Continuous Infusion of Iohexol to Monitor Perioperative Glomerular Filtration Rate.

Continuous monitoring of the glomerular filtration rate (GFR) in the perioperative setting could provide valuable information about acute kidney injury risk for both clinical and research purposes. This pilot study aimed to demonstrate that GFR measurement by a continuous 72 hrs iohexol infusion in patients undergoing colorectal cancer surgery is feasible. Four patients undergoing robot-assisted colorectal cancer surgery were recruited from elective surgery listings. GFR was determined preoperatively by the single-sample iohexol clearance method, and postoperatively at timed intervals by a continuous iohexol infusion for 72 hrs. Plasma concentrations of creatinine and cystatin C were measured concurrently. GFR was calculated as (iohexol infusion rate (mg/min))/(plasma iohexol concentration (mg/mL)). The association of the three different filtration markers and GFR with time were analysed in generalized additive mixed models. The continuous infusion of iohexol was established in all four patients and maintained throughout the study period without interfering with ordinary postoperative care. Postoperative GFR at 2 hours were elevated compared to the preoperative measurements for patients 1, 2, and 3, but not for patient 4. Whereas patients 1, 2, and 3 had u-shaped postoperative mGFR curves, patient 4 demonstrated a linear increase in mGFR with time. We conclude that obtaining continuous measurements of GFR in the postoperative setting is feasible and can detect variations in GFR. The method can be used as a tool to track perioperative changes in renal function.

Vitamin K catabolite inhibition of ovariectomy-induced bone loss: structure-activity relationship considerations.

SCOPE: The potential benefit of vitamin K as a therapeutic in osteoporosis is controversial and the vitamin K regimen being used clinically (45 mg/day) employs doses that are many times higher than required to ensure maximal gamma-carboxylation of the vitamin K-dependent bone proteins. We therefore tested the hypothesis that vitamin K catabolites, 5-carbon (CAN5C) and 7-carbon carboxylic acid (CAN7C) aliphatic side-chain derivatives of the naphthoquinone moiety exert an osteotrophic role consistent with the treatment of osteoporosis. METHODS AND RESULTS: Osteoblast-like MG63 cell cultures were challenged with lipopolysaccharide and the levels of interleukin-6, an osteoclastogenic cytokine, measured with and without catabolites; low concentrations of CAN7C significantly inhibited interleukin-6 release, but CAN5C did not. In models of bone loss induced by ovariectomy or sciatic neurectomy in C57BL/6 mice, we found that the rarer CAN7C catabolite markedly restricted ovariectomy-induced bone loss and possibly limited sciatic neurectomy-induced bone loss. CAN7C activity depends on a free carboxylic acid and its particular side-chain structure. CONCLUSION: These in vivo data indicate for the first time that the clinical utility of vitamin K for osteoporosis may reside in an unusual catabolite.

L-ornithine and phenylacetate synergistically produce sustained reduction in ammonia and brain water in cirrhotic rats.

UNLABELLED: Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L-ornithine and phenylacetate/phenylbutyrate (administered as the pro-drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L-ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine-derived glutamine as phenylacetylglutamine in the urine. Sprague-Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L-ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham-operated controls, which was significantly improved in the OP-treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L-ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups. CONCLUSION: The results of this study provide proof of the concept that L-ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats.

Ammonia impairs neutrophil phagocytic function in liver disease.

UNLABELLED: Hyperammonemia is a feature of liver failure, which is associated with increased risk of infection. The aims of the present study were to determine in vitro, in rats fed an ammoniagenic diet and in patients with cirrhosis, whether induction of hyperammonemia results in neutrophil dysfunction. As hyperammonemia produces cell swelling, we explored the role of the osmoregulating, p38 mitogen-activated protein kinase (p38(MAPK)) pathway in mediating this neutrophil dysfunction. Neutrophils were isolated from blood of healthy volunteers and incubated with either 75 microM ammonia or phosphate-buffered saline. Both groups were studied under hyponatremic conditions and/or with the addition of p38(MAPK) modulators. Neutrophil phagocytosis was measured in naive rats and rats fed an ammoniagenic diet and in patients with stable cirrhosis given placebo (n = 8) or an amino acid solution inducing hyperammonemia (n = 8). Cell volume and phagocytosis was analyzed by fluorescent-activated cell sorting using fluorescein isothiocyanate-labeled E. coli. p38(MAPK) phosphorylation was measured by western blotting. In healthy neutrophils incubated with ammonia and in rats fed an ammoniagenic diet, neutrophils showed evidence of swelling, impaired phagocytosis, and increased spontaneous oxidative burst compared to controls. Phagocytosis was significantly impaired in patients with induced hyperammonemia compared to placebo. The effects of hyperammonemia and hyponatremia were synergistic. The p38(MAPK) intracellular signaling pathways were activated in healthy neutrophils exposed to ammonia in association with increased burst activity. Neutrophil phagocytic dysfunction was abrogated by the addition of a p38(MAPK) agonist. CONCLUSION: Ammonia produces neutrophil swelling and impairs neutrophil phagocytosis. The p38(MAPK) intracellular signaling pathway has been shown to be important in mediating the ammonia-induced neutrophil dysfunction.

Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome.

UNLABELLED: Mortality in patients with alcoholic hepatitis (AH) remains high, and although corticosteroids are widely used for treatment, the results vary considerably. In AH, neutrophils are primed and infiltrate the liver to produce injury, but paradoxically, the main cause of death in such patients is infection. Our prospective study addressed this paradox of primed neutrophils on the one hand and increased risk of infection on the other. We hypothesized that the full activation of neutrophils by a humoral factor such as endotoxin renders them unable to respond to further bacterial challenge. We analyzed neutrophil oxidative burst and phagocytosis in whole blood by fluorescence-activated cell sorting analysis in 63 alcoholic patients with cirrhosis and patients with cirrhosis with superimposed AH (cirrhosis+AH). In 16 patients, ex vivo studies determined whether the removal of endotoxin restored neutrophil function. A resting burst greater than or equal to 55[corrected]%, indicating neutrophil activation and a reduced phagocytic capacity lower than 42%, was associated with significantly greater risk of infection, organ failure, and mortality. This defective neutrophil function was transmissible through patients' plasma to normal neutrophils, and patients' neutrophil function could be restored by normal plasma. The ex vivo removal of endotoxin from patients' plasma decreased the resting burst and increased the phagocytic function. CONCLUSIONS: Our study provides the rationale for a goal-directed approach to the management of patients with cirrhosis and AH, in which the assessment of neutrophil function may be an important biomarker to select patients for immunosuppressive therapy. The neutrophil dysfunction in cirrhosis and AH is reversible, with endotoxin-removal strategies providing new targets for intervention.

Increased gene and protein expression of the novel eNOS regulatory protein NOSTRIN and a variant in alcoholic hepatitis.

BACKGROUND & AIMS: Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers. METHODS: Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation. RESULTS: eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1. CONCLUSIONS: An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients.

Endotoxemia produces coma and brain swelling in bile duct ligated rats.

UNLABELLED: This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood-brain barrier. Adult Sprague-Dawley rats 4 weeks after bile duct ligation (BDL)/Sham-operation, or naïve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham-operated groups significantly (P < 0.05), but this was associated with progression to pre-coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood-brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham-operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro-inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF-alpha) and IL-6 significantly increased in LPS-treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non-LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). CONCLUSION: Injection of LPS into cirrhotic rats induces pre-coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre-coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins.

Inflammation is an important determinant of levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in acute liver failure.

Acute liver failure (ALF) is characterized by rapid progressive organ failure and poor outcome. The pathophysiology of multiorgan dysfunction in ALF remains unclear but increased systemic inflammatory response is believed to be an important determining factor. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, results from proteolysis and the liver is a major site for its metabolism. ADMA has been shown to independently predict outcome in multiorgan failure associated with severe liver dysfunction. In this study, we tested the hypothesis that proinflammatory cytokine driven responses are important in modulating ADMA levels in patients with acetaminophen-induced ALF. Blood samples were collected from 10 ALF patients (grade IV encephalopathy) from admission until the time of transplantation or death, and assayed for cytokines and ADMA. A total of 8 patients required treatment for raised intracranial pressure and all patients were managed with standard of care, including full mechanical ventilation and veno-venous hemofiltration. ADMA levels were markedly higher in ALF patients compared to age-matched controls (P < 0.001) and correlated with the levels of proinflammatory cytokines. In pretransplantation patients undergoing hepatic venous catheterization, we demonstrated no significant uptake of ADMA across the failing liver. However, following liver transplantation, ADMA levels reduced acutely. A timed study of ADMA levels during transplantation demonstrated a slight increase during the anhepatic phase but a marked and sustained reduction in ADMA following liver reperfusion. In conclusion, our data show a significant correlation between ADMA levels and proinflammatory cytokines, supporting a hypothesis that proinflammatory cytokines may regulate ADMA metabolism in ALF.

Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis.

UNLABELLED: Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function. CONCLUSION: Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis.

Hepatic guanylate cyclase activity is decreased in a model of cirrhosis: a quantitative cytochemistry study.

