Received: 8 November 2022 | Accepted: 27 December 2022 DOI: 10.1097/HC9.0000000000000072 RESEARCH LETTER A multistrain probiotic increases the serum glutamine/ glutamate ratio in patients with cirrhosis: a metabolomic analysis Luca Laghi1 | Eva Román2,3,4 | Qiuyu Lan1 | Juan Camilo Nieto5 | Aleix Canalda-Baltrons 6 | Maria Poca 2,4 | Maria B. Sánchez-Rodríguez 7 | Joan Clària 4,7,8 | Edilmar Alvarado 2,4 | Berta Cuyàs 2 | Elisabet Sánchez4 | Sílvia Vidal5,9 | Carlos Guarner2,4 | Àngels Escorsell2 | Chaysavanh Manichanh 4,6 | German Soriano2,4 1 Department of Agricultural and Food Sciences, University of Bologna, Cesena, Italy 2 Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 3 Escola Universitària d’Infermeria EUI-Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 4 CIBERehd, Instituto de Salud Carlos III, Madrid, Spain 5 Institut de Recerca IIB-Sant Pau, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 6 Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain 7 Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain 8 European Foundation for the Study of Chronic Liver Failure (EF Clif), Barcelona, Spain 9 Department of Immunology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain Correspondence German Soriano, Department of Abstract Gastroenterology, Hospital de la Santa Creu i Sant Pau, Mas Casanovas, 90, Barcelona To explore the potential mechanisms underlying the effects of a probiotic in 08041, Spain. cirrhotic patients, we analyzed the blood metabolome using proton nuclear E-mail: gsoriano@santpau.cat magnetic resonance (1H-NMR) spectroscopy in 32 patients with cirrhosis Luca Laghi, Department of Agricultural and Food Sciences, University of Bologna, P.za and cognitive dysfunction or falls. Patients were randomized to receive a Goidanich, 60, Cesena 47521, Italy. multistrain probiotic or placebo for 12 weeks. Among the 54 metabolites E-mail: l.laghi@unibo.it identified, the only significant changes in the probiotic group were an increase in glutamine, a decrease in glutamate, and an increase in the glutamine/glutamate ratio. In the placebo group, glutamate increased and the glutamine/glutamate ratio decreased. Our results suggest the multistrain probiotic could influence glutamine/glutamate metabolism, increasing the capacity of ammonia detoxification. Abbreviation: 1H-NMR, proton nuclear magnetic resonance; FABP-6, fatty-acid binding protein 6; FDR, false discovery rate; FGF-21, fibroblast growth factor 21; MELD, model for end-stage liver disease; PHES, psychometric hepatic encephalopathy score. Luca Laghi and Eva Román are co-first authors. Luca Laghi and German Soriano are co-corresponding authors. Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s, www. hepcommjournal.com. ------------------------------------------------------------------------------------------------------- This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases. Hepatology Communications. 2023;7:e0072. www.hepcommjournal.com | 1 2 | HEPATOLOGY COMMUNICATIONS In a previous double-blinded, placebo-controlled, the 30 samples at the genus level, we observed this randomized clinical trial, we observed that a multistrain ratio was associated with an abundance of Para- probiotic improved cognitive function and risk of falls, prevotella and Oscillospira in feces (coefficient: −0.53, reinforced the intestinal barrier, and modulated the p = 0.002, q = 0.07, and coefficient: −0.29, p = 0.02, proinflammatory state in patients with cirrhosis.[1] How- q = 0.21, respectively). The abundance of Oscillospira ever, the mechanisms underlying these effects are not was also associated with gait speed (coefficient: −1.05, well known. One approach that can help identify such p = 0.01, q = 0.15). mechanisms is metabolomics. The increase in the serum glutamine/glutamate ratio We analyzed serum metabolome in samples from 32 observed in the present study in probiotic-treated outpatients with cirrhosis included in the study mentioned patients could reflect a higher capacity of ammonia above.[1] All patients had cognitive dysfunction (psycho- clearance because ammonia binds to glutamate to metric hepatic encephalopathy score [PHES] <−4) and/ generate glutamine.[3] Unfortunately, we did not measure or falls during the previous year. Seventeen patients serum ammonia levels, but other authors have demon- were treated for 12 weeks with the multistrain probiotic strated a decrease in ammonemia associated with a De Simone Formulation (Vivomixx) 450×109 cfu bid for decrease in hepatic encephalopathy (HE) episodes in 12 weeks, and 15 were treated with placebo. We patients with cirrhosis treated with this probiotic performed untargeted metabolomics by proton nuclear formulation.