Research Article Changes in ADMA/DDAH Pathway after ...downloads.hindawi.com/journals/bmri/2014/627434.pdf · citrulline as follows. Solution A (diacetyl monoxime mM, thiosemicarbazide

Research ArticleChanges in ADMADDAH Pathway after HepaticIschemiaReperfusion Injury in Rats The Role of Bile

Andrea Ferrigno1 Vittoria Rizzo2 Alberto Bianchi1 Laura G Di Pasqua1

Clarissa Berardo1 Plinio Richelmi1 and Mariapia Vairetti1

1 Department of Internal Medicine andTherapeutics University of Pavia 27100 Pavia Italy2 Department of Molecular Medicine Fondazione IRCCS Policlinico S Matteo 27100 Pavia Italy

Correspondence should be addressed to Mariapia Vairetti mariapiavairettiunipvit

Received 22 May 2014 Revised 17 July 2014 Accepted 17 July 2014 Published 27 August 2014

Academic Editor Gjumrakch Aliev

Copyright copy 2014 Andrea Ferrigno et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We investigated the effects of hepatic ischemiareperfusion (IR) injury on asymmetric dimethylarginine (ADMA a nitric oxidesynthase inhibitor) protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved respin ADMA synthesis and degradation) and the cationic transporter (CAT) MaleWistar rats were subjected to 30 or 60min hepaticischemia followed by 60min reperfusion ADMA levels in serum and bile were determined Tissue ADMA DDAH activityDDAH-1 and CAT-2 protein DDAH-1 and PRMT-1 mRNA expression GSHGSSG ROS production and lipid peroxidationwere detected ADMA was found in bile IR increased serum and bile ADMA levels while an intracellular decrease was detectedafter 60min ischemia Decreased DDAH activity mRNA and protein expression were observed at the end of reperfusion Nosignificant difference was observed in GSHGSSG ROS lipid peroxidation and CAT-2 a decrease in PRMT-1 mRNA expressionwas found after IR Liver is responsible for the biliary excretion of ADMA as documented here for the first time and IR injuryis associated with an oxidative stress-independent alteration in DDAH activity These data are a step forward in the understandingof the pathways that regulate serum tissue and biliary levels of ADMA in which DDAH enzyme plays a crucial role

1 Introduction

Nitric oxide (NO) is abundantly synthesized from the aminoacid arginine by the action of NO-synthase (NOS) a familyof enzymes with an endothelial neuronal and inducibleisoform [1] Asymmetric dimethylarginine (ADMA) is anendogenous inhibitor of these enzymes because it competeswith L-arginine for each of the three isoforms of NOS it isconsidered an important marker of endothelial dysfunctionbecause of its inhibiting role in NO synthesis An increasein ADMA leads to vasoconstriction increases platelet aggre-gation increases cell adhesion to the endothelium andincreases vascular muscle cell proliferation [2] The first stepin the synthesis of methylarginines is the methylation ofprotein arginine residues by intracellular enzymes termedprotein methyltransferases (PRMTs) The second step relates

to the proteolytic degradation of the methylated proteinwhich produces free ADMA and symmetric dimethylargi-nine (SDMA) and the latter is not biologically active [3]The liver and kidneys represent the main sites of ADMAmetabolism and excretion The kidney plays an importantrole in the elimination of dimethylarginine from the bodysince ADMA is found in human urine [4] An additionalpathway was found for ADMA namely metabolic degrada-tion by dimethylarginine dimethylaminohydrolase (DDAH)an enzyme that is widely distributed in rats and humansubjects but in particular in the liver kidney and pancreas[5 6] Nijveldt et al provide a detailed insight into the liverrsquoshandling of dimethylarginine demonstrating that it playsa crucial role in ADMA metabolism with DDAH takingup a large amount of ADMA from the systemic circulation[7] Over the past few years types 1 and 2 isoforms of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 627434 11 pageshttpdxdoiorg1011552014627434

