Citric acid-d4-1, being a tracer, targets the same enzymes as non-labeled citric acid. Citric acid is an allosteric regulator of several key enzymes in carbohydrate and lipid metabolism. It is a negative feedback inhibitor of the glycolytic enzyme phosphofructokinase-1 (PFK-1). When the cellular energy charge is high and the citric acid cycle is running efficiently, citrate accumulates and is exported from the mitochondria to the cytosol, where it inhibits PFK-1, thereby slowing down the rate of glycolysis. In the cytosol, citrate is also the substrate for the enzyme ATP-citrate lyase (ACLY), which cleaves citrate into oxaloacetate and acetyl-CoA. The acetyl-CoA produced by ACLY is the building block for fatty acid synthesis and cholesterol synthesis. Therefore, citrate links glucose catabolism to lipid anabolism. Citrate also acts as an allosteric activator of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, further promoting lipid production under conditions of high energy availability. Additionally, citrate is a chelator of calcium ions, and it is used in anticoagulant solutions (e.g., ACD, acid-citrate-dextrose) for blood collection, where it binds calcium and prevents the coagulation cascade. As a deuterated tracer, citric acid-d4-1 can be used to study the metabolic flux through these pathways without interfering with the endogenous pool of unlabeled citric acid.

Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.[1]

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In vitro studies using citric acid-d4-1 are primarily focused on its use as a tracer for metabolic flux analysis. For example, in a study investigating the citric acid cycle (TCA cycle) in cultured cells, a labeled substrate such as [U-¹3C]-glucose or [U-¹3C]-glutamine can be added to the cell culture medium. After a period of metabolism, the cells are harvested, and metabolites are extracted. Citric acid-d4-1 (and other labeled internal standards) are added to the extract at known concentrations, and the sample is analyzed by LC-MS/MS. The incorporation of the ¹3C label into citric acid (and other TCA cycle intermediates) is measured, and the isotopologue distribution is analyzed. This allows researchers to calculate the relative contributions of different carbon sources to the TCA cycle and to determine metabolic fluxes. As an internal standard, citric acid-d4-1 is added to the sample at a fixed concentration (e.g., 10-100 ng/mL), and its peak area is used to normalize the peak area of the unlabeled or ¹3C-labeled citric acid. In addition to its role as a tracer, citric acid itself has been shown to have biological activities in vitro. In HaCaT human keratinocyte cells, citric acid has been demonstrated to induce apoptosis (programmed cell death) and cause cell cycle arrest at the G2/M phase and S phase. In these studies, cells are typically treated with citric acid at concentrations ranging from 1 to 20 mM for 24-72 hours. Apoptosis is assessed by flow cytometry using Annexin V-FITC/PI staining or by measuring caspase-3/7 activity. Cell cycle distribution is analyzed by flow cytometry after staining with propidium iodide. The mechanisms underlying these effects are not fully understood but may involve changes in intracellular pH, chelation of metal ions, or oxidative stress.

Citric acid (120, 240, and 480 mg/kg; i.p.) significantly decreases GSH-Px activity and induces an increase in the MDA (malonyldialdehyde) levels in mouse liver[1]. Citric acid (120, 240, and 480 mg/kg; i.p.) induces apoptosis by increases caspase-3 activity in a dose-dependent manner in mouse hepatocytes[1]. Citric acid (120, 240, and 480 mg/kg; i.p.; weekly for 3 weeks) causes renal toxicity in mice[2].

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In vivo studies with citric acid have been conducted to evaluate its metabolic effects and toxicity. In rodent models, citric acid has been shown to cause oxidative damage to the liver by decreasing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and by increasing lipid peroxidation, measured as malondialdehyde (MDA) levels. Citric acid has also been reported to induce nephrotoxicity (kidney damage) in mice, as evidenced by increased serum levels of blood urea nitrogen (BUN) and creatinine, and histopathological changes in the kidney tissue, such as tubular necrosis and inflammation. These toxic effects are typically observed at high oral doses (e.g., 1-3 g/kg) in acute studies or with repeated dosing over several weeks. At the lower concentrations typically used as a food additive or excipient (milligrams to low grams), citric acid is considered safe and is generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA). The citric acid cycle is a central metabolic pathway, and the steady-state concentration of citric acid in cells and tissues is tightly regulated. However, the administration of high doses of citric acid can overwhelm this regulation and lead to metabolic acidosis (a decrease in blood pH), which is the primary mechanism of toxicity. Citric acid-d4-1, as a tracer, is typically used at very low concentrations (microgram to milligram levels) and is unlikely to cause any of these toxic effects. Instead, it is used as a safe and accurate internal standard for quantitative analysis.

