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Norfloxacin Modulates the Inflammatory Response and Directly Affects Neutrophils in Patients With Decompensated Cirrhosis

Patients with cirrhosis undergoing selective intestinal decontamination with norfloxacin show a reduction in serum cytokine levels, probably because of a combined effect of norfloxacin on bowel flora and neutrophils.Thirty-one patients with cirrhosis
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  Norfloxacin Modulates the Inflammatory Response and Directly AffectsNeutrophils in Patients With Decompensated Cirrhosis PEDRO ZAPATER,* ,‡ ROCÍO CAÑO, § LUCÍA LLANOS,* ANTONIO J. RUIZ–ALCARAZ,  SONIA PASCUAL, ‡,§ CLAUDIA BARQUERO, § ROCÍO MOREU,* PABLO BELLOT, § JOSÉ F. HORGA,* CARLOS MUÑOZ, ¶ JARA PÉREZ, § PILAR GARCÍA–PEÑARRUBIA,  MIGUEL PÉREZ–MATEO, § JOSÉ SUCH, ‡,§ and RUBÉN FRANCÉS ‡,§ *Sección de Farmacología Clínica, Hospital General Universitario, Alicante, and Universidad Miguel Hernández, Elche;  ‡ CIBERehd, Instituto de Salud Carlos III,Madrid;  § Unidad Hepática, Hospital General Universitario, Alicante, and Universidad Miguel Hernández, Elche;   Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Campus de Espinardo, Universidad de Murcia, Murcia; and   ¶ Sección de Inmunología, Hospital General Universitario, Alicante, and Universidad Miguel Hernández, Elche, Spain See related article, Guy J et al, on page 1236 in  CGH  . BACKGROUND & AIMS:  Patients with cirrhosis un-dergoing selective intestinal decontamination with nor-floxacin show a reduction in serum cytokine levels, prob-ably because of a combined effect of norfloxacin on bowelflora and neutrophils.  METHODS:  Thirty-one patientswith cirrhosis receiving norfloxacin (400 mg/day) wereincluded. Blood samples were collected at 0.5–4 hours(peak samples group, n  47) and at 22–24 hours (troughsamples group, n  84) after dose. Fifty-nine ascitic fluidsamples were obtained. Single doses of norfloxacin andtrimethoprim/sulfamethoxazole were administered to 13and 5 patients, respectively, (temporal profile group) andsamples were collected at 0, 0.5, 1, 1.5, 2, 4, and 24 hours.Norfloxacin, trimethoprim/sulfamethoxazole, cytokines,nitric oxide, expression levels of nuclear factor (NF)-  Band inhibitor of NF-  B (IkB-  ), neutrophil oxidativeburst, and rate of apoptotic events were determined. RESULTS:  All samples were bacterial DNA negative andhad no significant levels of lipopolysaccharide. Serumand ascitic levels of tumor necrosis factor-  , interferon-   ,interleukin-12, and nitric oxide were significantly lowerin peak than in trough samples. A correlation was presentbetween serum norfloxacins concentrations and tumornecrosis factor-   ( r   0.68;  P   .001), interferon-    ( r   0.66;  P     .001), interleukin-12 ( r     0.66;  P     .001),and nitric oxide ( r     0.68;  P     .001). Serum norflox-acin’s highest concentrations (1    0.5   g/mL) wereachieved at 1–2 hours and concurred in time with thelower levels of cytokines and nitric oxide. Intracellularnorfloxacin’s highest levels (2  1   g/mL/10 7 cells) wereobserved at 2 hours and concurred with a lower NF-  Bexpression, a reduced anion superoxide generation, andapoptotic rate in response to phorbol myristate acetate.Trimethoprim/sulfamethoxazole did not significantly modulate cytokine expression.  CONCLUSIONS: Nor-floxacin but not trimethoprim/sulfamethoxazolemodulatesinflammatoryresponseanddirectlyaffectsneutrophils in patients with cirrhosis. B acterial infections are common complications of cir-rhosis, and, among them, spontaneous bacterialperitonitis (SBP) is probably the most representative andclinically relevant. 