2 BioMed Research International

DDAH emerged as critical regulators of NO bioavailability[8] Studies of gene silencing or deletion in rodents led tothe conclusion that plasma levels of ADMA are regulatedby DDAH-1 whereas the significance of DDAH-2 lies inpreserving the endothelial function [8]

ADMA is also able to interfere with NO synthesis bycompeting with arginine and SDMA for cellular transportacross cationic amino acid transporters (CATs) Interestinglythe liver expresses CATs abundantly especially CAT-2A andCAT-2B suggesting a higher uptake of ADMA in this organas compared with the heart lungs and kidneys [9]

ADMA has been shown to correlate with cardiovascularrisk factors [10 11] and is considered a predictor of cardio-vascular events [12] Moreover ADMAplasma concentrationincreases in patients suffering from hepatic dysfunction[13] and end-stage kidney disease [14] and in situations ofendothelial dysfunction and increased atherosclerotic risk[15 16] Furthermore in ischemiareperfusion (IR) injuryADMA which is increased by reducing DDAH activity maywell influence NO production by competing with argininefor the binding site in the active NOS centre [17] Indeedevidence for the protective effects of NO synthesis was seenwhen NOS inhibitors dramatically worsened liver necro-sis and apoptosis [18] Correlation between methylargininederivatives and liver function and survival after liver trans-plantation was also observed [19] However this does notexhaust the relationship between ADMA and hepatic IRinjury Indeed the molecular mechanisms involved in IRinjury are not completely understood and only a few workshave reported changes induced by hepatic IR injury on theADMADDAH pathway which needs to be considered as apoint of interest potentially capable of reducing the effects ofIR In addition a previous studymerely reported cirrhosis bybile duct ligature (BDL) which induced an increase in plasmaADMA levels this did not happen with thioacetamide-(TAA-) induced cirrhosis [20] Yet no explanation aboutthis event has been reported Understanding themechanismsinvolved in ADMA elimination increases the possibility ofunderstanding its modulation which particularly crucial inseveral pathological conditions [10ndash14]

The present study was carried out to clarify whether(1) ADMA clearance also occurs through the bile and (2)hepatic IR induces changes in ADMA levels by quantifyingits content in hepatic tissue serum and bile samples Inaddition enzymatic activity and protein expression ofDDAHand mRNA expression of DDAH-1 and PRMT-1 and CAT-2protein at the end of reperfusion were examined

2 Materials and Methods

21 Animals The use of animals in this experimental studywas approved by the National Institute for Research and theanimals were cared for in accordancewith its guidelinesMaleWistar rats (200ndash250 gr 2-3months old Charles River CalcoLecco Italy) with free access to water and food were used

22Materials All reagentswere of the highest grade of purityavailable and were purchased from Sigma (Milan Italy)

23 IschemiaReperfusion (IR) Procedure The rats wereanesthetized with sodium pentobarbital (40mgkg ip) theabdomenwas opened via amidline incision and the bile ductwas cannulated (PE-50) Ischemia to the left andmedian lobewas induced by clamping the branch of the portal vein andthe branch of the proper hepatic artery after bifurcation tothe right lobe for 30 or 60min with microvascular clips [21]with the abdomen temporarily closed with a suture After30 or 60min of ischemia (119899 = 8 each group) the abdomenwas reopened the clips were removed the abdomen wasclosed again and the liverwas allowed to reperfuse for 60min(Figure 1) By using partial rather than total hepatic ischemiaportal vein congestion and subsequent bacterial translocationinto the portal venous blood were avoided A set of 60minischemia experiments followed by 60min reperfusion wasperformed after 5 hours of BDL (119899 = 6) (Figure 1) All theanimals were maintained on warm support to prevent heatloss rectal temperature was maintained at 37 plusmn 01∘C Toprevent postsurgical dehydration and hypotension 1mL ofsaline was injected in the inferior vena cava Sham-operatedcontrol animals (119899 = 8 each group) underwent similarmanipulation of the liver hilum without vascular occlusionor BDL and were kept under anesthesia for an equal length oftime