The production of nitric oxide (NO) in liver disease and its role in vascular control has been a subject of much interest in recent years. However, the activity of guanylate cyclase (GC), the enzyme activated by NO has received little attention with regard to liver disease. In this study we have utilised a quantitative cytochemical technique to examine the activity of GC on a per cell basis in a rat model of cirrhosis. Our results show a significant reduction in GC activity, indicating that vascular regulation is likely to be substantially affected irrespective of NO generation in this disease model.

Effect of albumin dialysis on intracranial pressure increase in pigs with acute liver failure: a randomized study.

BACKGROUND: Increased intracranial pressure (ICP) worsens the outcome of acute liver failure (ALF). This study investigates the underlying pathophysiological mechanisms and evaluates the therapeutic effect of albumin dialysis in ALF with use of the Molecular Adsorbents Recirculating System without hemofiltration/dialysis (modified, M-MARS). METHODS: Pigs were randomized into three groups: sham, ALF, and ALF + M-MARS. ALF was induced by hepatic devascularization (time = 0). M-MARS began at time = 2 and ended with the experiment at time = 6. ICP, arterial ammonia, brain water, cerebral blood flow (CBF), and plasma inflammatory markers were measured. RESULTS: ICP and arterial ammonia increased significantly over 6 hrs in the ALF group, in comparison with the sham group. M-MARS attenuated (did not normalize) the increased ICP in the ALF group, whereas arterial ammonia was unaltered by M-MARS. Brain water in the frontal cortex (grey matter) and in the subcortical white matter at 6 hrs was significantly higher in the ALF group than in the sham group. M-MARS prevented a rise in water content, but only in white matter. CBF and inflammatory mediators remained unchanged in all groups. CONCLUSION: The initial development of cerebral edema and increased ICP occurs independently of CBF changes in this noninflammatory model of ALF. Factor(s) other than or in addition to hyperammonemia are important, however, and may be more amenable to alteration by albumin dialysis.

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.

Pathophysiological effects of albumin dialysis in acute-on-chronic liver failure: a randomized controlled study.

The pathophysiological basis of acute-on-chronic liver failure (ACLF) is unclear but systemic inflammatory response is thought to be important. In patients with ACLF, the molecular adsorbents recirculating system (MARS) improves individual organ function, but the effect of MARS on the proposed mediators of systemic inflammatory response is unclear. The present study was designed to determine the effect of MARS on the cytokine profile, oxidative stress, nitric oxide, and ammonia. A total of 18 patients with alcohol-related ACLF due to inflammation-related precipitants were randomized to receive standard medical therapy (SMT) alone, or with MARS therapy over 7 days. Plasma cytokines, malondialdehyde (MDA), free radical production, nitrate / nitrite (NOx), and ammonia were measured. Encephalopathy improved significantly with MARS (P < .01), but not with SMT. Mean arterial pressure and renal function remained unchanged. No significant change of plasma cytokines and ammonia levels were observed in either group. Plasma MDA levels did not change either. There was a fall in NOx (P < .05) with MARS, but not with SMT. In conclusion, in inflammation-related ACLF patients, albumin dialysis using MARS results in improvement of encephalopathy, independent of changes of ammonia or cytokines, without improving blood pressure or renal function. These results should temper the liberal use of MARS until further data is available.

Anti-tumor necrosis factor-alpha monoclonal antibody therapy in severe alcoholic hepatitis.

BACKGROUND/AIMS: Severe alcoholic hepatitis (AH) is associated with high mortality. Tumor necrosis factor-alpha (TNFalpha) has been demonstrated to play an important role in its pathophysiology. METHODS: Twelve patients with biopsy-confirmed AH and a Maddrey discriminant factor >32 were treated with a single infusion of the anti-TNF monoclonal antibody Infliximab at a dose of 5mg/kg body weight. Serial measurements were made for various cytokines using specific enzyme-linked immunoassays (ELISA). In four patients, liver biopsy samples were available pretreatment and on day+28 of therapy. RESULTS: Ten of the 12 patients are alive at a median of 15 (12-20) months. Two patients died within 30 days from septicemia. Serum bilirubin levels, Maddrey score, neutrophil count and C-reactive protein fell significantly within the first month. There was an early, though not significant, decrease in plasma levels of proinflammatory cytokines (interleukins (IL)-1beta, IL-6, IL-8, interferon-gamma), whereas plasma levels of TNFalpha remained near the sensitivity limit of the assay throughout the treatment course. While TNFalpha mRNA expression in the liver did not change, expression of IL-8, a cytokine regulated mainly by TNFalpha, was almost absent on day+28. CONCLUSIONS: Our data suggest that randomized controlled trials of anti-TNF antibody in severe AH are warranted.