O into corresponding oxygen-containing products | Water-involving reactions and oxygen exchange | |
Sodium bicarbonate-(¹³C) | ≥99 atom% 13C,≥98% | The inorganic carbon of bicarbonate is labeled with ¹³C; following carboxylation, this carbon enters products such as oxaloacetate, malonyl-CoA, or carbamoyl phosphate | Inorganic carbon fixation and carboxylation | |
Urea-15N₂ | ≥99 atom%,≥98.5% | Both nitrogen atoms in urea are labeled with ¹⁵N, forming the ¹⁵N₂ urea isotopologue, with the labeling signal located at both nitrogen positions of urea | Urea production, clearance, and whole-body nitrogen balance |
Stable isotope tracing can resolve in vivo substrate sources, pathway allocation, and metabolic turnover. With the continued development of high-resolution mass spectrometry, spatial metabolomics, and dynamic flux modeling, its applications will extend further into tissue heterogeneity, interorgan metabolic coordination, and disease-associated metabolic reprogramming.
References
[1] Fernández-García J, Altea-Manzano P, Pranzini E, Fendt SM. Stable Isotopes for Tracing Mammalian-Cell Metabolism In Vivo. Trends Biochem Sci. 2020 Mar;45(3):185-201
[2] Jang C, Hui S, Lu W, Cowan AJ, Morscher RJ, Lee G, Liu W, Tesz GJ, Birnbaum MJ, Rabinowitz JD. The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids. Cell Metab. 2018 Feb 6;27(2):351-361.e3.
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