A common cell-free enzyme assay involving citric acid is the measurement of citrate synthase (CS) activity, which is the first and rate-limiting enzyme of the citric acid cycle. In this assay, the conversion of oxaloacetate and acetyl-CoA to citrate is monitored. A typical protocol is as follows: Prepare a reaction mixture containing 100 mM Tris-HCl (pH 8.0), 0.5 mM oxaloacetate (freshly prepared), 0.3 mM acetyl-CoA, and 0.1 mM 5,5‘-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent). Citrate synthase (purified or in a crude tissue homogenate, e.g., from rat liver mitochondria) is added to the cuvette to initiate the reaction. The reaction proceeds at 30degC, and the release of free CoASH from acetyl-CoA as it condenses with oxaloacetate is measured by the increase in absorbance at 412 nm (due to the reaction of CoASH with DTNB). Alternatively, the product of the reaction, citrate, can be quantified by a coupled enzymatic assay using citrate lyase and malate dehydrogenase. For research involving stable isotope-labeled compounds, the activity of ACLY (ATP-citrate lyase) can be measured by LC-MS. In this assay, a reaction mixture containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 10 mM ATP, 0.5 mM CoASH, and 0.5 mM citric acid-d4-1 (or unlabeled citric acid) is incubated with purified ACLY or a cytosolic extract at 37degC for 30 minutes. The reaction is terminated by adding an equal volume of ice-cold acetonitrile, and the products (oxaloacetate-d4 and acetyl-CoA) are analyzed by LC-MS/MS. The use of the deuterated substrate allows for the specific quantification of the enzymatic product without interference from endogenous unlabeled metabolites.

Cell-based studies using citric acid-d4-1 to investigate metabolic flux are common in the field of metabolomics. A typical protocol for a cell culture metabolic flux analysis experiment is as follows: Cells (e.g., cancer cell lines such as HCT116, HepG2, or MCF-7) are seeded in 6- or 12-well plates and grown to 70-90% confluence in standard growth medium (e.g., DMEM supplemented with 10% FBS). To initiate the metabolic labeling experiment, the medium is replaced with fresh medium containing a ¹3C-labeled tracer, such as 5 mM [U-¹3C₆]-glucose or 2 mM [U-¹3C₅]-glutamine. The cells are then incubated for a defined period, typically 1-24 hours, depending on the kinetics of the pathway of interest. At the end of the labeling period, the medium is removed, and the cells are rapidly washed with ice-cold PBS. Metabolites are extracted by adding 1 mL of ice-cold extraction solvent (e.g., 80% methanol or 50% acetonitrile/50% water) directly to the cell monolayer. The plate is placed on dry ice or at -80degC for 10-15 minutes. The cell extract is then scraped, transferred to a microcentrifuge tube, and centrifuged at 12,000 × g for 10 minutes at 4degC to pellet the debris. The supernatant is transferred to a new tube and dried down under a stream of nitrogen gas. The dried extract is then reconstituted in an appropriate volume (e.g., 50-100 uL) of water or mobile phase, and a fixed amount (e.g., 1-10 ng/sample) of citric acid-d4-1 is added as an internal standard. The sample is then analyzed by LC-MS/MS. The isotopologue distribution of citric acid (and other TCA cycle metabolites such as succinate, fumarate, malate, and alpha-ketoglutarate) is determined. This approach allows for the calculation of metabolic fluxes, such as the relative contributions of glucose and glutamine to the TCA cycle. It can also be used to identify metabolic bottlenecks and to assess the effects of genetic or pharmacological perturbations on metabolism.

Animal studies using citric acid-d4-1 as a tracer are typically performed in the context of metabolic research. A common protocol is as follows: Adult male or female rodents (e.g., C57BL/6 mice, 8-12 weeks old) are fasted for 4-6 hours to stabilize baseline blood glucose levels. A solution containing citric acid-d4-1 (or another stable isotope-labeled tracer) is administered via intraperitoneal (IP) injection or by oral gavage at a dose of, for example, 10-50 mg/kg body weight. For more precise control of plasma concentrations, a catheter can be surgically implanted in the jugular vein for intravenous (IV) administration. After administration, blood samples (e.g., 10-20 uL) are collected at various time points (e.g., 0, 5, 15, 30, 60, 120, and 240 minutes post-injection) from the tail vein or from the IV catheter. The blood samples are immediately mixed with an anticoagulant (e.g., EDTA or heparin) and centrifuged to separate the plasma. A fixed volume of plasma (e.g., 10-50 uL) is then mixed with extraction solvent (e.g., 3 volumes of ice-cold acetonitrile containing 0.1% formic acid) and centrifuged. The supernatant is analyzed by LC-MS/MS to measure the concentration of citric acid-d4-1 and its potential metabolites. For tissue distribution studies, animals are euthanized at a specific time point (e.g., 30 or 60 minutes post-dose), and tissues of interest (e.g., liver, kidney, heart, brain, and muscle) are harvested, snap-frozen in liquid nitrogen, and stored at -80degC. The tissues are weighed, homogenized in extraction solvent, and processed similarly to the plasma samples. The concentrations of the labeled compound in plasma and tissues are plotted against time to derive pharmacokinetic parameters such as AUC, Cmax, Tmax, t½, CL, and Vd. This information can be used to determine the absorption, distribution, metabolism, and excretion (ADME) of citric acid and related compounds, and to assess the impact of disease states or pharmacological interventions on these processes.