1,2 The main pathogenic mechanismleading to bacterial infections in these patients is consid-ered to be translocation of viable bacteria from the in-testinal lumen. Hence, attempts have been made to re-duce the incidence of this complication in high-riskpatients by acting on intestinal flora: those with uppergastrointestinal bleeding receive primary prophylaxiswith oral norfloxacin (400 mg twice a day) for 1 week,and those surviving a previous episode of SBP are treatedindefinitely with oral norfloxacin (400 mg daily) as sec-ondary prophylaxis of SBP. In both circumstances, nor-floxacin administration significantly reduces the inci-dence of bacterial infections. 3,4 Selective intestinal decontamination (SID) of thebowel flora as primary prophylaxis of SBP, althoughbased on clinical characteristics of patients, has not beenuniversally accepted in clinical guides. 5 However, we haverecently shown that translocation of bacterial genomicfragments is associated with an increased risk of mortal-ity, 6 and it has also been reported that primary prophy-laxis of SBP with norfloxacin in a certain group of high-risk patients not only significantly decreases theincidence of SBP episodes but also improves survival by   Abbreviations used in this paper:  AF, ascitic fluid; LPS, lipopolysac-charide; NF-   B, nuclear factor-   B; PMN, polymorphonuclear leuko-cytes; QR, quinolone resistant; ROS, reactive oxygen species; SBP,spontaneous bacterial peritonitis; SID, selective intestinal decontami-nation. © 2009 by the AGA Institute0016-5085/09/$36.00doi:10.1053/j.gastro.2009.07.058      C     L     I     N     I     C     A     L     A     D     V     A     N     C     E     S     I     N     L     I     V     E     R  ,     P     A     N     C     R     E     A     S  ,     A     N     D     B     I     L     I     A     R     Y     T     R     A     C     T GASTROENTEROLOGY 2009;137:1669–1679  decreasing hepatorenal syndrome, one of the most deadly complications in these patients. 7 It is likely then that thecurrent criteria of primary prophylaxis may be expandedand become more common in the future. In this regard,the study of norfloxacin effects in patients with cirrhosis,besides its selective effect on intestinal bacterial flora,becomes relevant.Previous publications in small series of patients haveshown a marked reduction of proinflammatory cytokinelevels in patients undergoing SID. 8 However, immuno-logic effects of norfloxacin such as modulation of phago-cytic capacity or intraleukocytic killing 9 reported in otherclinical areas remain to be studied in this clinical setting.Neutrophil activation and development of an acute in-flammatory response are related to the production of reactive oxygen species (ROS) through activation of nic-otinamide adenine dinucleotide phosphate (NADPH) ox-idase. 10 ROS species such as superoxide anion (O 2  ) canact as bactericidal molecules 11 and acting through a p38 mitogen-activated protein kinase (MAPK)-dependentmechanism can result in the enhanced expression of nuclear factor-  B (NF-  B)-dependent proinflammatory cytokines. 10,12 These effects, however, need to be counter-balanced to minimize other detrimental effects of thesefree radicals, such as induction of apoptosis. 13 The present investigation was conducted in a series of patients with decompensated cirrhosis to determine (1)the influence of serum norfloxacin levels on the solubleinflammatory status of patients and (2) the relationshipbetween intracellular norfloxacin concentrations andneutrophil NF-  B signaling, oxidative burst, and numberof apoptotic events in response to stress. Patients and Methods  Patients  We conducted a prospective study in blood andascitic fluid (AF) of patients with cirrhosis. Inclusioncriteria were the presence of cirrhosis and culture-nega-tive AF from patients receiving norfloxacin (400 mg daily)as secondary prophylaxis of SBP. Cirrhosis was diagnosedby histology or by clinical, laboratory, and/or ultrasono-graphic findings. Exclusion criteria were the presence of culture-positive blood or AF, upper gastrointestinalbleeding, or intake of antibiotics in the preceding 2 weeksother than norfloxacin for secondary prophylaxis of SBP,hepatocellular carcinoma (1 nodule bigger than 5 cm indiameter, or 3 