24 Bile Serum and Tissue Sampling Bile duct was cannu-lated as follows bile duct was closed with a 40 silk sutureligation placed in distal position This causes the bile ductto swell upstream becoming more discernible Then a cutwas opened in the bile duct with spring scissors and aslant-shaped polyethylene tubing was inserted When thetubing was completely filled with bile it was fixed with40 silk suture ligation This operation was conducted usingmagnifying spectacles Bile was collected in darkened vialsduring the reperfusion period Blood and tissue samples werealso collected after 60min reperfusion Blood was drawnfrom the vena cava and centrifuged at 3000 g for 10min at4∘C Hepatic biopsies were quickly removed from the centrallobe and immediately frozen in liquid nitrogen as were bileand serum samples until they were analyzed

25 Biochemical Assays Liver injury was assessed by serumrelease of alanine transaminase (ALT) aspartate transami-nase (AST) and alkaline phosphatase (ALP) by an automatedHitachi 747 analyzer (RocheHitachi Indianapolis IN USA)

ADMA levels in serum bile and tissue were evalu-ated by direct ELISA kit according to the manufactur-ing procedure (Immundiagnostik AG Germany) Quanti-tative analysis of ADMA was also performed in depro-teinized rat bile by reversed-phase high-performance liquidchromatography (RP-HPLC) with o-phthalaldehydebeta-mercaptoethanol (OPAbetaME) and fluorescence detectionas previously described with some modifications [22] Thechromatography was carried out on the HPLCHT400Esystem (ESSECI-Group Como Italy) equipment routinelyused for amino acid analysis that allows automatic onlinemixing of all reagents for OPA derivatizationThe derivativeswere separated on a Teknokroma Mediterranean sea C18

BioMed Research International 3

Group 1

Group 2

Group 3

Reperfusion (1 hour)


Reperfusion (1 hour)Bile duct ligation (5 hours)

Ischemia (60min)

Ischemia (60min)

Ischemia (30min)

Figure 1 Schematic illustration of the experimental groups

column (4 6times150mm 3 120583mparticle size) by binary gradientelution Fluorescence was measured at an excitation andemission wavelength of 330 and 450 nm respectively

Proteins were measured according to Lowryrsquos methodusing albumin as standard [23]

DDAHactivity was evaluated using themethod proposedby Tain and Baylis [24] Tissue samples were homogenizedin cold phosphate buffer 100mM pH 65 urease (100UmL)was added and samples were incubated at 37∘C for 15minADMA 1mM in phosphate buffer was added (final ADMAconcentration 08mM) and samples were incubated at 37∘Cfor 60min the reaction was stopped by mixing 1 1 with4 sulphosalicylic acid and samples were centrifuged for10min at 3000 g Finally the supernatants were assayed forcitrulline as follows Solution A (diacetyl monoxime 80mMthiosemicarbazide 2mM) and solution B (H2PO4 3MH2SO4 6M NH4Fe(SO4)2 175mM) were prepared mixed1 3 and added 1 1 to the samples Samples were incubated at60∘C for 110min and read spectrophotometrically at 528 nmagainst citrulline standards

Both DDAH-1 and CAT-2 protein expressions were eval-uated using Rat DDAH-1 and CAT-2 ELISA kit (CusabioWuhan University Science Park Wuhan China)

DDAH-1 and PRMT-1 mRNA were analyzed by a real-time polymerase chain reaction (RT-PCR) (Table 1) totalRNA was isolated from the liver samples with Trizol reagentin accordance with the method of Chomczynski and Mackey[25] RNA was quantified by measuring the absorbanceat 260280 nm cDNA was generated using the iScriptcDNA Synthesis kit (BIO-RAD) following the supplierrsquosinstructions Gene expression was analyzed using the SsoAdvanced SYBR Green supermix (BIO-RAD) As regardshousekeeping gene ubiquitin C (UBC) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were used (Table 1)DDAH-1 PMRT-1 UBC and GAPDH gene amplificationefficiency was 928 935 986 and 974 respectivelyin a cDNA concentration range of 10ndash01 ng120583L The expres-sion of the house-keeping gene remained constant in allthe experimental groups considered The amplification wasperformed through two-step cycling (95ndash60∘C) for 40 cyclesin a CFX Connect RT-PCR Detection System (BIO-RAD)following the supplierrsquos instructions All sampleswere assayedin triplicate Gene expression was calculated using the ΔCtmethod Comparison between groups was calculated usingthe ΔΔCt method