The pharmacokinetics of citric acid-d4-1 are expected to closely mirror those of unlabeled citric acid. Orally administered citric acid is rapidly absorbed from the gastrointestinal tract, and it is naturally present in many foods, so it is unlikely to be extensively metabolized before absorption. Once absorbed, citric acid enters the portal circulation and is transported to the liver. In the liver, it is taken up by hepatocytes and can enter the TCA cycle, where it is metabolized to isocitrate by the enzyme aconitase, and then to alpha-ketoglutarate, succinyl-CoA, and eventually to CO2 and water, with the generation of energy in the form of ATP. Citric acid is also present in the systemic circulation in healthy individuals, typically in the low micromolar range. It is freely filtered by the glomeruli in the kidneys and then actively reabsorbed in the proximal tubules via a sodium-dicarboxylate cotransporter (NaDC-1). A small fraction of the filtered load is excreted unchanged in the urine. The elimination half-life of citric acid is relatively short, on the order of 20-60 minutes in humans and rodents. At the high oral doses used in toxicity studies (grams per kg), citric acid can transiently increase the plasma concentration and acidify the blood (lower the pH). This can lead to a condition known as metabolic acidosis, which is characterized by a decrease in blood pH, compensatory hyperventilation, and in severe cases, cardiovascular depression and coma. The acidifying effect of citric acid is used therapeutically in some formulations to adjust the pH of the urine to prevent the formation of certain types of kidney stones. As a stable isotope-labeled internal standard, citric acid-d4-1 is used at very low concentrations (typically less than 1 ug per sample), and its pharmacokinetic properties are not directly studied; rather, it is used as a tool to quantify the endogenous levels of citric acid. The compound is not intended for therapeutic use, and its administration to animals is for research purposes only.

The non-deuterated parent compound, citric acid, is a natural preservative and food tartness enhancer widely used in the food, beverage, and pharmaceutical industries. It is a weak tricarboxylic acid that is a key intermediate in the citric acid cycle (Krebs cycle), the central metabolic pathway for energy production in aerobic organisms. In addition to its role in metabolism, citric acid is also used as an excipient in pharmaceutical formulations due to its antioxidant, pH-adjusting, and chelating properties. It can be used to prevent the degradation of active pharmaceutical ingredients, to adjust the pH of solutions for enhanced drug stability or solubility, and to chelate metal ions that could catalyze oxidative reactions. Citric acid is also used in combination with sodium citrate as an anticoagulant for blood collection (ACD solution). The U.S. Food and Drug Administration (FDA) has designated citric acid as Generally Recognized as Safe (GRAS). Citric acid-d4-1 is a deuterated version of citric acid. The primary application of citric acid-d4-1 is as a stable isotope-labeled internal standard for the quantification of citric acid in biological samples using LC-MS/MS. The use of a stable isotope-labeled internal standard is considered the gold standard for quantitative mass spectrometry because it corrects for matrix effects, variations in sample preparation, and instrumental drift, leading to the highest level of accuracy and precision. Citric acid-d4-1 is essential for metabolomics studies, where the precise and accurate measurement of citric acid (and other TCA cycle intermediates) is needed to understand metabolic health and disease. For example, perturbations in the TCA cycle are seen in cancer (the Warburg effect), diabetes, and various metabolic and mitochondrial disorders. Citric acid-d4-1 can also be used as a tracer in metabolic flux analysis, where it is administered to cells or animals, and its metabolic fate is tracked by mass spectrometry. This allows researchers to determine the rates of metabolic reactions and the contributions of different substrates to the TCA cycle. However, as a fully deuterated molecule, citric acid-d4-1 may exhibit a kinetic isotope effect (KIE) if a C-D bond is broken in a rate-limiting step of an enzymatic reaction. In the TCA cycle, the conversion of citrate to isocitrate by aconitase involves the removal of a proton (a hydrogen atom) from the citrate molecule. This proton is abstracted from one of the carbons of the citrate molecule to form the intermediate cis-aconitate. If this hydrogen is replaced with deuterium, the reaction may proceed more slowly due to the higher bond strength of the C-D bond compared to the C-H bond. This could potentially alter the rate of citrate metabolism in a tracer experiment. Therefore, for metabolic flux analysis, it is often preferable to use a ¹3C-labeled tracer (such as [U-¹3C₆]-citrate or a ¹3C-labeled precursor like [U-¹3C₆]-glucose) rather than a deuterated one, to avoid potential KIE. However, for use as an internal standard (where only a small amount is added, and it is not expected to be significantly metabolized during the short sample preparation and analysis time), citric acid-d4-1 is perfectly adequate and widely used. Citric acid-d4-1 is intended for research use only and is not for diagnostic or therapeutic applications in humans.

[1]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019;53(2):211-216.

[2]. Study on injury effect of food additive citric acid on liver tissue in mice. Cytotechnology. 2014 Mar;66(2):275-82.

[3]. Chen X, Lv Q, Liu Y, Deng W. Effects of the food additive, citric acid, on kidney cells of mice. Biotech Histochem. 2015 Jan;90(1):38-44.

[4]. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways. Anticancer Res. 2013 Oct;33(10):4411-20.

C6H4D4O7

196.15

Solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)