Acid Excretion Research Articles

Tryptophan Metabolism-Regulating Probiotics Alleviate Hyperuricemia by Protecting the Gut Barrier Integrity and Enhancing Colonic Uric Acid Excretion.

The balance of gut microbiota affects uric acid synthesis and excretion, influencing the development of hyperuricemia. This study aimed to investigate the effects and mechanisms of probiotics on hyperuricemia and adenine- and potassium oxonate-induced colonic damage. After two months of gavage at 109 CFU/day, the probiotic strains Lactobacillus rhamnosus UA260 and Lactobacillus plantarum YU28, identified through in vitro screening, significantly reduced serum uric acid levels in hyperuricemia mice from 109.71 ± 56.33 to 38.76 ± 15.06 and 33.22 ± 6.91 μmol/L, respectively. These strains attenuated inflammatory, repaired gut barrier damage, and enhanced colonic uric acid transporter function, thereby promoting uric acid excretion. Furthermore, the probiotics significantly reshaped gut microbiota by increasing the abundance of beneficial bacteria, including Lactobacillus and Coprococcus, while modulating tryptophan, purine, and riboflavin metabolism. Changes in tryptophan metabolites, specifically indole-3-propionic acid and indole-3-acetic acid, were correlated with xanthine oxidase activity, colonic injury, and the expression of the uric acid transporter protein ABCG2 during treatment. Probiotics intervention activated aryl hydrocarbon receptor pathways. These findings suggest that probiotics alleviate hyperuricemia and colonic inflammatory by regulating gut microbiota composition and tryptophan microbial metabolite pathways. Probiotics that modulate tryptophan microbial metabolism may provide a potential strategy for treating or preventing hyperuricemia.

The potential role of alkaline diets in prevention of calcium oxalate kidney stone formation.

Formation of kidney stones is considered a major global problem. Diet plays an important role in the management of kidney stone formation. The main goal of the present research was to evaluate the protective role of fruit and vegetable mixtures as models of an alkaline diet on formation of kidney stones in rats and to conduct molecular docking study. The chemical compositions, phenolic compound profile, β-carotene content, vitamin C and antioxidant activity of both mixtures were assessed. Fruit (-42.419 ) and vegetable (-11.13) mixtures recorded a negative potential renal acid load in the presence of macro-/micro-nutrients, β-carotene and phenolic compounds; chlorogenic acid was the major content in both mixtures. Both mixtures exhibited high antioxidant activity. Molecular docking study proved that rutin displayed the highest binding affinities for glycolate oxidase (-11.8 kcal mol-1) and lactate dehydrogenase (-10.1 kcal mol-1). The kidney stone model in rats exhibited metabolic acidosis in the urinary profile through reduction of citrate; Ca, Mg and K excretion and elevation of oxalate, creatinine, creatinine clearance, uric acid, urea and protein. Additionally, there was a significant reduction in plasma Ca, Mg and K levels, while liver and kidney function parameters improved significantly. Fruit and vegetable mixtures as models of an alkaline diet proved improvement in all the parameters. Histopathological examination of kidney sections of the kidney stone model showed crystal deposition, inflammation, and severe necrosis. Kidney sections of alkaline diet models indicated mild and moderate changes. Conclusion: The results of this study proved that both alkaline diet models were effective in protecting against kidney stone formation in vivo and in molecular docking studies.

Investigation of renal tubular function with newly diagnosed type 1 diabetes mellitus during diabetic ketoacidosis