The hepatic concentration of total glutathione was mea-sured by an enzymatic method (Cayman Chemical CoAnn Arbor MI) Oxidized glutathione (GSSG) was deter-mined after derivatization of reduced glutathione (GSSG)

with 2-vinylpyridine Determination of hepatic reactiveoxygen species (ROS) was followed by the conversion of2101584071015840-dichlorofluorescein diacetate (H2DCFDA) to fluores-cent 2101584071015840-dichlorofluorescein (DCF) Tissue samples werehomogenized (50mgmL) in Lockersquos buffer (120mM NaCl25mM KCl 5mM NaHCO3 6mM g-glucose 1mM CaCl2and 10mMHEPES pH 74) and incubated for 20min at roomtemperaturewith 10mMH2DCFDA(Molecular Probes Inc)Production of the fluorescent derivative DCF as a functionof time (min) was measured using a microplate reader(Perkin Elmer Life Science Monza Italy) The extent of lipidperoxidation in terms of thiobarbituric acid reactive sub-stances (TBARS) formation was measured using Esterbauerand Cheesemanrsquos method [26] TBARS concentrations werecalculated using malondialdehyde (MDA) as standard

26 Statistical Analysis Data are presented as themeanplusmn SEStatistical analysis for multiple comparisons was performedby the one-way ANOVA test with Bonferronirsquos corrections

3 Results

31 Biliary Serum and Tissue Levels of ADMA InterestinglyADMA was found in bile for the first time and a time-dependent increase was observed after 30 and 60min ofischemia (Figure 2(a))

To confirm biliary ADMA levels bile samples were alsoanalyzed by the HPLC method equipped with a fluorescencedetector and no significant differences when comparing dataobtained by the ELISA kit were detected (ie 031 plusmn 004versus 029 plusmn 002 nmolmL resp in bile obtained after30min ischemia followed 60min reperfusion)

The ADMA levels in the serum of control rats confirmedpreviously reported levels in rats [27] (Figure 2(b)) The IRinjury induced a moderate increase in serum ADMA only in60min ischemic rats

Moreover using samples obtained after 60min ofischemia the evaluation of tissue ADMA showed a decreaseat the end of reperfusion (Figure 2(c))

After 5 hours of bile duct ligation no changes in serumADMA were found (Figure 2(b)) a significant increase inintracellular ADMA was recorded when compared with theunaltered bile production IR group (Figure 2(c))

32 Liver IR Injury As expected serum AST ALT and ALPincreased in animals submitted to ischemia and reperfusionas compared with the sham-operated group (Figure 3) Liverdamage depends on the ischemia period considered and


0

01

02

03

04

05

Bilia

ry A

DM

A (n

mol

mL)

30 60(min)

ShamIR

lowastP le 0002

lowastP le 003

N = 8

(a)

0

02

04

06

08

1

12

14

Seru

m A

DM

A (n

mol

mL)

30 60(min)

ShamIR

BDL IR

lowastP le 005

N = 6ndash8

(b)

0

3

6

9

12

15

18

30 60

Hep

atic

AD

MA

(pm

olm

g pr

ot)

(min)

ShamIR

BDL IR

lowastP le 005

lowastP le 003

N = 6ndash8

(c)

Figure 2 Biliary serum and tissue levels of ADMA at the end of reperfusion Livers were submitted to 30 or 60min ischemia followedby 60min reperfusion (119899 = 8) A set of 60min ischemia experiments followed by 60min reperfusion was performed after 5 hours of bileduct ligation (BDL) (119899 = 6) Sham-operated control animals had similar manipulation without vascular occlusion At the end of reperfusionbiliary blood and hepatic samples were collected from all groups The results are reported as the mean plusmn SE of 6ndash8 different experiments

