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KREBS CYCLE METABOLITES

STUDY DETAILS

Title: Tricarboxylic cycle intermediates in combination with calcium phosphate chelators and sodium bicarbonate increase eGFR in patients with stages 3b, 4 and 5 CKD: a retrospective observational study [see Ref. 1].

Year of publication: 2024

Journal: Revista Colombiana de Nefrología (Colombian Journal of Nephrology)

Study type: retrospective observational study

Sponsored by: Virtus Humanitatis

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Number of patients: 55

Evaluation period: average of 11 months

Inclusion criteria: CKD stages 3b, 4, or 5; patients older than 18 years

Exclusion criteria: patients on renal replacement therapy and/or those taking alpha-ketoanalogues and/or pre/probiotics

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Patient Characteristics:

  • Mean age: 67.2 years (range: 31–88 years)

  • Sex distribution: 27 women and 28 men

  • Etiology of CKD:

69% Type 2 Diabetes Mellitus   |   23.6% Hypertension   |   7% Systemic Lupus Erythematosus   |   5.6% Other glomerulopathies

  • Stage distribution:  

52.7% Stage 5 (eGFR <15 mL/min)  |  36.4% Stage 4 (eGFR 15–30 mL/min)  |  10.9% Stage 3b (eGFR 30–45 mL/min)

  • Baseline mean eGFR: 16.73 mL/min/1.73 m² (Stage 4)

RESULTS

With respect to eGFR, the mean baseline value prior to administration of the Krebs Cycle intermediate metabolites formulation was 16.73 mL/min, and after 11 months of follow-up it reached 19.18 mL/min—representing an improvement, an increase in eGFR of 2.45 mL/min over nearly one year. This result was expected, as 69% of patients showed a decrease in creatinine levels, compared to only 31% who exhibited an increase.

Regarding creatinine, mean serum levels decreased from a baseline of 4.26 mg/dL to 3.77 mg/dL, corresponding to an annual reduction of 0.49 mg/dL. Notably, the reduction in creatinine occurred primarily in patients with Stage 5 CKD, with an average decrease of 0.92 mg/dL (close to 1 mg/dL).

In addition to the benefits of increased eGFR and reduced serum creatinine—findings not previously reported in the global literature—other important benefits of administering Krebs Cycle intermediate metabolites included:

  • Reduction in urea: Mean baseline value of 136.9 mg/dL decreased to 113 mg/dL.

  • Reduction in phosphorus: Mean baseline value of 5.04 mg/dL decreased to 3.87 mg/dL, resulting in 8 out of 10 patients not requiring additional phosphate binders.

  • Increase in serum hemoglobin: Mean baseline value of 11.16 g/dL increased to 11.68 g/dL, which allowed some patients to intermittently discontinue erythropoietin therapy.

  • Maintenance of normoalbuminemia, despite low-protein diets and the chronic inflammatory state characteristic of CKD.

  • Control of chronic metabolic acidosis due to its sodium bicarbonate content. A daily dose of 30 grams of the formulation (two 15 g servings) provides 1.95 grams of sodium bicarbonate. In this regard, the study by de Brito, “Sodium bicarbonate supplementation slows CKD progression and improves nutritional status,” used an average daily dose of 1.82 ± 0.8 grams [see Ref. 2]. Note: Serum bicarbonate was not measured in the present study.

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Prior to this clinical study, another open-label, observational, retrospective study was conducted in 20 patients with CKD stages 3b, 4, and 5 who received treatment with the same Krebs Cycle intermediate metabolites formulation. Follow-up ranged from a minimum of 1 month to a maximum of 5.5 years, with a mean patient age of 61.2 years.

Study results showed a reduction in urea from a mean baseline value of 148.5 mg/dL to 106.17 mg/dL, and for phosphorus, a decrease from a mean baseline of 6.7 mg/dL to 4.7 mg/dL [see Ref. 3].

MECHANISM OF ACTION

he Krebs Cycle intermediate metabolites in the studied formulation include citric acid, succinic acid, fumaric acid, and malic acid, combined with sodium bicarbonate and calcium-based phosphate binders: calcium carbonate and calcium lactate.

These acids, in the body’s aqueous environment, dissociate into their corresponding anions and enter the cell—more specifically the mitochondria (where the Krebs Cycle takes place)—where they participate in anaplerotic pathways (replenishment of metabolic intermediates). This process generates two molecules with a keto functional group at the alpha carbon: oxaloacetate (4 carbons) and alpha-ketoglutarate (5 carbons).

These two metabolic intermediates, through transamination reactions, capture NH₃ (ammonia) groups, leading to the formation of non-essential amino acids. Thus, oxaloacetate gives rise to aspartic acid, asparagine, and related amino acids; alpha-ketoglutarate generates glutamic acid, glutamine, and related amino acids. In total, 10 non-essential amino acids are produced through these reactions.