BackgroundProximal renal tubular dysfunction occurs during diabetic ketoacidosis (DKA) in type 1 diabetes. However, only a few studies have reported on the multiple proximal renal tubular functions simultaneously. Moreover, to the best of our knowledge, distal renal tubular function has not yet been investigated.MethodsPatients with newly diagnosed type 1 diabetes mellitus were classified into those with DKA and those without DKA, and their proximal and distal renal tubular functions were investigated. The diagnostic criteria for DKA were blood glucose > 200 mg/dL, blood pH < 7.3 or HCO3– < 15 mEq/L, and urine ketone body positivity.ResultsSix patients with DKA and five patients without DKA were included. In patients with DKA, urinary β2-microglobulin levels were significantly higher, while blood pH, HCO3–, and tubular reabsorption of phosphorus were significantly lower than in those without DKA. There were no significant differences in blood glucose, HbA1c, serum phosphorus, urinary N-acetyl-beta-glucosaminidase, and urinary amino acid excretion between patients with and without DKA. Elevated NH3 levels and impaired urinary acidification were not observed in patients with and without DKA.ConclusionsIn patients with newly diagnosed type 1 diabetes mellitus complicated with DKA, multiple proximal renal tubular dysfunctions occur simultaneously, suggesting transient Fanconi syndrome. Distal renal tubular acidosis was unlikely. The diagnostic criteria for DKA are appropriate also in the view of proximal renal tubular dysfunction and are considered suggestive of pathophysiological factors that may cause proximal renal tubular dysfunction.

Natural Bioactive Compounds: Emerging Therapies for Hyperuricemia.

Hyperuricemia is a crucial feature of metabolic syndrome, characterized by elevated uric acid that causes urate crystal deposits in joints, kidneys, and subcutaneous tissues, resulting in gout and hyperuricemic nephropathy. The primary causes of uric acid metabolism disorder include overproduction and reduced excretion. The majority of uric acid in human body is derived from the breakdown of purine nucleotides. Overproduction of uric acid can result from increased concentration or activity of xanthine oxidase, the key enzyme responsible for uric acid synthesis. Alterations in the activity of proteins responsible for uric acid reabsorption and excretion can also affect serum uric acid. Many bioactive compounds derived from natural plants have been shown to inhibit xanthine oxidase activity to reduce uric acid production, modulate the activity of transport proteins to promote uric acid excretion, or alleviate oxidative stress and inflammation through various signaling pathways. These properties have garnered significant attention from researchers. In this paper, we first introduce the pathophysiological mechanisms of hyperuricemia, then summarize bioactive compounds with urate-lowering effects, and discuss their potential applications in treating hyperuricemia and its complications.

Phyllanthi Fructus ameliorates hyperuricemia and kidney injure via inhibiting uric acid synthesis, modulating urate transporters, and alleviating inflammation

Phyllanthi Fructus, known as Yuganzi (YGZ), is a unique medicine and food homologous fruit with both medicinal and nutritional properties. Its historical use in treating hyperuricemia (HUA) and gout is well-documented. However, the precise therapeutic effects and potential molecular mechanisms remain unclear. In this study, an experimental rat modelling by a high-fat/high-sugar diet and potassium oxonate/adenine oral administration was used to evaluate the pharmacodynamic effects of YGZ. Network pharmacology, molecular docking and molecular dynamics simulation were utilized to elucidate the potential mechanisms. Supplementation with YGZ effectively ameliorated HUA by inhibiting xanthine oxidase activity, and enhancing uric acid excretion through up-regulating of OAT1 and ABCG2, while down-regulating of URAT1. Furthermore, YGZ supplementation enhanced superoxide dismutase activity, reduced malondialdehyde content, and inhibited the secretion of IL1B, IL6, TNFα, ICAM1, VCAM1, TGFβ1, and NF-κB protein expression. Network pharmacology analysis indicated that YGZ influences 138 targets, modulating the disease network via lipid and atherosclerosis, insulin resistance, HIF-1, TNF, IL-17, TLRS, and NF-κB signaling pathways. Molecular docking analysis suggested that organic acids (e.g. ellagic acid, gallic acid) and flavonoids (e.g. quercetin, delphinidin, luteolin, epigallocatechin gallate) exhibited superior binding abilities to key targets (e.g. XDH, ABCG2, URAT1, OAT1, IRS1, PTGS2, TLR4). Noteworthy, molecular dynamics simulations confirmed that epigallocatechin gallate binds to URAT1 with the greatest stability. These results provide substantial evidence for the therapeutic efficacy of YGZ and establish a theoretical foundation for the development of natural products in treating hyperuricemia.

SLC6A19 inhibition facilitates urinary neutral amino acid excretion and lowers plasma phenylalanine.