Table 1 List of forward and reverse primers used in experiments

Gene Sequence

Rat DDAH-1 Forward 51015840-CAA CGA GGT CCT GAG ATC TTG GC-31015840

Reverse 51015840-GCA TCA GTA GAT GGT CCT TGA GC-31015840

Rat PRMT-1 Forward 51015840-TGC TGC ACG CTC GTG ACA AGT-31015840

Reverse 51015840-TCC ACC ACG TCC ACC AGG GG-31015840

Rat UBC Forward 51015840-CAC CAA GAA CGT CAA ACA GGA A-31015840

Reverse 31015840-AAG ACA CCT CCC CAT CAA ACC-51015840

Rat GAPDH Forward 51015840-AAC CTG CCA AGT ATG ATG AC-31015840

Reverse 51015840-GGA GTT GCT GTT GAA GTC GTC A-31015840


a time-dependent increase was recorded serum AST ALTand ALP levels increase especially after 60 minutes ofischemia (Figure 3)

Direct bilirubin concentrations in serum were signifi-cantly higher in the IR group as compared with the shamgroup In particular a threefold increase in serum directbilirubin was detected in the 60min ischemia group (Fig-ure 3)

The analysis of bile shows a time-dependent increase inenzyme (AST ALT and ALP) release high levels appeared inrats submitted to 60min ischemia (Figure 3) particularlyThesame trend was observed when comparing direct bilirubinlevels in bile (Figure 3)

33 DDAH Activity Protein and Expression in IR InjuryDecreased DDAH activity was observed at the end of reper-fusion and higher reduction was found after 30 and 60minischemia as reported in Figure 4(a)

DDAH contains SH groups in the catalytic site and weevaluated the oxidative stress so as to provide an explanationfor the reduction in its activity observed after IR Nosignificant changes in GSHGSSG ratio were observed at theend of reperfusion after both 30 and 60min of ischemia(Table 2) The evaluation of TBARS and ROS formationshowed the same trend and no significant difference in anyof the experimental groups considered was found (Table 2)

Evaluation of the protein and mRNA expression of iso-formDDAH-1 was performed after 60min reperfusion in the60min ischemic group The amount of DDAH-1 decrease atthe end of reperfusion and the detected results were evaluatedusing ELISA assay (Figure 4(b)) The same was also true forthe mRNA expression of DDAH-1 in the sample collected atthe end of reperfusion and compared with the control group(Figure 4(c))

34 PRMT-1 Expression in IR Injury HepaticmRNA expres-sion of isoform PMRT-1 was performed after 60min reper-fusion in the 60min ischemic group The amount of PMRT-1 decreased at the end of reperfusion as compared with thesham-operated group (Figure 5)

35 ADMA Transport via CAT-2 No significant changeswere detected for protein expression of CAT-2 transportersin rat liver samples obtained from the sham or IR group(Figure 6)

4 Discussion

This work demonstrated that the liver is responsible for thebiliary excretion of ADMA as documented here for the firsttime and also that IR injury induces changes in serumbiliary and hepatic levels of ADMA Notably a significantoxidative stress-independent decrease in DDAH activity thatassociates with low mRNA and protein expression occurs atthe end of the reperfusion period supporting the hypothesisthat the ADMADDAH pathway plays a crucial role in acutehepatic IR injury

41 Biliary Clearance of ADMA The liverrsquos ability as demon-strated in this work to eliminate ADMA through the bilesupports the previously published results an increase inplasma ADMA levels is induced only by cirrhosis causedby bile duct ligature (BDL) they are not increased bythioacetamide- (TAA-) induced cirrhosis [20] Laleman etal reported that the BDL rats exhibited a decreased rate ofADMA removal of about 50 probably due to blocked bileexcretion thus inducing an increase in circulating ADMAThe liver necrosis found in BDL rats was lower than thatin thioacetamide- (TAA-) induced cirrhosis thus excludingthe involvement of hepatic damage in the accumulationof serum ADMA concentration [20] In the present workIR following a short BDL period induced an increase inintracellular ADMA levels Moreover high ADMA plasmaconcentrations have been found in human alcoholic cirrhosisonly in association with high levels of plasma bilirubin [28]suggesting a possible relationship between the compromisedbiliary excretion of bilirubin and the increase in plasmaADMA Furthermore the induction of cirrhosis by BDLcaused an increase in plasma levels of both bilirubin andADMA and not in thioacetamide- (TAA-) induced cirrhosis[20] The present work reports that when compared withthe sham group a marked increase in serum bilirubin andsignificantly higher serum levels of ADMA were found with60min ischemia but not 30min ischemia