or more nodules 3 cm in diameter each),portal thrombosis, previous liver transplantation, tran-sjugular intrahepatic portosystemic shunt, alcoholic hep-atitis, older than 80 or younger than 18 years, and refusalto participate in the study. The Hospital General Univer-sitario’s Ethics Committee approved the study protocol,and all patients gave informed consent for inclusion inthe study.Blood was obtained for routine hematologic, biochem-ical, and coagulation tests. AF samples were obtainedwhen a large volume paracentesis was needed as a part of the patient’s treatment. Time interval between the lastdose of norfloxacin and blood/AF sampling was re-corded, and samples were classified accordingly in 2groups: (1) peak samples, obtained from 0.5 to 4 hoursafter drug intake, which is expected to reach the maxi-mum serum concentration of norfloxacin; and (2) troughsamples, drawn before the next dose, in which the daily minimum serum concentration of norfloxacin is ex-pected. Paracentesis was performed in aseptic conditionsfollowing the usual procedures when clinically needed.Total protein, albumin, leukocyte count, and polymor-phonuclear leukocytes (PMN) count were performed inall AF specimens. Both blood and AF were inoculated atbedside in aerobic and anaerobic blood culture bottles,10 mL each. Finally, separate blood and AF samples wereinoculated, under aseptic conditions, in rubber-sealedsterile Vacutainer SST II tubes (BD Diagnostics, Belgium)that were never exposed to free air.To study the acute effects of norfloxacin peak andtrough concentrations on proinflammatory mediatorsand cellular metabolic events, a time-course blood sam-pling was followed in a subgroup of 8 norfloxacin-naïvepatients. In these patients, blood samples were obtainedimmediately before and 30, 60, 90, 120, and 240 minutesafter the administration of 400 mg of oral norfloxacin.The effects of single doses of trimethoprim/sulfame-thoxazole (160/800 mg) and norfloxacin (400 mg) oncytokines and cellular events were compared in a 2-way crossover design, open-label, 2-period, 2-sequence study in 5 additional norfloxacin-naïve patients. Antibiotic ad-ministration sequence, with a minimum washout periodof 2 weeks, was randomly assigned. Schedule of samplesdrawing was at 0, 30, 60, 90, 120, 240 minutes, and 24hours after drug administration.  Detection of Bacterial DNA Fragments and  Measurement of Serum and AF LPS Levels  To detect the presence of bacterial DNA frag-ments in blood and AF, a broad-range polymerase chainreaction (PCR) was performed according to the method-ology described elsewhere. 14  A quantitative chromogeniclimulus amebocyte lisate (LAL) test (BioWhittaker, Notting-ham, UK) was followed to evaluate LPS levels in blood and AF samples as previously described. Samples and reagentswere handled in an airflow chamber and processed withpyrogen-free material tested by manufacturers.  Isolation of Human PMN Cells  PMNs from peripheral blood samples were isolatedwith PolymorphPrep (Axis–Shield PoC, Oslo, Norway) ac-cording to manufacturer’s instructions. After PMNs, isola-tion, cells were washed twice with freshly made phosphate-  C L  I   NI    C AL  ADVAN C E   S I   NL  I   VE  R  ,P AN C R E  A S  ,ANDB I   L  I   AR Y T  R A C T   1670 ZAPATER ET AL GASTROENTEROLOGY Vol. 137, No. 5  buffered saline (PBS) at 4°C. Cell viability was evaluated by trypan blue (Sigma, Madrid, Spain). Cell Cultures, Oxidative Burst Test, and  Annexin V Measurement  Cells were resuspended in phenol red free RPMI1640 medium (Gibco BRL, Life Technologies, Pais-ley, UK) supplemented with 10% human serum AB(BioWhittaker, Walkersville, MD), 100 IU/mL penicillin/streptomycin, and 2.5 mg/mL amphotericin B (GibcoBRL). For the respiratory burst test, the production of O 2  was evaluated by using a colorimetric assay based onthe intracellular production of blue formazan depositsformed by the reduction of membrane permeable ni-troblue tetrazolium (NBT). One million cells