BACKGROUNDThe toxic accumulation of phenylalanine (Phe) in the brain underlies the neurological presentation of phenylketonuria (PKU). Solute carrier family 6 member 19 (SLC6A19) is the major transporter responsible for the (re)absorption of Phe in the kidney and intestine. Here, we describe the characterization of the first small molecule SLC6A19 inhibitor to enter clinical development for the treatment of PKU.METHODSC57Bl/6J WT and Pahenu2 mice were dosed with an inhibitor of SLC6A19 to investigate the effects on urinary amino acids and plasma Phe. In a phase 1 study, healthy human volunteers were dosed with JNT-517, an investigational oral inhibitor of SLC6A19. The primary objective of the study was safety. Secondary objectives included pharmacokinetic and pharmacodynamic studies.RESULTSInhibition of SLC6A19 increased the urinary excretion of Phe in a mouse model of PKU, thereby reducing plasma Phe levels. JNT-517, an investigational oral SLC6A19 inhibitor, was found to be safe and well tolerated and increased the urinary excretion of Phe in a phase 1 healthy volunteer study.CONCLUSIONSThese data indicate that pharmacological inhibition of SLC6A19 presents a promising approach to lower toxic elevated levels of amino acids found in PKU and related amino acid metabolism disorders by facilitating their renal elimination.TRIAL REGISTRATIONAustralian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12622001222730.FUNDINGThe studies in this paper were funded by Jnana Therapeutics.

Fat Distribution and Urolithiasis Risk Parameters in Uric Acid Stone Formers and Patients with Type 2 Diabetes Mellitus.

Key Points Under a controlled diet, uric acid stone formers (UASFs) and diabetic patients have higher endogenous net acid production.Under a controlled diet, UASFs have lower ammonium-to-net acid excretion ratio.Body fat inversely correlates with urine buffer capacity in normal individuals, but this relationship is lost in diabetic patients and UASFs. Background Uric acid (UA) nephrolithiasis affects approximately 10% of kidney stones, with a greater preponderance among patients with obesity and diabetes mellitus (DM). UA lithogenicity is driven by abnormally acidic urine pH. Distinguishing the contribution of intrinsic (e.g., body adiposity) versus external (e.g., dietary) factors to UA stone propensity is challenging because of uncontrolled diets in outpatients in previously published studies. Methods This compilation of metabolic studies with body composition examined by dual-energy x-ray absorptiometry scan and blood and urine biochemistry collected under a controlled metabolic diet was conducted across three distinct populations: 74 UA stone formers (UASF group), 13 patients with type 2 DM without kidney stones (DM group), and 51 healthy volunteers (HV group). Results Compared with HVs, both UASFs and patients with DM exhibited higher levels of net acid excretion (NAE) and significantly lower urine pH and lower proportion of NAE excreted as ammonium (NH4 +/NAE), all under controlled diets. UASFs exhibited significantly lower NH4 +/NAE compared with patients with DM. UASFs also showed higher total body and truncal fat compared with HVs. Among the HVs, lower NH4 +/NAE ratio correlated with higher truncal and total fat. However, this association was abolished in the UASF and DM groups who exhibit a fixed low NH4 +/NAE ratio across a range of body and truncal fat. Conclusions The findings suggest a dual defect of diet-independent increase in acid production and impaired kidney NH4 + excretion as major contributors to the risk of UA stone formation. There is an inverse physiologic association between body fat content and NH4 +/NAE in HVs, whereas NH4 +/NAE is persistently low in UASFs and patients with DM, regardless of body fat, representing pathophysiology.

Liraglutide and Colesevelam Changes Serum and Fecal Bile Acid Levels in a Randomized Trial with Patients with Bile Acid Diarrhea.

