Citrate Science

Welcome to the Citrate Science website.

People have often heard about the Citric Acid Cycle or Krebs Cycle. This is the metabolic cycle that runs in the mitochondria and is used to generate the cellular level energy supply of Adenosine TriPhosphate (ATP). However, citrate Metabolism is a lot more than that. Citrate can leave the Mitochondria and enter into the Cytosol where it is used to Generate Acetyl-CoA. Citrate can leave the cell (as it does in Prostate cells) and it can get into the Cell from Blood Serum. (Site Maintained by John A. M. Hemming).

There are a number of ways that Citrate can get through the cell membrane as well as a protein that is placed in the mitochondrial membrane that enables swapping Citrate for Malate.

Interesting Recent Papers (plus a really old one)
Mitochondrial citrate metabolism and efflux regulate BeWo differentiation May 6 2023
This is an interesting paper because it shows how Citrate is taken out of the mitochondria in relatively large amounts during the process of differentiation, but then the volume of citrate movements is turned down once differentiated.

This has broader relevance because it is differentiation failure that lies behind a range of age related diseases and in essence this is part of a corpus of evidence that shows that citrate metabolism is at the core of the aging process.

The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse
Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

Mitochondrial citrate carrier SLC25A1 is a dosage-dependent regulator of metabolic reprogramming and morphogenesis in the developing heart
The developing mammalian heart undergoes an important metabolic shift from glycolysis toward mitochondrial oxidation, such that oxidative phosphorylation defects may present with cardiac abnormalities. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mice with systemic loss of the mitochondrial citrate carrier SLC25A1. Slc25a1 null embryos displayed impaired growth, cardiac malformations, and aberrant mitochondrial function. Importantly, Slc25a1 haploinsufficient embryos, which are overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 dose-dependent effects. Supporting clinical relevance, we found a near-significant association between ultrarare human pathogenic SLC25A1 variants and pediatric congenital heart disease. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of PPARγ to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.

Effect of Pre-exercise Sodium Citrate Ingestion on Repeated Sprint Performance in Soccer Players
Kuru, D, Aktitiz, S, Atakan, MM, Köse, MG, Turnagöl, HH, and Koşar, ŞN. Effect of pre-exercise sodium citrate ingestion on repeated sprint performance in soccer players. J Strength Cond Res XX(X): 000-000, 2023-This study aimed to test the hypothesis that sodium citrate (CIT) administered 180 minutes before exercise improves repeated sprint performance in athletes within a field-based setting. Twenty male soccer players (mean ± SD: age = 20.9 ± 2.3 years; body mass [BM] = 73.8 ± 5.9 kg) performed a running-based anaerobic sprint test (RAST) with 0.5 g·kg-1 BM of CIT or with placebo (PLC; NaCl) ingestion 180 minutes before exercise in a randomized, crossover, and double-blind design, with at least 6 days between the trials. Blood samples were collected before exercise and at first, third, fifth, and seventh minutes after exercise to analyze blood pH, bicarbonate, and lactate levels. Gastrointestinal symptoms were also monitored at 30-minute intervals for 180 minutes after CIT and PLC ingestion. Pre-exercise blood pH (CIT = 7.49 ± 0.03 vs. PLC = 7.41 ± 0.02) and bicarbonate (CIT = 30.57 ± 1.33 vs. PLC = 25.25 ± 1.52) increased with CIT compared with PLC (p < 0.001). Blood pH, bicarbonate, and lactate at the first, third, fifth, and seventh minutes after RAST with CIT were higher than PLC (p < 0.05), except for lactate at first minute (p > 0.05). Compared with PLC, CIT ingestion significantly improved minimum power output (p = 0.024) and percentage decrement score (p = 0.023). Gastrointestinal symptoms were significantly higher after CIT ingestion vs. PLC at 30th (p = 0.003) and 60th minutes (p = 0.010). However, there were no significant differences at 90th, 120th, 150th, or 180th minutes (p > 0.05). The ingestion of 0.5 g·kg-1 BM of CIT 180 minutes before exercise is an effective ergogenic aid for improving repeated sprint ability as evidenced by improvements in minimum power output and percentage decrement score.

Bone citrate content as an indicator of post-mortem interval
The citrate levels in the bone start to decrease after death. Therefore, it may be possible to estimate the post-mortem interval if the citrate content decreases at regular and predictable intervals. To verify this, it was used twelve fresh porcine tibiae and femora, fifteen recently buried porcine bones, two samples of human bones curated from the anatomical teaching collection from Bournemouth University, UK (never been buried) and fifteen samples of archaeological human bones from Saint Augustine the Less, Bristol, UK. The results obtained in this study align together with the data available in the literature, with higher citrate content in the fresh porcine bones and lower in the archaeological samples, however, a statistical significance was not found. Moreover, the formula used to estimate the time since death largely overestimated the known PMI. Further research should be done with a larger sample and a new formula to estimate the post-mortem interval is required.