were cul-tured with 100   L of nitroblue tetrazolium (Sigma) andwith or without 0.5   g/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma) on a 24-well plate for 1 hour at37°C in a humidified atmosphere with 5% CO 2 . Afterincubation, cells were washed twice with warm PBS, oncewith methanol, and then air-dried to completely removeextracellular NBT. The NBT deposited inside the cellswas dissolved by adding first, 120   L of 2 mol/L KOH tosolubilize cell membranes and then 140   L of dimethylsulfoxide (DMSO) to dissolve blue formazan with gen-tle shaking for 10 minutes at room temperature. Thedissolved NBT solution was transferred to a 96-well plate,and absorbance was read on a Sunrise microplate readerat 620 nm (Tecan, Männedorf, Switzerland). Determina-tion of O 2  production by this colorimetric assay is notinterfered by nitric oxide metabolites, according to pre- vious specificity assays. Total endogenous annexin V lev-els were evaluated as an indirect measurement of apopto-tic activity in neutrophils by handling a human Annexin V ELISA kit from PromoKine (Heidelberg, Germany). Cellular Lysates and Immunoblotting  Cells were lysated with a commercial lysis buffer(Tris-HCl, pH 7.5, 20 mmol/L, NaCl 150 mmol/L,Na  2 EDTA 1 mmol/L, EGTA 1 mmol/L, Triton 1%, so-dium pyrophosphate 2.5 mmol/L,   -glycerophosphate 1mmol/L, Na  3  VO 4  1 mmol/L, leupeptin 1   g/mL; CellSignaling Technology, Boston, MA), 1 mmol/L phenyl-methylsulfonyl fluoride (PMSF). Protein concentrationwas obtained by Bradford assay. Protein extracts (15-  gprotein/lane) were resolved under reducing conditions on12% SDS-polyacrylamide gels and then transferred toImmobilon-P membranes (Millipore, Billerica, MA).Membranes were blocked with 5% milk in PBS withTween 20 0.1% for 1 hour at room temperature thenincubated with primary antibodies overnight at 4°C andfinally for 1 hour at room temperature with the corre-spondent horseradish peroxidase (HRP)-conjugated sec-ondary antibody. The primary antibodies used againstphosphorylated p65-nuclear factor (NF-  B (Ser 536), in-hibitor of NF-  B (I  B-  ), and   -actin were purchasedfrom Santa Cruz Biotechnology (Heidelberg, Germany).The activity of membrane-bound peroxidase was detectedusing the ECL chemiluminescent system from AmershamPharmacia Biotech (Piscataway, NJ). Protein bands werescanned and quantified by densitometry using Scion Im-age software (Scion Corp, Frederick, MD). Band densitieswere expressed relative to total   -actin protein.  Norfloxacin Norfloxacin in plasma samples was analyzedusing a high-pressure liquid chromatography (HPLC)method. Chromatographic separation was performed us-ing a reverse-phase Eclipse XDB-C18 column (5   m;4.6    150 mm). The mobile phase was an acetonitrile-tetrabutyl ammonium hydroxi-phosphate buffer pre-pared by adding 1.67 mL of   o -phosphoric acid 85% (J. T.Baker Chemical Co, Phillipsburg, NJ), 15 mL of tetrabu-tyl ammonium hydroxide 1.54 mol/L (Sigma ChemicalCo, St. Louis, MO) and 300 mL of acetonitrile to 1 L of deionized water. The mobile phase was eluted at a flow rate of 1 mL/min, and effluent was monitored at excita-tion and emission wavelengths of 278 and 456 nm, respec-tively. The limit of detection was 0.06   g/mL (signal tonoise ratio, 3:1). The calibration graph was found to belinear from 0.06 to 4   g/mL. All solvents used were HPLCgrade; norfloxacin was obtained from Sigma Chemical Co. Serum and AF samples preparation for HPLCinjection.  A 100-  L plasma sample was diluted with 100  L of deionized water and vortexed for 30 seconds. Atotal of 200   L of acetonitrile were added, and the mix-ture shaken and centrifuged at 6000 rpm for 10 minutes. A 20-  L aliquot of supernatant was injected, and peakareas were recorded. Serum norfloxacin concentrationswere expressed in micrograms/milliliters. Cell sample preparation for HPLC injection. Eight to 12 million cells were resuspended in 200   L HCl1.6% for 10 minutes and centrifuged at 6000 rpm for 10minutes. An aliquot of 100   L supernatant was dilutedwith 100   L deionized water and 200 mL acetonitrile, vortexed, and centrifuged at 6000 rpm for 10 minutes. A60-  L aliquot supernatant was injected, and peak areaswere recorded. Intracellular norfloxacin concentrationswere expressed in   g/mL/10 7 cells. Trimethoprim/Sulfamethoxazole Plasma samples were analyzed for trimethoprimand sulfamethoxazole according to an HPLC methodpreviously described. 