This process enables the reutilization of serum ammonium derived from amino acid catabolism and other molecules containing NH₂ and NH₃ groups, preventing ammonium from entering the urea cycle for subsequent elimination. As a result, the neoformation of non-essential amino acids improves the patient’s nutritional status.

Through this mechanism, serum urea levels decrease, while metabolic acidosis is simultaneously mitigated by the sodium bicarbonate content. Additionally, hyperphosphatemia is controlled through intestinal phosphate binding by calcium carbonate and calcium lactate.

Regarding the reduction in serum creatinine and the consequent increase in eGFR, the proposed hypothesis involves pleiotropic effects, as the formulation exerts multiple simultaneous benefits: reduction in urea, reduction in phosphorus, improvement in nutritional status, and control of metabolic acidosis.

The results of these two studies allow clinicians to consider incorporating Krebs Cycle intermediate metabolites as part of standard therapy in patients with CKD where preservation of renal function is a priority. There is both scientific and clinical evidence supporting their use, with the aim of delaying, as much as possible, the need for renal replacement therapies as a last-line management strategy.

Special consideration should be given to the use of this formulation in patients who, based on personal beliefs, decline renal replacement therapies, and for whom the clinician opts for a conservative, palliative treatment approach.

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THE KEY LIES IN ANAPLEROSIS

The term anaplerosis was first coined by Kornberg in 1966, describing mechanisms that replenish metabolic cycles. Under conditions of catabolic oxidation, the concentrations of Krebs Cycle intermediates are very low (10⁻⁵–10⁻⁴ mol/L). In this state, intermediates are neither significantly depleted nor newly synthesized.

However, beyond substrate oxidation, Krebs Cycle intermediates also serve as starting points for anabolic pathways. These intermediates are essential for multiple biosynthetic processes, including: fatty acid synthesis (citrate), heme biosynthesis (succinyl-CoA), gluconeogenesis (oxaloacetate), and the synthesis of non-essential amino acids (α-ketoglutarate and oxaloacetate) [see Ref. 4].

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Anaplerosis should be understood as a systemic process occurring in every cell of the body, rather than as an isolated phenomenon. In disease states, there is clear evidence that these Krebs Cycle substrates or intermediates become depleted.

Based on the metabolic pathways of the cycle—and as supported by the literature—replenishing these intermediates is expected to result in:

  • Improved heme synthesis, via succinyl-CoA / fumarate

  • Increased synthesis of non-essential amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine, tyrosine, and histidine

From this, one can infer an improvement in nutritional status and a reduction in the rate of ammonium formation, and consequently, urea production.

It is important to always view ammonium metabolism as an integrated system rather than in isolation: as ammonium levels decrease, urea levels also decrease. The urea cycle occurs primarily in the liver and involves the conversion of ammonium into urea for excretion.

KrebsCycle image.png

METABOLOMICS

Metabolomic and gene expression analyses have revealed a downregulation of Krebs Cycle intermediates in individuals with CKD. Furthermore, pathway analyses show that this cycle is among the most affected, with reductions in citrate and succinate levels ranging from 40% to 68%. These findings support the hypothesis that several pathologies may be rooted in impaired mitochondrial function.

Reduced Krebs Cycle activity may result from decreased mitochondrial biogenesis, reduced expression of genes encoding cycle enzymes, or diminished availability of cycle substrates.

In conclusion, a pattern of significantly altered metabolites has been observed, along with reduced cycle activity, decreased fatty acid oxidation, and increased ketone body metabolism [see Ref. 5].

Certain amino acids and Krebs Cycle intermediates also contribute to the cardioprotective response of the myocardium to ischemia. Studies have reported that the hypoxic-ischemic myocardium converts aspartate and glutamate into succinate via the Krebs Cycle, generating ATP and GTP. Fumarate has been shown to stabilize and increase the Nrf2 protein [see Ref. 6].

Under physiological conditions, the main substrates of the anaplerotic pathway of the Krebs Cycle include pyruvate, glutamate/glutamine, and precursors of propionyl-CoA (long-chain fatty acids, specific amino acids, and five-carbon ketone bodies).

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There is strong evidence that several pathological conditions may benefit from replenishment of anaplerotic substrates. Conditions associated with tissue reperfusion (myocardial infarction, embolism, organ transplantation) are linked to cellular membrane damage and a potential decrease in certain Krebs Cycle intermediates [see Ref. 7].

OTHER BENEFITS ASSOCIATED WITH
REDUCTION OF SERUM UREA

Urea can exert direct toxicity on various tissues, including the intestinal epithelium, vascular walls, pancreatic β-cells, and adipocytes, as well as indirect toxicity through carbamylation [see Ref. 8,9].