Lessons for the clinical nephrologist: vascular access recirculation during continuous renal replacement therapy and regional citrate anticoagulation (case notes)
Should the citrate used in continuous renal replacement therapy be taken into account as a source of calories?
Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate
Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.

Recent advance of ATP citrate lyase inhibitors for the treatment of cancer and related diseases
ATP citrate lyase (ACLY), a strategic metabolic enzyme that catalyzes the glycolytic to lipidic metabolism, has gained increasing attention as an attractive therapeutic target for hyperlipidemia, cancers and other human diseases. Despite of continual research efforts, targeting ACLY has been very challenging. In this field, most reported ACLY inhibitors are "substrate-like" analogues, which occupied with the same active pockets. Besides, some ACLY inhibitors have been disclosed through biochemical screening or high throughput virtual screening. In this review, we briefly summarized the cancer-related functions and the recent advance of ACLY inhibitors with a particular focus on the SAR studies and their modes of action. We hope to provide a timely and updated overview of ACLY and the discovery of new ACLY inhibitors.

Loss of the mitochondrial citrate carrier, Slc25a1/CIC disrupts embryogenesis via 2-Hydroxyglutarate
Biallelic germline mutations in the SLC25A1 gene lead to combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a fatal systemic disease uniquely characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). How SLC25A1 deficiency contributes to D/L-2HGA and the role played by 2HG is unclear and no therapy exists. Both enantiomers act as oncometabolites, but their activities in normal tissues remain understudied. Here we show that mice lacking both SLC25A1 alleles exhibit developmental abnormalities that mirror human D/L-2HGA. SLC25A1 deficient cells undergo premature mitochondrial dysfunction associated senescence, suggesting that loss of proliferative capacity underlies the pathogenesis of D/L-2HGA. Remarkably, D- and L-2HG directly induce mitochondrial respiratory deficit and treatment of zebrafish embryos with the combination of D- and L-2HG phenocopies SLC25A1 loss, leading to developmental abnormalities in an additive fashion relative to either enantiomer alone. Metabolic analyses demonstrated that loss of SLC25A1 leads to global remodeling towards glutamine metabolism, with glutamine serving as a source for 2HG synthesis. Therefore, we explored the pre-clinical relevance of phenylbutyrate, an FDA-approved drug that reduces the blood glutamine levels, and found that it reduces 2HG accumulation reversing metabolic abnormalities in patients affected by D/L-2HGA. These results reveal pathogenic and growth suppressive activities of 2HG in the context of SLC25A1 deficiency and expose metabolic vulnerabilities for the clinical management of this disease.

The effect of citrate in cardiovascular system and clot circuit in critically ill patients requiring continuous renal replacement therapy
We aimed to evaluate the impact of citrate on hemodynamic responses and secondary outcomes, including the filter life span, metabolic complications, and levels of inflammatory cytokines, in critically ill patients who required CRRT compared with those who underwent the heparin-free method. This prospective, multicenter, open-label randomized trial compared regional citrate anticoagulation (RCA) with a heparin-free protocol in severe acute kidney injury (AKI) patients who received continuous venovenous hemodiafiltration (CVVHDF) in the postdilution mode. We measured hemodynamic changes using the FloTrac Sensor/EV1000™ Clinical Platform at certain time points after starting CRRT (0, 6, 12, 24, 48, and 72 h.). The levels of inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-ɑ) were measured on days 1 and 3. Forty-one patients were recruited and randomized into the heparin (n = 20) and citrate groups (n = 21). The cardiac performances were not significantly different between the 2 groups at any time point. The inflammatory cytokines declined similarly in both treatment arms. The maximum filter survival time was insignificantly longer in the RCA group than in the heparin-free group (44.64 ± 26.56 h. vs p = 0.693 in citrate and heparin free group). No serious side effects were observed for either treatment arm, even in the group of liver dysfunction patients. RCA did not affect hemodynamic changes during CRRT. Inflammatory cytokines decreased similarly in both treatment arms.The filter life span was longer in the citrate group. RCA is a valid alternative to traditional anticoagulation and results in stable hemodynamic parameters.

Experiments on the Action of certain Diuretics (Citrate and Acetate of Potash, Spiritus Ætheris Nitrosi, and Oil of Juniper) on the Urine in Health - 1870
Let's stop talking about 'citrate toxicity'
Purpose of review: Continuous renal replacement therapy (CRRT) is a vital medical intervention used in critically ill patients with acute kidney injury (AKI). One of the key components of adequate clearance with CRRT is the use of anticoagulants to prevent clotting of the extracorporeal circuit. Regional citrate anticoagulation is the most often recommended modality. The term 'citrate toxicity' is used to describe potential adverse effects of accumulation of citrate and subsequent hypocalcemia. However, citrate is itself not inherently toxic. The term and diagnosis of citrate toxicity are questioned in this review.