42 Changes in Tissue Serum and Biliary Levels of ADMAafter IR Injury Rat and human livers have been shownto play an important role in ADMA metabolism by takingup large amounts from the systemic circulation [7] Thepresent work shows that higher serum levels of ADMAas compared with control livers were found after 60minischemia the same trend was previously reported by Trochaet al [27] PlasmaADMA levels are increased in patients withliver cirrhosis [28] alcoholic hepatitis [29] and acute liverfailure [13] In keeping with the increase in serum ADMAlevels we recorded a tissue time-dependent decrease in tissueADMA content after IR Little is known about the changesin tissue ADMA during IR injury Interestingly Cziraki etal have reported myocardial ADMA during IR damagedemonstrating that ADMA concentrations are markedlyreduced during ischemia and increased during reperfusion[30] They began the evaluation after 2 hours of reperfusionand this could explain our low ADMA levels observed inliver after 1 hour of reperfusion Recently He et al reporteda significant decrease in tissue ADMA levels during acutemyocardial infarction [11] In the present work after both30min and 60min of ischemia we recorded a markedincrease in biliaryADMAwhen comparedwith the respectivesham-operated groups Recently the plasma concentrationof ADMA has been reported to increase significantly untilthe first postoperative day after cardiopulmonary bypass dueto extensive IR damage suggesting the use of ADMA as areliable and feasible marker of early IR injury [31] On theother hand in the 85 of patients who rejected the liver grafta clear increase in ADMA concentration preceded the onsetof the first episode of rejection [32] Thus serum ADMA


0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile

Figure 3 Serum and biliary levels of AST ALT and AP and direct bilirubin after 60min reperfusion Livers were submitted to 30 or 60minischemia followed by 60min reperfusion Sham-operated control animals had similar manipulation without vascular occlusion At the end ofreperfusion blood and biliary samples were collected from all groups The results are reported as the mean plusmn SE of 8 different experiments


0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)

Figure 4 Hepatic DDAH activity and protein and mRNA expression of DDAH-1 at the end of reperfusion Livers were submitted to 30or 60min ischemia followed by 60min reperfusion panel (a) Livers were submitted to 60min ischemia followed by 60min reperfusionpanels (b) and (c) Sham-operated control animals had similar manipulation without vascular occlusion After reperfusion liver sampleswere collected from all groups The results are reported as the mean SE of 8 different experiments

Table 2 GSHGSSG ratio MDA and ROS levels after 30 or 60min ischemia followed by 60min reperfusion

30min ischemia and 60min reperfusion 60min ischemia and 60min reperfusionSham IR Sham IR

GSHGSSG 91 plusmn 09 81 plusmn 05 93 plusmn 13 83 plusmn 07MDA (nmolmg liver) 19 plusmn 03 18 plusmn 05 23 plusmn 04 24 plusmn 09ROS (Arbitrary Unit) 1849 plusmn 39 1982 plusmn 71 2019 plusmn 34 2055 plusmn 56The results are reported as the mean plusmn SE of 8 different experiments

levels together with biliary ADMA concentrations could bea potential marker of dysfunction of the liver graft in theposttransplantation period In particular the increased levelsof biliary ADMA already detected after 30min ischemiapreceded the increase in serum levels of ADMA suggesting

that the evaluation of ADMA in bile could represent an earlymarker of liver injury It is worth noting that previous resultsdemonstrated that plasmaADMAevaluation appears to be anearly predictor for survival in patients with sepsis associatedwith acute liver failure [33]