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Urea slowly dissociates into cyanate, which is rapidly converted into isocyanate. Carbamylation is recognized as a spontaneous post-translational modification of amino acids and proteins mediated by cyanate, leading to biochemical alterations [see Ref. 10].

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Urea induces the production of reactive oxygen species (ROS) in adipocytes, contributing to insulin resistance [see Ref. 11].

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Insulin secretion defects associated with CKD arise from elevated circulating urea levels, which increase islet protein O-GlcNAcylation and impair glycolysis [see Ref. 12].

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Carbamylated proteins are associated with both all-cause mortality and cardiovascular mortality in patients with end-stage CKD [see Ref. 13,14,15].

Cited References

  1. Hernández-Miramontes JA, Méndez-Durán A, Hernández-Villanueva JA. Tricarboxylic cycle intermediates in combination with calcium phosphate chelators and sodium bicarbonate increase eGFR in patients with stages 3b, 4 and 5 CKD: a retrospective observational study. Rev Colomb Nefrol. 2024;11(2):1-18. https://revistanefrologia.org/index.php/rcn/article/view/778/1115

  2. de Brito-Ashurst I, Varagunam M, Raftery MJ, Yaqoob MM. Bicarbonate supplementation slows progression of CKD and improves nutritional status. J Am Soc Nephrol. 2009;20(9):2075-2084. doi:10.1681/ASN.2008111205 

  3. Hernández-Miramontes JA, Hernández-Villanueva JA, Pacifuentes-Orozco A, Méndez-Durán A. Ácidos carboxílicos en combinación con quelantes cálcicos de fósforo y bicarbonato de sodio para el tratamiento de la uremia e hiperfosfatemia en pacientes con ERC estadios 3, 4 y 5. Gac Med Bilbao. 2019;116(3):104-109. https://gacetamedicabilbao.eus/index.php/gacetamedicabilbao/article/view/707​

  4. Werner C, Doenst T, Schwarzer M. Metabolic Pathways and Cycles. The Scientist's Guide to Cardiac Metabolism. 2016;39-55.

  5. Hallan S, Afkarian M, Zelnick LR, Kestenbaum B, Sharma S, Saito R, Darshi M, Barding GA, Raftery D, Ju W, Kretzler M, Sharma K, & De Boer IH. Metabolomics and gene expression analysis reveal down-regulation of the citric acid (TCA) cycle in non-diabetic CKD patients. EBioMedicine. 2017;26: 68-77. https://doi.org/10.1016/j.ebiom.2017.10.027

  6. Ashrafian H, Czibik G, Bellahcene M, Aksentijevic D, Smith A C, Mitchell SJ, Dodd MS, Kirwan J, Byrne J, Ludwig C, Isackson H, Yavari A, Støttrup NB, Contractor H, Cahill TJ, Sahgal N, Ball DR, Birkler RID, Hargreaves IP, Watkins H. Fumarate is cardioprotective via activation of the NRF2 antioxidant pathway. Cell Metabolism. 2012;15(3):361-371. https://doi.org/10.1016/j.cmet.2012.01.017

  7. Brunengraber H, Roe CR. Anaplerotic molecules: current and future. Journal of Inherited Metabolic Disease. 2005;29(2-3):327-331. https://doi.org/10.1007/s10545-006-0320-1

  8. Lau WL, Vaziri ND. Urea, a true uremic toxin: the empire strikes back. Clin Sci (Lond). 2017;131:3–12. 

  9. Vanholder R, Gryp T, Glorieux G. Urea and chronic kidney disease: the comeback of the century?. Nephrol Dial Transplant. 2018;33:4–12. 

  10. Seki M, et al. Blood urea nitrogen is independently associated with renal outcomes in Japanese patients with stage 3–5 chronic kidney disease: a prospective observational study. BMC Nephrology. 2019;20:115.

  11. D’Apolito M, Du X, Zong H, Catucci A, Maiuri L, Trivisano T, et al. Urea-induced ROS generation causes insulin resistance in mice with chronic renal failure. J Clin Invest. 2010;120:203–13. 

  12. Koppe, L. et al. Urea impairs β cell glycolysis and insulin secretion in chronic kidney disease. The Journal of Clinical Investigation. 2016;126(9):3598-3612.

  13. Berg AH, Drechsler C, Wenger J, Buccafusca R, Hod T, Kalim S, et al. Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure. Sci Transl Med. 2013;5:175ra29.

  14. Drechsler C, Kalim S, Wenger JB, et al. Protein carbamylation is associated with heart failure and mortality in diabetic patients with ESRD. Kidney Int. 2015;87:1201–8.

  15. Koeth RA, Kalantar-Zadeh K, Wang Z, Fu X, Tang WH, Hazen SL. Protein carbamylation predicts mortality in ESRD. J Am Soc Nephrol. 2013;24:853–61.

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