[4] A higher ammonia clearance capacity magnetic resonance (1H-NMR) using a procedure and the subsequent decrease in ammonemia, together previously described.[2] A subgroup of 9 patients in the with a decrease in the proinflammatory state, would probiotic group and 6 in the placebo group also provided explain the improvement in cognitive function and risk of fecal samples for the study of microbiota composition falls observed in patients treated with the probiotic.[1] This using 16S rRNA gene sequencing.[1] hypothesis is further supported by the positive correlation Patients had a mean age of 64.5 years, 65.6% were between the change in the glutamine/glutamate ratio and women, the etiology of cirrhosis was alcohol in 50%, the cognitive function, and the negative correlation between mean model for end-stage liver disease (MELD) score the ratio and the risk of falls. was 9.3, 81.2% had previous decompensations, 84.3% Our findings are in line with a recent study in rats with had previous falls, and 28.1% presented a PHES < −4. bile duct ligation, which showed the multistrain probiotic There were no statistical differences between the 2 used here ameliorated locomotor activity and improved the groups at baseline. neurometabolic profile assessed by in vivo brain ultrahigh The untargeted observation of the serum metabolome field 1H-NMR spectroscopy.[5] The authors found an by 1H-NMR allowed the unambiguous assignment of 54 attenuation in the increase in brain glutamine and a lesser metabolites (Supplemental Fig. 1, http://links.lww.com/HC9/ decrease in myo-inositol and glutamate. These changes A166, and Supplemental Table 1, http://links.lww.com/HC9/ indicate a reversal of the characteristic neurometabolic A167). Pairwise comparisons between values at baseline alterations observed in hyperammonemia and HE. and at 12 weeks (Figure 1) showed blood glutamine The exact mechanisms underlying the increase in increased in the probiotic group (p = 0.002, FDR p = the glutamine/glutamate ratio cannot be elucidated from 0.007) while glutamate decreased (p = 0.03, FDR p = the present study. One possible mechanism is that the 0.03), leading to an increase in the glutamine/glutamate probiotic could induce a decrease in glutaminase activity ratio (p = 0.009, FDR p = 0.01). In contrast, the placebo or an increase in glutamine synthetase activity, as has group showed an increase in glutamate concentration (p = been observed in experimental models with other 0.01, FDR p = 0.02) and a decrease of the glutamine/ treatments for HE, such as rifaximin[6] or ornithine glutamate ratio (p = 0.02, FDR p = 0.03). No statistically phenylacetate.[7] Another potential explanation for the significant changes were observed in any of the other increase in the glutamine/glutamate ratio is an improve- metabolites identified. We found a correlation between the ment in mitochondrial dysfunction. Mitochondrial dysfunc- change in the glutamine/glutamate ratio and the change in tion can lead to a decrease in glutamine due to its utilization the PHES (r = 0.34, p = 0.05), the change in the risk of to fuel the mitochondria through anaplerotic reactions to falls evaluated by the gait speed (r = 0.47, p = 0.008), obtain energy.[8] However, the lack of correlation between and the change in the intestinal barrier integrity assessed the change in the glutamine/glutamate ratio and the change by the fatty-acid binding protein 6 (FABP-6) (r = −0.37, p in FGF-21 does not support this hypothesis. = 0.04). We evaluated mitochondrial dysfunction by Regarding gut microbiota composition, with the limita- determining serum fibroblast growth factor 21 (FGF-21), tions of the small sample size, here we focused on the but we did not find a correlation between the change in glutamine/glutamate ratio and found that higher values in FGF-21 and the change in glutamine or the change in the blood were associated with lower abundance of Para- glutamine/glutamate ratio. prevotella and Oscillospira in feces. We failed to find a Regarding metagenomic results, when we focused relationship between these bacteria and ammonia metab- on the serum glutamine/glutamate ratio, analyzing all olism in previous literature. However, the abundance of SERUM GLUTAMINE/GLUTAMATE RATIO IN CIRRHOSIS | 3 F I G U R E 1 (A) Volcano plots showing the change between baseline and 12 weeks in all metabolites identified in the probiotic group and in the placebo group. (B) Changes in glutamine/glutamate ratio in patients treated with the probiotic and patients treated with placebo. Results are expressed as mean (SD). Abbreviation: FDR, false discovery rate. Paraprevotella has been associated with sedentarism,[9] could help in the design of new approaches to improve and the abundance of Oscillospira with physical frailty and patients’ outcomes and health-related quality of life. sarcopenia in older people.