0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8

Figure 5 Hepatic mRNA expression of PMRT-1 at the end ofreperfusion Livers were submitted to 60min ischemia followedby 60min reperfusion Sham-operated control animals had similarmanipulation without vascular occlusion After reperfusion liversamples were collected from all groups The results are reported asthe mean SE of 8 different experiments

43 DDAH and IR Injury DDAH is a key regulator ofintrahepatic ADMA homeostasis and to gain more insightsinto the role of tissue DDAH in the regulation of intracellularserum and biliary ADMA levels the present study evaluatesthe activity and expression of this enzyme using a rat modelof IR In particular DDAH is a cysteine hydrolase enzymethat may be inhibited by oxidative stress [8] and can be sup-pressed by both superoxide and H

2O2[34] To support this

hypothesis we evaluated hepatic MDA and ROS productionand the GSHGSSG ratio No changes after 30 or 60minischemia were found in any of these parameters As suggestedby Hu et al a more prolonged reperfusion time is neededto obtain a significant increase in MDA levels after hepaticIR damage [35] Based on the published results and thepresent data this work supports the hypothesis that oxidativestress-independent changes in DDAH activity occur in theliver after acute IR injury The decrease in DDAH activityobserved after hepatic IR is associated with a reduction inmRNA and protein expression and an increase in serumADMA levels Interestingly the reduction in DDAH activityby deleting the gene or by inhibiting its transcription RNAshas been found to lead to an increase in plasma ADMA levels[36 37] whereas an overexpression of DDAH in transgenicmice led to a decrease in ADMA levels [38] Our resultssupport the previous findings that the serum concentrationof ADMA is negatively associated with DDAH activity inliver [39] One possible explanation for the contrasting resultsobtained by Volti et al could be connected with reperfusiontime they evaluated the tissue DDAH after 3 hours of

0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8

Figure 6 Hepatic CAT-2 protein at the end of reperfusion Liverswere submitted to 60min ischemia followed by 60min reperfusionSham-operated control animals had similar manipulation withoutvascular occlusion After reperfusion liver samples were collectedfrom all groups The results are reported as the mean SE of 8different experiments

reperfusion while we quantified the DDAH activity afteronly 1 hour of reperfusion [40] Recent data in rat liversubjected to IR has demonstrated a decrease in DDAHactivity related to the age of rats [17] We also used youngrats and this could justify the decrease of DDAH activity inaccordance with Trocha et al [17] Moreover the evaluationof renal IR injury has demonstrated a reduction in DDAHactivity and expression after 1 hour of reperfusion leadingto an increase in ADMA levels and cardiovascular risks [41]Thus we suggest that while intracellular ADMA levels willdrop during reperfusion a concomitant decrease in DDAHactivity and expression and a simultaneous ADMA release inthe circulation and bile will occur

DDAH-1 gene regulation in ischemia can be attributed tofarnesoid X receptor (FXR) FXR plays critical roles in thetranscriptional regulation of various genes Sequence analysisof the DDAH-1 gene reveals the presence of an FXR responseelement (FXRE) and a dose-dependent response to FXRantagonist GW4064 in terms of DDAH-1 gene expressionhas been shown [42] Furthermore FXR suppression hasbeen demonstrated in HEPG2 cells exposed to ischemiaThissuppression was p38 MAPK and not HIF1alpha dependent[43]

DDAH-2 is the predominant isoform of these enzymesin the vasculature as it is found in endothelial cells Genesilencing of DDAH-2 reduces vascular NO generation andproduces vasoconstriction DDAH-2 is also expressed inthe kidney in the macula densa and distal nephron [8]During IR injury the main role of degradation of ADMA


is conducted by DDAH-1 and the role of DDAH-2 is not soclear It is known that renin-angiotensin-aldosterone system(RAAS)may promote the progression of IR injury especiallyin the kidney [44] Furthermore angiotensin type 1 receptoractivation in kidneys reduces the expression of DDAH-1 butincreases the expression of DDAH-2 This isoform-specificdistribution and regulation of DDAH expression in liverkidney and blood vessels provides a potential mechanism forsite-specific regulation of NO production [8]