Both liraglutide and colesevelam improve bile acid diarrhea (BAD) symptoms. Colesevelam binds excess amounts of diarrhea-causing bile acids in the colon whereas the mode of action for liraglutide remains elusive. Here, we examined the impact of colesevelam and liraglutide treatment on the concentrations of bile acids in serum and feces and the fecal microbiota composition to better understand the two drugs' modes of action. Bile acid species were analyzed in serum and fecal samples from a randomized, double-blind, double-dummy trial at baseline and after three and six weeks of orally administered colesevelam (1,875 mg twice daily, n = 26) or subcutaneously administered liraglutide (uptitrated by weekly increments of 0.6 mg from 0.6 to 1.8 mg daily, n = 26) in patients with 75selenium-homotaurocholic acid test-verified, idiopathic, or post-cholecystectomy BAD. Fecal microbiota composition was analyzed by 16S rRNA gene amplicon sequencing at the same time points. Colesevelam increased the fecal concentrations of all bile acid species while it decreased serum concentrations of secondary bile acids. Liraglutide induced a small increase in serum unconjugated bile acid concentrations without affecting fecal bile acid concentrations. No changes in fecal microbiota composition were observed with either treatment. Colesevelam and liraglutide exhibit distinct effects on serum and fecal bile acid concentrations with colesevelam reducing serum concentrations of secondary bile acids and promoting fecal bile acid excretion while liraglutide enhances serum concentrations of unconjugated bile acids, potentially through deceleration of small intestinal transit time allowing more time for passive absorption of bile acids.

Lactobacillus Kefir M20 Adaptation to Bile Salts: A Novel Pathway for Cholesterol Reduction.

(1) Background: This study investigated the impact of in vitro adaptations to acid and bile stress on the cholesterol-lowering activity of the probiotic Lactobacillus kefir M20. (2) Methods: Lactobacillus kefir M20 was extracted from fermented dairy products in Xinjiang, China, and isolated using MRS medium. The lactic acid bacteria were cultured for stress resistance to acid and bile salts and then gavaged into mice for animal experiments. (3) Results: The adaptation to bile stress treatment resulted in a notable enhancement of the cholesterol-lowering capacity of Lactobacillus kefir M20, with reductions of 16.5% and 33.1% in total and non-HDL cholesterol, respectively, compared to the untreated strain. Furthermore, the daily fecal total bile acid excretion was 9.2, 5.4 and 5.0 times higher in the M20-BSA group compared to the HC, M20 and M20-ASA groups, respectively. (4) Conclusions: This study suggests that targeted probiotics have the potential for application in the next generation of functional foods and probiotic formulations aimed at combating hypercholesterolemia.

NUMB dysfunction defines a novel mechanism underlying hyperuricemia and gout

Defective renal excretion and increased production of uric acid engender hyperuricemia that predisposes to gout. However, molecular mechanisms underlying defective uric acid excretion remain largely unknown. Here, we report a rare genetic variant of gout-unprecedented NUMB gene within a hereditary human gout family, which was identified by an unbiased genome-wide sequencing approach. This dysfunctional missense variant within the conserved region of the NUMB gene (NUMBR630H) underwent intracellular redistribution and degradation through an autophagy-dependent mechanism. Mechanistically, we identified the uric acid transporter, ATP Binding Cassette Subfamily G Member 2 (ABCG2), as a novel NUMB-binding protein through its intracellular YxNxxF motif. In polarized renal tubular epithelial cells (RTECs), NUMB promoted ABCG2 trafficking towards the apical plasma membrane. Genetic loss-of-function of NUMB resulted in redistribution of ABCG2 in the basolateral domain and ultimately defective excretion of uric acid. To recapitulate the clinical situation in human gout patients, we generated a NUMBR630H knock-in mouse strain, which showed marked increases of serum urate and decreased uric acid excretion. The NUMBR630H knock-in mice exhibited clinically relevant hyperuricemia. In summary, we have uncovered a novel NUMB-mediated mechanism of uric acid excretion and a functional missense variant of NUMB in humans, which causes hyperuricemia and gout.

Urine Metabolomics during a Legume Diet Intervention Suggests Altered Metabolic Signatures and Potential New Intake Markers: First Insights.