[10] In agreement with these data, we observed a negative association between the ACKNOWLEDGMENTS abundance of Oscillospira and gait speed—a parameter The authors thank Carolyn Newey for English language related to frailty.[1] Moreover, the change in gait speed revision. correlated positively with the change in the glutamine/ glutamate ratio. These data suggest a possible relationship between Oscillospira abundance in feces, frailty and FUNDING INFORMATION ammonia metabolism in patients with cirrhosis. This study was partially funded by grant PI12/00629 In conclusion, metabolomic analysis in patients with from the Instituto de Salud Carlos III. Mendes S.A. cirrhosis treated with this multistrain probiotic showed (Lugano, Switzerland) also partially funded the study. an increase in the serum glutamine/glutamate ratio. This finding contributes to the understanding of the C O N F L I C TS O F IN T E R E S T mechanisms involved in the effects of this probiotic and The authors have no conflicts to report. 4 | HEPATOLOGY COMMUNICATIONS ORCID chronic fatigue in COVID-19 patients. Front Nutr. 2022;8: Luca Laghi https://orcid.org/0000–0003–1593–7376 756177. Eva Román https://orcid.org/0000–0001–9084–8555 3. Wright G, Noiret L, Olde Damink SW, Jalan R. Interorgan ammonia metabolism in liver failure: the basis of current and Qiuyu Lan https://orcid.org/0000–0002–7812–0171 future therapies. Liver Int. 2011;31:163–75. Juan Camilo Nieto https://orcid.org/0000–0002– 4. Lunia MK, Sharma BC, Sharma P, Sachdeva S, Srivastava S. 3400–1488 Probiotics prevent hepatic encephalopathy in patients with Aleix Canalda-Baltrons https://orcid.org/0000–0001– cirrhosis: a randomized controlled trial. Clin Gastroenterol 8366–7377 Hepatol. 2014;12:1003–8. 5. Rackayová V, Flatt E, Braissant O, Grosse J, Capobianco D, Maria Poca https://orcid.org/000–0002–0235–1395 Mastromarino P, et al. Probiotics improve the neurometabolic Maria B. Sánchez-Rodríguez https://orcid.org/0000– profile of rats with chronic cholestatic liver disease. Sci Rep. 0003–0528–0294 2021;11:2269. Joan Clària https://orcid.org/0000–0003–4333–7749 6. Kang DJ, Kakiyama G, Betrapally NS, Herzog J, Nittono H, Hylemon PB, et al. Rifaximin exerts beneficial effects independ- Edilmar Alvarado https://orcid.org/0000–0003–2036–6133 ent of its ability to alter microbiota composition. Clin Transl Berta Cuyàs https://orcid.org/0000–0001–9988–7163 Gastroenterol. 2016;7:e187. Elisabet Sánchez https://orcid.org/0000–0002–3280– 7. Jover-Cobos M, Noiret L, Lee K, Sharma V, Habtesion A, 9301 Romero-Gomez M, et al. Ornithine phenylacetate targets Sílvia Vidal https://orcid.org/0000–0002–3909–6682 alterations in the expression and activity of glutamine synthase Carlos Guarner https://orcid.org/0000–0001–5957–9481 and glutaminase to reduce ammonia levels in bile duct ligated rats. J Hepatol. 2014;60:545–53. Àngels Escorsell https://orcid.org/0000–0001–5083–8467 8. Zhang IW, Curto A, López-Vicario C, Casulleras M, Duran-Güell Chaysavanh Manichanh https://orcid.org/0000– M, Flores-Costa R, et al. Mitochondrial dysfunction governs 0002–2287–4003 immunometabolism in leukocytes of patients with acute-on-chronic German Soriano https://orcid.org/0000–0002–9267–6811 liver failure. J Hepatol. 2022;76:93–106. 9. Bressa C, Bailén-Andrino M, Pérez-Santiago J, González- Soltero R, Pérez M, Montalvo-Lominchar MG, et al. Differences REFERENCES in gut microbiota profile between women with active lifestyle and 1. Román E, Nieto JC, Gely C, Vidal S, Pozuelo M, Poca M, et al. sedentary women. PLoS One. 2017;12:e0171352. Effect of a multistrain probiotic on cognitive function and risk of 10. Picca A, Ponziani FR, Calvani R, Marini F, Biancolillo A, falls in patients with cirrhosis: a randomized trial. Hepatol Coelho-Junior HJ, et al. Gut microbial, inflammatory and Commun. 2019;3:632–45. metabolic signatures in older people with physical frailty and 2. Santinelli L, Laghi L, Innocenti GP, Pinacchio C, Vassalini P, sarcopenia: results from the BIOSPHERE Study. Nutrients. Celani L, et al. Oral bacteriotherapy reduces the occurrence of 2019;12:65.