15 Briefly, a Beckmann ultrasphereODS C18 column (5   m; 4.6    150 mm; at 40°C) wasused with a mobile phase consisting of 20 mmol/L so-dium hydrogen phosphate buffer, pH 3.0, and acetoni-trile (89/11, vol/vol) at a flow rate of 1.5 mL/min. Awavelength of 230 nm was used. Detection was linearfrom 0.5 to 60   g/mL for sulfamethoxazole and from0.05 to 5   g/mL for trimethoprim. Sulfamethoxazoleand trimethoprim were obtained from Sigma ChemicalCo.      C     L     I     N     I     C     A     L     A     D     V     A     N     C     E     S     I     N     L     I     V     E     R  ,     P     A     N     C     R     E     A     S  ,     A     N     D     B     I     L     I     A     R     Y     T     R     A     C     T November 2009 NORFLOXACIN CELLULAR EFFECTS IN CIRRHOSIS 1671  Serum and AF samples preparation for HPLCinjection.  Twenty microliters of 65% perchloric acid wereadded to 250   L plasma, vortexed, and centrifuged for 5minutes at 11,300  g  . Fifty milliliters of 2 mol/L dipotas-sium hydrogen phosphate was added to supernatant (100  L), mixed, and centrifuged for 1 minute at 11,300  g  . A20-  L aliquot of supernatant was injected, and peak areaswere recorded. Serum sulfamethoxazole and trimethoprimconcentrations were expressed in micrograms/milliliters. Cell sample preparation for HPLC injection.  Cellswere resuspended in 200   L HCl 1.6% for 10 minutes andcentrifuged at 6000 rpm for 10 minutes. An aliquot of 100  L supernatant was diluted with 100   L deionized waterand 20   L 65% perchloric acid, vortexed, and centrifuged at11,300  g   for 5 minutes. Fifty milliliters of 2 mol/L dipotas-sium hydrogen phosphate was added to supernatant (100  L), mixed, and centrifuged for 3 minutes at 11,300  g  . A60-  Laliquotsupernatantwasinjected,andpeakareaswererecorded. Intracellular sulfamethoxazole concentrationswere expressed in   g/mL/10 7 cells.  Measurement of Serum and Ascitic Fluid Cytokine Levels  Enzyme-linked immunosorbent assays (ELISAs)for the quantitative measurement of tumor necrosis fac-tor (TNF)-  , interferon (IFN)-   , and interleukin (IL)-12levels were carried out in basal serum and AF of patients,handling Human Quantikine kits (R&D Systems, Min-neapolis, MN), according to manufacturer’s instructions. All samples were tested in triplicate and read at 490 nmin a microplate reader. Lower limits of detection of allcytokine assays were between 5 and 10 pg/mL. Standardcurves were generated for each plate, and the average zerostandard optical densities were subtracted from the restof standards, controls, and samples to obtain a correctedconcentration for all cytokines.  Nitric Oxide Metabolite Levels in Serum and  Ascitic Fluid  The sum of the nitric oxide metabolites nitrite(NO 2  ) and nitrate (NO 3  ) is widely used as an index of nitric oxide generation and expressed as NOx levels. 16 NOxlevels in serum samples were calculated by measuring con- version of NO 3  to NO 2  by the enzyme nitrate reductaseusing an ELISA assay (R&D Systems) based on the Griessreaction that absorbs visible light at 540 nm. All sampleswere tested in triplicate; standard curves were generated foreach plate, and the average zero standard optical densitieswere subtracted from the rest of standards, controls, andsamples to obtain a corrected NOx concentration. Statistical Analysis  Continuous variables are reported as mean   standard deviation and categorical variables as frequen-cies or percentages. Differences in basal characteristicsbetween groups were analyzed