Given the general increase in legume consumption worldwide, there is a need to characterize the resulting human metabolic adaptations in order to demonstrate potential legume diet/health relationships. A nuclear magnetic resonance (NMR) metabolomics urine study was carried out on a small cohort (n = 18) to characterize the excretory effects of a pilot longitudinal 8-week legume-based dietary intervention. Despite the expected high interindividual variability in the excreted metabolome, the results suggested a nonlinear metabolic response, with higher metabolic activity in the first 4 weeks and a tendency toward baseline at the end of the intervention. The excretion of isoleucine, leucine, and threonine increased, along with metabolite changes suggestive of activation of the tricarboxylic acid cycle (through anaplerosis), ketogenesis, fat catabolism, and glycoprotein biosynthesis. Gut microbiota adaptations were also suggested based on the increased excretion of 2-hydroxyisobutyrate, allantoin, and hippurate. Increased levels of trigonelline were consistent with its role as a legume intake marker, whereas malonate and pseudouridine were suggested as possible additional markers. Correlation of NMR data with nutritional parameters aided putative explanatory hypotheses to be advanced. Our results suggest a dynamic response to legume consumption, mainly through increased amino acid excretion and altered energy metabolism, while advancing potential new markers of legume intake. These results require confirmation in larger cohorts but pave the way for an informed interpretation of the effects of legume-based diets on human health.

The alleviating effects and mechanisms of Lactiplantibacillus plantarum MC14 on hyperuricemia in mice

Hyperuricemia (HUA) is a chronic disorder resulting from purine metabolic dysregulation. This research explored the alleviating effects and potential mechanisms of Lactiplantibacillus plantarum MC14 on HUA in a mouse model. Further, it investigated the impact of MC14 intervention on the intestinal flora and metabolites of HUA model mice. Results indicated that the activity of MC14 in alleviating HUA may be linked to its abilities to lower serum levels of uric acid, creatinine, and blood urea nitrogen, reduce inflammation, alleviate renal tissue damage, enhance uric acid excretion, inhibit the overactivation of the PI3K/AKT/mTOR signaling pathway, suppress hepatic adenosine deaminase and xanthine oxidase activities, competitively absorb nucleosides and nucleobases, and regulate the intestinal flora. Our research demonstrates that MC14 is a potential candidate for developing novel therapeutics for hyperuricemia.

Bioavailability of mango (poly)phenols: An evaluation of the impact of the colon, and phenylalanine and tyrosine on the production of phenolic catabolites

A mango pulp purée was ingested by ileostomists, whose colon had been removed surgically, and subjects with a full gastrointestinal (GI) tract, after which ileal fluid, urine and feces were collected over a 24 h period and analysed by UHPLC-HR-MS. The main (poly)phenols in the purée were gallotannins (356 μmol) and two hydroxy-methoxy-cinnamoyl glucose esters (43 μmol) together with the aromatic amino acids phenylalanine (22 μmol) and tyrosine (209 μmol). Analysis of ileal fluid revealed almost all the ingested gallotannins appeared to have broken down in the upper GI tract with the released benzoic acids being rapidly absorbed into the circulatory system prior to urinary excretion mainly as phase-2 metabolites. Likewise, the glucose moiety of the cinnamic acid conjugates was cleaved and the released cinnamic acids absorbed efficiently from the proximal GI tract and subjected to phase II metabolism prior to excretion. Among the main phenolics excreted after mango intake were phenylacetic and benzoic acids and hydroxybenzene catabolites which were present in lower, but none-the-less, substantial amounts, in the urine of ileostomists. This indicates that a portion of these phenolics, including the hydroxybenzene derivatives, originate from substrates absorbed in the upper GI tract and are principally products of endogenous metabolism rather than being derived from colonic microbiota-mediated catabolism. 1,2,3-Trihydroxybenzene (aka pyrogallol) was the dominant urinary catabolite in both groups. Hippuric acid excretion exceeded (poly)phenol intake indicating a significant contribution from phenylalanine and tyrosine. The aromatic amino acids, while present in the ingested pulp, can also originate from several sources including breakdown of dietary proteins in the GI tract, and endogenous breakdown of surplus mammalian proteins independent of the GI tract. The trial was registered at clinical trials.gov as NCT06182540.