Recent findings: Citrate is being increasingly used for regional anticoagulation of the CRRT circuit. Citrate accumulation is infrequent and can cause hypocalcemia and metabolic alkalosis, which are potential adverse effects. Citrate itself, however, is not a toxic molecule. The term 'citrate toxicity' has been used to denote hypocalcemia and metabolic acidosis. However, citrate administration is well known to cause systemic and urinary alkalinization and under certain circumstances, metabolic alkalosis, but is not associated itself with any 'toxic' effects.We review the existing literature and debunk the perceived toxicity of citrate. We delve into the metabolism and clearance of citrate and question current data suggesting metabolic acidosis occurs as the result of citrate accumulation.

Summary: In conclusion, this article calls into question prevailing concerns about 'citrate toxicity'. We emphasize the need for a more nuanced understanding of its safety profile. We recommend discarding the term 'citrate toxicity' in favor of another frequently used, but more meaningful term: 'citrate accumulation'.

Mapping the Metabolic Niche of Citrate Metabolism and SLC13A5
The small molecule citrate is a key molecule that is synthesized de novo and involved in diverse biochemical pathways influencing cell metabolism and function. Citrate is highly abundant in the circulation, and cells take up extracellular citrate via the sodium-dependent plasma membrane transporter NaCT encoded by the SLC13A5 gene. Citrate is critical to maintaining metabolic homeostasis and impaired NaCT activity is implicated in metabolic disorders. Though citrate is one of the best known and most studied metabolites in humans, little is known about the consequences of altered citrate uptake and metabolism. Here, we review recent findings on SLC13A5, NaCT, and citrate metabolism and discuss the effects on metabolic homeostasis and SLC13A5-dependent phenotypes. We discuss the "multiple-hit theory" and how stress factors induce metabolic reprogramming that may synergize with impaired NaCT activity to alter cell fate and function. Furthermore, we underline how citrate metabolism and compartmentalization can be quantified by combining mass spectrometry and tracing approaches. We also discuss species-specific differences and potential therapeutic implications of SLC13A5 and NaCT. Understanding the synergistic impact of multiple stress factors on citrate metabolism may help to decipher the disease mechanisms associated with SLC13A5 citrate transport disorders.
Quantification of Intracellular Citrate Concentrations with Genetically Encoded Biosensors
Citrate is a central intracellular metabolite with roles in a variety of normal and aberrant biological processes. The methods for quantifying citrate concentration in cells can enable the study of the molecular mechanisms of citrate-related biological processes and diseases. Compared to existing analytical methods such as enzymatic assays and mass spectrometry, genetically encoded biosensors based on fluorescent proteins (FPs) offer the advantage of noninvasively tracking intracellular ion and small molecule dynamics with high spatial-temporal resolution. These biosensors are less toxic than chemosensors and can be targeted to specific organelles for subcellular imaging. Here we present a protocol for quantification of cytosolic and mitochondrial citrate in mammalian cells with recently reported genetically encoded biosensors for citrate.

Urine pH and Citrate as Predictors of Calcium Phosphate Stone Formation
Key Points:
The occurrence of calcium phosphate stones has increased over the past five decades, and this is most notable in female stone formers.
High urine pH and hypocitraturia are the most discriminatory urine parameters between calcium phosphate and calcium oxalate stone formers.
High urine pH in calcium phosphate stone formers is independent of the effect of dietary alkali and acid.
Background: Urinary parameters, including urine pH and citrate, are recognized as critical in the pathophysiology of calcium-based stones. The factors contributing to variation in these parameters between calcium oxalate (CaOx) and calcium phosphate (CaP) stone formers (SFs) are, however, not well-understood. In this study, using readily available laboratory data, we explore these differences to delineate the odds of forming CaP versus CaOx stones. Methods: In this single-center retrospective study, we compared serum and urinary parameters between adult CaP SFs, CaOx SFs, and non–stone formers.
Results: Urine pH was higher and urine citrate lower in CaP SFs compared with same-sex CaOx SFs and non–stone formers. In CaP SFs, higher urine pH and lower citrate were independent of markers of dietary acid intake and gastrointestinal alkali absorption, suggesting abnormal renal citrate handling and urinary alkali excretion. In a multivariable model, urine pH and urine citrate were most discriminatory between CaP SFs and CaOx SFs (receiver-operating characteristic area under the curve of 0.73 and 0.65, respectively). An increase in urine pH by 0.35, a decrease in urine citrate by 220 mg/d, a doubling of urine calcium, and female sex all independently doubled the risk of CaP stone formation compared with CaOx stones.
Conclusions: High urine pH and hypocitraturia are two clinical parameters that distinguish the urine phenotype of CaP SFs from CaOx SFs. Alkalinuria is due to intrinsic differences in the kidney independent of intestinal alkali absorption and is accentuated in the female sex.
Mitochondrial citrate accumulation triggers senescence of alveolar epithelial cells contributing to pulmonary fibrosis in mice
Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of pulmonary fibrosis (PF). However, the exact mechanism underlying AEC senescence during PF remains poorly understood. Here, we reported an unrecognized mechanism for AEC senescence during PF. We found that, in bleomycin (BLM)-induced PF mice, the expressions of isocitrate dehydrogenase 3α (Idh3α) and citrate carrier (CIC) were significantly down-regulated in the lungs, which could result in mitochondria citrate (citratemt) accumulation in our previous study. Notably, the down-regulation of Idh3α and CIC was related to senescence. The mice with AECs-specific Idh3α and CIC deficiency by adenoviral vector exhibited spontaneous PF and senescence in the lungs. In vitro, co-inhibition of Idh3α and CIC with shRNA or inhibitors triggered the senescence of AECs, indicating that accumulated citratemt triggers AEC senescence. Mechanistically, citratemt accumulation impaired the mitochondrial biogenesis of AECs. In addition, the senescence-associated secretory phenotype from senescent AECs induced by citratemt accumulation activated the proliferation and transdifferentiation of NIH3T3 fibroblasts into myofibroblasts. In conclusion, we show that citratemt accumulation would be a novel target for protection against PF that involves senescence.