using the    2 test for cate-gorical data and the Student  t   test for quantitative data.Bivariate correlations between continuous variables werecalculated using the Pearson test. Differences in cyto-kines and norfloxacin levels according to study groupand time point of sample collection were analyzed usingthe ANOVA test with Bonferroni correction for multiplecomparisons. All reported  P   values are 2-sided, and  P   values lower than .05 are considered to indicate signifi-cance. All calculations were performed using the SPSS15.0 software (SPSS, Inc, Chicago, IL). Results Clinical and Analytical Characteristics of Patients  Thirty-one patients with cirrhosis and ascites un-dergoing SID with norfloxacin were included in thisstudy. Male/female proportion was 24/7, and mean agewas 55.7  10.9 years old. At inclusion in the study, 58%of patients were classified as Child–Pugh B and 42% asChild–Pugh C. Mean Model for End-Stage Liver Diseasescore was 14.3  5.3. The etiology of cirrhosis was alco-hol in 21 patients, hepatitis C virus (HCV) in 6, andalcohol  HCV in 4 patients. A total of 31 serum and 14 AF samples were collected atinclusion. A second set of samples was collected from 27patients (27 serum 12 AF samples) in the following 15 days.The remaining samples were obtained after 1 month (18serum and 8 AF samples), 2 months (15 serum and 7 AFsamples),4months(12serumand5AFsamples),7months(10 serum and 5 AF samples), 8 months (6 serum and 3 AFsamples), 9 months (6 serum and 3 AF samples), and 10months (6 serum and 2 AF samples) of follow-up. Nosignificantchangesinclinicaloranalyticalstatusofpatientswere observed during the sample collection period. According to the moment in which the samples wereobtained, 47 serum and 14 AF samples were consideredas peak samples, and 84 serum and 45 AF samples wereclassified as trough samples. Analytical characteristics of both groups are detailed in Table 1. None of the serum and AF samples from patients included in this study showed the presence of bacterial DNA. Serum meanendotoxin values for the overall series of patients in-cluded were 0.36  0.04 UE/mL for the peak group and0.35  0.05 UE/mL for the trough group (  P   ns). As of August 2008 (end of the follow-up), 3 out of the31 (9.7%) patients included in the study had died. Causesof death included graft dysfunction after liver transplan-tation (n    1), liver insufficiency (n    1), and heartfailure (n  1).To investigate the acute effects of norfloxacin, 8 con-secutive norfloxacin-naïve patients undergoing a large- volume paracentesis were included in a protocol aimed atstudying the acute effects of norfloxacin concentrationson proinflammatory mediators and cellular metabolicevents. Out of the 5 patients included in the crossoverstudy to compare the effects of norfloxacin and tri-  C L  I   NI    C AL  ADVAN C E   S I   NL  I   VE  R  ,P AN C R E  A S  ,ANDB I   L  I   AR Y T  R A C T   1672 ZAPATER ET AL GASTROENTEROLOGY Vol. 137, No. 5  methoprim/sulfamethoxazole (see Patients and Meth-ods section), 3 were male, mean age was 60    10.3years. All of them were class Child–Pugh B. Etiology of cirrhosis was alcohol in 2 patients (both abstinent),HCV in 2 cases, and primary biliary cirrhosis in 1patient. All these 5 patients had presented ascites inthe previous 2 months and were currently receivingdiuretics as routine follow-up as outpatients. Theamount of AF was minimal and did not require para-centesis. No significant differences were observed be-tween these 5 patients and the remaining series of 8patients who received norfloxacin. These subgroups of patients did not show any statistically significant dif-ferences for any baseline analytical characteristics be-tween them or with the patients included in the gen-eral study (data not shown).  