FUNCTIONAL STATE OF RENAL PARENCHYMA IN CHILDREN WITH DYSMETABOLIC NEPHROPATHY

Abstract. Metabolic nephropathy in children is a relevant issue in our time, as the prevalence of this disease is increasing. Negative consequences of metabolic nephropathy include a gradual deterioration of kidney function. A significant aspect is the fact that metabolic nephropathy can lead to the development of chronic kidney disease in children. Chronicity requires continuous medical monitoring and treatment to support kidney function and prevent further complications, such as renal insufficiency and other metabolic disorders. There is no doubt that one of the leading pathogenetic factors in kidney dysfunction is hypoxia. It can be caused by both hemodynamic disturbances associated with renal pathology and tissue respiration disorders in congenital dysplasia of the kidney's connective tissue, which arises in the embryo under the influence of epigenetic factors in utero and is further exacerbated by various epigenetic factors in postnatal life. Purpose of the work to investigate the functional state of kidney parenchyma in children with dysmetabolic nephropathy. Materials and methods. Two groups of children were examined: those with a complicated course of dysmetabolic nephropathy, a history of inflammatory episodes of the urinary system organs (Group I - UN, 52 children), and dysmetabolic nephropathy with persistent crystalluria (Group II - DN, 56 children). A control group consisted of 65 healthy children. Tubular reabsorption, as a partial function of the proximal segment of the nephrons, was assessed based on urinolysis test data, specifically the levels of excretion of inorganic phosphates, proteinuria, amino acids, reduced sugars (maltose, sucrose, fructose by Benedict's test), glucose (by Gaines's test), and calcium (by Sulcovitch's test). The level of creatinine and glycosaminoglycan (GAG) excretion in daily urine in children with dysmetabolic nephropathy was determined and compared with data from healthy children in the control group. Research results. In children from both observation groups, a significantly high frequency of hyperphosphaturia (94.23% and 96.42%), calciuria (94.23% and 89.28%), glucosuria (78.85% and 73.21%), increased excretion of amino acids in the urine (40.38% and 37.5%), microproteinuria (32.69% and 28.57%), and increased excretion of reduced sugars (28.85% and 26.79%) were observed. The daily excretion of creatinine, glycosaminoglycans, and oxypyrrolidine in the urine of children with dysmetabolic nephropathy indicates a significantly reduced level of creatinine in the daily urine in the majority of children in Group I (0.56±0.19 g/L, 80.77% of those examined) and more than half of the children in Group II (0.83±0.08 g/L, 57.14% of those examined), compared to the data from healthy children in the control group (1.25±0.75 g/L, 0.0%). Conclusions. The analysis of the functional state of the kidney tissue in oxalate dysmetabolic nephropathy in children allows us to testify to the presence of tubular and glomerular partial renal tissue failure. Significant decreases in creatinine excretion and increased excretion of glycosoaminoglycans in the urine and significantly higher levels of oxyproline in the urine indicate the presence of glomerular type partial renal failure in oxalate dysmetabolic nephropathy in children and the presence of undifferentiated dysplasia of the renal connective tissue with the onset of its sclerosis with subsequent transition to interstitial nephritis. The presence of these changes in the vast majority of children with complicated course of oxalate dysmetabolic nephropathy in the period of clinical and laboratory remission of the inflammatory process indicates the priority of renal connective tissue dysplasia and paucity of renal failure in the pathogenesis of dysmetabolic nephropathy and its severity in children. Indicators of oxyproline creatinine, glycosoaminoglycans and oxyproline excretion can be used as epigenetic markers of susceptibility to dysmetabolic nephropathy in general and its more severe course in particular. Keywords: dysmetabolic nephropathy, functional state, kidney parenchyma, nephron.