The potential for citrate to reinforce epigenetic therapy by promoting apoptosis
Epigenetic drugs induce ATP depletion, promoting a glycolysis-to-oxidative phosphorylation (OXPHOS) shift which sometimes favors tumor growth by promoting necroptosis over apoptosis. To restore effective apoptosis in tumors, we propose that the administration of citrate could inhibit ATP production, activate caspase-8 (a key necroptosis inhibitor), and downregulate key anti-apoptotic proteins (Bcl-xL and MCL1).

Plasma Citrate Levels Are Associated with an Increased Risk of Cardiovascular Mortality in Patients with Type 2 Diabetes (Zodiac-64)
Circulating citrate may represent a proxy of mitochondrial dysfunction which plays a role in the development of vascular complications in type 2 diabetes (T2D). Here, we determined the associations between plasma citrate levels and cardiovascular (CV) mortality in T2D patients. In this prospective cohort study, 601 patients were included who participated in the Zwolle Outpatient Diabetes project Integrating Available Care (ZODIAC). Plasma citrate levels were measured by nuclear magnetic resonance spectroscopy. Cox proportional hazards regression models were used to evaluate the associations between plasma citrate and the risk of CV mortality. Over a median follow-up of 11.4 years, 119 (19.8%) of the 601 patients died from a CV cause. In multivariable Cox proportional hazards regression models, adjusting for conventional risk factors, plasma citrate was associated with an increased risk of CV mortality (the hazard ratio (HR) per 1-SD increment was 1.19 (95%CI: 1.00-1.40), p = 0.048). This association was prominent in males (n = 49 with CV mortality) (HR 1.52 (95%CI: 1.14-2.03), p = 0.005), but not in females (n = 70 with CV mortality) (HR 1.11 (95%CI: 0.90-1.37), p = 0.319) (age-adjusted Pinteraction = 0.044). In conclusion, higher plasma citrate levels are associated with an increased risk of CV mortality in patients with established T2D. Future studies are warranted to unravel the potential role of citrate-related pathways in the pathogenesis of T2D-related vascular complications.

Hypercitratemia is a mortality predictor among patients on continuous venovenous hemodiafiltration and regional citrate anticoagulation
The use of regional citrate anticoagulation (RCA) in liver failure (LF) patients can lead to citrate accumulation. We aimed to evaluate serum levels of citrate and correlate them with liver function markers and with the Cat/Cai in patients under intensive care and undergoing continuous venovenous hemodiafiltration with regional citrate anticoagulation (CVVHDF-RCA). A prospective cohort study in an intensive care unit was conducted. We compared survival, clinical, laboratorial and dialysis data between patients with and without LF. Citrate was measured daily. We evaluated 200 patients, 62 (31%) with LF. Citrate was significantly higher in the LF group. Dialysis dose, filter lifespan, systemic ionized calcium and Cat/Cai were similar between groups. There were weak to moderate positive correlations between Citrate and indicators of liver function and Cat/Cai. The LF group had higher mortality (70.5% vs. 51.8%, p = 0.014). Citrate was an independent risk factor for death, OR 11.3 (95% CI 2.74–46.8). In conclusion, hypercitratemia was an independent risk factor for death in individuals undergoing CVVHDF-ARC. The increase in citrate was limited in the LF group, without clinical significance. The correlation between citrate and liver function indicators was weak to moderate.