Norfloxacin Values in Peak and TroughSamples  Mean norfloxacin serum concentration in serumpeak samples was 1.0    0.5   g/mL (range, 0.4–2.6   g/mL) (Table 2). Norfloxacin was detected in all peak sam- ples. Among trough samples, mean norfloxacin concen-tration was 0.2  0.1   g/mL (range, 0.1–0.4   g/mL), andnorfloxacin was not detected in 25% of specimens. Thesedata are in agreement with concentrations previously described in healthy volunteers after the same dose of norfloxacin. 17 The mean peak norfloxacin concentration was signifi-cantly lower in AF than in serum (0.5  0.3  g/mL vs 1.0  0.5   g/mL, respectively;  P   .02). Peak values in AF were inthe range between 0.1 and 0.8   g/mL. Mean AF trough value was 0.2    0.2   g/mL, similar to serum value. Asignificant correlation between serum and AF norfloxacinconcentrations was observed (Pearson,  r   0.693;  P   .001).The peak AF/serum norfloxacin concentration relationshipwas 62.8%  45.1%, and the corresponding proportion fortrough samples was 93.2%  38.9%.The coefficient of variability of norfloxacin measure-ments in different serum and AF samples obtained froma same patient on different days was lower than 35% bothfor peak and trough samples. This result is according toa steady state treatment.  Proinflammatory Cytokines, NOx, and  Norfloxacin Levels Are Inversely Related in Patients With Cirrhosis and Ascites Undergoing SID Table 2 shows norfloxacin, proinflammatory cyto-kines, and NOx levels in serum and AF distributed ingroups according to peak and trough samples of nor-floxacin. Statistically significant differences were presentboth in serum and AF between groups for all studiedparameters. Overall, the higher the norfloxacin levels, theless the cytokine and NOx response is triggered. Simi- Table 1.  Analytical Characteristics of Patients According toGroup Distribution: Peak Samples, ObtainedBetween 0.5 and 4 Hours After Dose, and TroughSamples, Obtained Between 4 and 24 Hours AfterDose VariablePeak (0.5  4hours after dose)Trough (beforenext dose) P  valueSerum variablesNumber of samples 47 84Bilirubin ( mg/dL ) 3.1  3.6 2.5  2.3 .29Albumin ( g/dL ) 3.1  0.3 3.2  0.3 .93Quick ( %  ) 58.3  10.8 60.8  14.1 .47INR 1.5  0.3 1.5  0.4 .35Serum creatinine( mg/dL )1.3  0.6 1.1  0.5 .11Urea ( mg/dL ) 48.5  25.1 46.0  22.6 .54Serum sodium( mEq/L )135.3  4.1 134.0  4.8 .10Platelets/mm 3 78,233  46,811 92,740  51,258 .21Blood WBC/mm 3 5787  5713 4995  4638 .41AF variablesNumber of samples 14 45AF WBC/mm 3 63  72 98  168 .38% AF PMNs 5  16 8  18 .51AF total protein( g/dL )1.4  0.7 1.3  0.8 .88AF albumin ( g/dL ) 0.7  0.2 0.7  0.2 .78NOTE. Values represent mean  SD.INR, International normalized ratio; WBC, white blood cells; AF, asciticfluid; PMNs, polymorphonuclear cells. Table 2.  Norfloxacin, Cytokines, and NOx Levels in Serumand AF of Patients Included in the Study,According to Group Distribution: Peak Samples,Obtained Between 0.5 and 4 hours After Dose andTrough Samples, Obtained Between 4 and 24Hours After Dose VariablePeak (0.5  4hours afterdose)Trough (beforenext dose)  P   valueSerumNumber of samples 47 84Norfloxacin (  g/mL ) 1.0  0.5 0.2  0.1   .001Number of NDsamples0 (0%) 21 (25%)TNF-   ( pg/mL ) 74.7  43.3 304.4  157.2   .001 IFN-     (pg/mL)   156.0  44.0 242.8  79.7   .001 IL-12 (pg/mL)   176.7  76.0 407.8  195.8   .001NOx ( nmol/mL ) 11.1  3.8 24.0  8.0   .001AF Number of samples 14 45Norfloxacin (  g/mL ) 0.5  0.3 0.2  0.2   .001Number of NDsamples0 (0%) 12 (27%)TNF-   ( pg/mL ) 136.7  40.9 401.3  215.8 .002IFN-    ( pg/mL ) 196.6  43.7 309.4  78.3 .001IL-12 ( pg/mL ) 300.6  91.0 671.4  301.2 .003NOx ( nmol/mL ) 18.3  3.5 27.5  9.8 .019NOTE. Values represent mean  SD unless otherwise indicated.AF, ascitic fluid; ND, norfloxacin not detected.      C     L     I     N     I     C     A     L     A     D     V     A     N     C     E     S     I     N     L     I     V     E     R  ,     P     A     N     C     R     E     A     S  ,     A     N     D     B     I     L     I     A     R     Y     T     R     A     C     T November 2009 NORFLOXACIN CELLULAR EFFECTS IN CIRRHOSIS 1673
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