A rare and underestimated cause of anion gap metabolic acidosis in a pediatric patient

Abstract Recognized causes of anion gap metabolic acidosis include methanol, uremia, diabetic ketoacidosis, propylene glycol, isoniazid, iron, lactic acidosis, ethylene glycol, and salicylates, which is taught as the mnemonic, MUDPILES. While this mnemonic can be a useful learning tool, it is important to consider other, atypical causes. Here, we present the case of a 9-year-old male patient with a complex medical history, including trisomy 21 and Lennox-Gastaut syndrome who presented to the emergency department (ED) in hypotensive shock. Upon admission, he was noted to have a distended abdomen, evidence of acute pancreatitis and possible sepsis. Of note, his initial labs revealed an anion gap of 30.1 mmol/L, a whole blood lactate of 4.4 mmol/L, a pH of 7.2, and a normal pCO2, indicting an anion gap metabolic acidosis. Importantly, methanol, ethylene glycol, ketones, and salicylates were not detected, and renal function was normal. However, he did have an extensive medication history, including several antiepileptic drugs (AEDs). Over the coming weeks, he recovered from the acute pancreatitis, but developed fluid overload, electrolyte derangements (hypernatremia and hypokalemia), a worsening metabolic acidosis with an anion gap up to 54.6 mmol/L, and hyperammonemia (plasma NH3 of 232 μmol/L). At this point, the metabolism team was contacted for concerns regarding the rising ammonia levels, which subsequently responded to a combination of lactulose and Rifaximin. An inborn error of metabolism (IEM) was considered, and plasma amino acid analysis was performed (Waters Acquity MassTrak AAA solution) but was not diagnostic. Notably, the patient developed a significantly elevated gamma glutamyl transpeptidase (GGT) up to 4088 U/L, suggesting glutathione depletion, however, transaminases and alkaline phosphatase were normal. The laboratory was contacted by the metabolism providers for other possible causes of metabolic acidosis and it was suggested that the patient undergo urine organic acid testing. Urine specimens were analyzed using gas chromatography/mass spectrometry (GC/MS) (Aligent system). The results were notable for marked excretion of pyroglutamic acid (5-oxoproline), which is typically associated with acetaminophen, however, the patient’s acetaminophen levels were less than 5 μg/mL, indicating another, atypical cause of increased pyroglutamic acid. In this case, vigabatrin, an anticonvulsant known to cause glutathione depletion and secondary pyroglutamic aciduria was suspected, and subsequently withdrawn. The patient was also started on a trial of N-acetylcysteine (NAC) to replenish glutathione, which resolved the pyroglutamic aciduria and anion gap, and corrected the metabolic acidosis. This case highlights the importance of recognizing pyroglutamic acid as a rare cause of anion gap metabolic acidosis, even in the setting of normal acetaminophen levels, and the importance of organic acid testing in the work-up of such patients. Wider use of the mnemonic GOLD MARK, where the “O” stands for oxoproline, may help in the differential diagnosis of anion gap metabolic acidosis.

Diet rich in soluble dietary fibres increases excretion of perfluorooctane sulfonic acid (PFOS) in male Sprague-Dawley rats

Perfluorooctane sulfonic acid (PFOS) belongs to a large group of anthropogenic compounds with high persistency named per- and polyfluorinated substances (PFAS). Widespread use from industry to household appliances and food-contact materials contributes to PFAS exposure with food as the primary source. Association studies suggest that vegetables and fibre rich diet may reduce PFOS levels in humans, but experimental data remain limited. Here, we investigated PFOS uptake and wash-out after seven days of PFOS (3 mg/kg/day) in two groups of rats (N = 12 per group) fed diets either high (HF) or low (LF) in soluble dietary fibres. Two control groups (N = 12/group) were fed the same diets without PFOS. Changes in pH and transit time were monitored alongside intestinal and faecal microbiota composition. We quantified systemic and excreted, linear and branched PFOS. Results revealed significantly lower pH and faster intestinal transit in the HF groups. Importantly, HF rats had lower serum PFOS concentrations and higher PFOS concentrations in caecal content and faeces, indicating a more efficient excretion on the fibre rich diet. In both dietary groups, PFOS affected the gut microbiota composition. Our results suggest that a diet rich in soluble dietary fibres accelerates excretion of PFOS and lowers PFOS concentration in serum.

9210 Nephron specific ATP6AP2 knockout reduces renal fat accumulation in mice consuming high fat diet

Abstract Disclosure: S.A. Culver: None. J. Balugo: None. M. Jansen: None. T. Harris: None. H.M. Siragy: None. Diet induced obesity causes increased renal fat accumulation in both humans and mice which contributes to obesity related kidney injury. We have previously shown that mice with nephron specific ATP6AP2 knockout (KO) consuming high fat diet (HFD) are resistant to weight gain due to increased urinary excretion of glucose, albumin, and fatty acids. Magnetic resonance imaging (MRI) is a useful imaging modality for quantifying organ fat content in live animals though there are few reports of its use quantifying fat in murine kidney. We hypothesized that nephron specific ATP6AP2 knockout inhibits renal fat accumulation with HFD and that we would be able to detect this using MRI. Eight-week-old male wild type (WT) or inducible nephron specific ATP6AP2 KO mice underwent either induction with oral doxycycline or received sucrose water as controls before being placed on either normal diet (ND) (12% fat) or HFD (45% fat) for 3 months. Mice were then imaged using a 9.4 Tesla Bruker BioSpin MRI to determine both liver and kidney cortex fat content. The mean weight of WT HFD mice was 36.03 g compared to 29.03 g in WT ND controls. ND fed and HFD fed KO mice weighed 22.74 g and 23.03 g respectively. MRI showed significant 67% (p&amp;lt;0.05) increase in liver fat content in WT HFD compared to WT ND fed controls, confirming the ability of our MRI technique to detect expected differences in organ fat content. In renal cortex, MRI detected a 54% (p&amp;lt;0.05) increase in lipid content for WT HFD mice compared to WT ND controls. Nephron specific ATP6AP2 KO on HFD demonstrated reduced renal cortical lipid content by 36% (p&amp;lt;0.05) compared to WT HFD controls. These results indicate that ATP6AP2 KO is associated with reduced renal fat accumulation that is measurable with in vivo MRI. MRI therefore represents a valuable tool for studying renal sequelae of obesity in live murine models. Future investigation should examine whether ATP6AP2 KO prevents renal lipotoxicity and kidney injury in the setting of obesity. Presentation: 6/3/2024

Pyruvate stimulates transamination of leucine into α-ketoisocaproic acid and supports 3-methylbutanal production by Lactococcus lactis.

To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity. Lactococcus lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3-4 times after 72 h of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolizing cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 h than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal. These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production.

A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion

Abstract Background Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine. Methods Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined. Results Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient. Conclusions Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.

Paclitaxel chemotherapy disrupts microbiota-enterohepatic bile acid metabolism in mice

ABSTRACT Balanced interactions between the enteric microbiota and enterohepatic organs are essential to bile acid homeostasis, and thus normal gastrointestinal function. Disruption of these interactions by cancer treatment instigates bile acid malabsorption, leading to treatment delays, malnutrition, and decreased quality of life. However, the nature of chemotherapy-induced bile acid malabsorption remains poorly characterized with limited treatment options. Therefore, this study sought to characterize changes in hepatic, enteric, and microbial bile acid metabolism in a mouse model of chemotherapy-induced toxicity. Consistent with clinical bile acid malabsorption, chemotherapy increased fecal excretion of primary bile acids and water, while diminishing microbiome diversity, secondary bile acid formation, and small intestinal bile acid signaling. We identified new contributors to pathology of bile acid malabsorption in the forms of lipopolysaccharide-induced cholestasis and colonic crypt hyperplasia from reduced secondary bile acid signaling. Chemotherapy reduced markers of hepatic bile flow and bile acid synthesis, elevated markers of fibrosis and endotoxemia, and altered transcription of genes at all stages of bile acid metabolism. Primary hepatocytes exposed to lipopolysaccharide (but not chemotherapy) replicated chemotherapy-induced transcriptional differences, while gut microbial transplant into germ-free mice replicated very few differences. In the colon, chemotherapy-altered bile acid profiles (particularly higher tauromuricholic acid and lower hyodeoxycholic acid) coincided with crypt hyperplasia. Exposing primary colonoids to hyodeoxycholic acid reduced proliferation, while gut microbiota transplant enhanced proliferation. Together, these investigations reveal complex involvement of the entire microbiota-enterohepatic axis in chemotherapy-induced bile acid malabsorption. Interventions to reduce hepatic lipopolysaccharide exposure and enhance microbial bile acid metabolism represent promising co-therapies to cancer treatment.