PMRTs involved in the demethylation reaction can bedivided into two groups type I producing ADMA and typeII producing SDMA [45] We evaluated mRNA expressionof PMRT-1 at the end of IR and our results support therecently published data the highest value was recorded insham-operated animals not in the IR group [45]

The kidneys and the liver play a key role in the regulationof the systemic serum concentrations of ADMA The kidneyis mainly involved in urinary excretion and the liver has amajor role in ADMAmetabolism In this study we also foundthat biliary excretion plays a role in ADMA elimination andthat biliary excretion increases during IR injury ADMAcan be released in extracellular space by cationic amino acidtransporters (CATs) that are also involved in the removal ofcirculating ADMA by the liver [46] In the present studyno changes in CAT-2 involved in ADMA transport weredetected after IR injuryThis confirms the recently publisheddata obtained using an ischemic acute kidney injury model[47] Cellular disposal of ADMA can take place by twomechanisms degradation by DDAH and export from the cellvia CAT [46] Diminished activity and expression of DDAH-1 and no changes in CAT-2 activity may be involved in theincrease in extracellular ADMA levels after IR

The hepatoprotective effects of NO have been well doc-umented in several models of liver injury including IR [18]thus the understanding of ADMADDAH pathway may beconsidered as a potential point of interest to reduce the effectof IR injury

In conclusion the main findings of this study are asfollows (1) ADMA is excreted in the bile as we havedocumented for the first time (2) biliary ADMA excretionincreases after IR and (3) hepatic IR injury is associatedwith an oxidative stress-independent alteration in DDAHactivity Although the relationship between the changes inADMA concentration in serum bile and hepatic tissueand hepatic injury needs to be clarified these data area fundamental step forward in the development of noveltherapeutic strategies that regulate serum tissue and biliarylevels of ADMA in which the DDAH enzyme plays a crucialrole

Abbreviations

ADMA Asymmetric dimethylarginineBDL Bile duct ligatureCATs Cationic transportersDDAH Dimethylarginine dimethylaminohydrolasePRMT Protein methyltransferaseSDMA Symmetric dimethylarginine

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Mr Massimo Costa for his skillful tech-nical assistance Professor Anthony Baldry for revising theEnglish and Mrs Nicoletta Breda for the editing assistanceThis work has been supported by Fondazione Cariplo Grantno 2011-0439

References

[1] U Forstermann H H Schmidt J S Pollock et al ldquoIsoformsof nitric oxide synthase Characterization and purification fromdifferent cell typesrdquo Biochemical Pharmacology vol 42 no 10pp 1849ndash1857 1991

[2] M C Richir R H Bouwman T Teerlink M P C Siroen T PG M de Vries and P A M van Leeuwen ldquoThe prominent roleof the liver in the elimination of asymmetric dimethylarginine(ADMA) and the consequences of impaired hepatic functionrdquoJournal of Parenteral and Enteral Nutrition vol 32 no 6 pp613ndash621 2008

[3] D Zakrzewicz and O Eickelberg ldquoFrom arginine methylationto ADMA a novel mechanism with therapeutic potential inchronic lung diseasesrdquo BMC Pulmonary Medicine vol 9 article5 2009

[4] C Zoccali ldquoAsymmetric dimethylarginine in end-stage renaldisease patients a biomarker modifiable by calcium blockadeand angiotensin II antagonismrdquo Kidney International vol 70no 12 pp 2053ndash2055 2006

[5] M Kimoto G SWhitley H Tsuji and T Ogawa ldquoDetection ofNGNG-dimethylarginine dimethylaminohydrolase in humantissues using a monoclonal antibodyrdquo The Journal of Biochem-istry vol 117 no 2 pp 237ndash238 1995

[6] M Kimoto H Tsuji T Ogawa and K Sasaoka ldquoDetection ofN119866N119866-dimethylarginine dimethylaminohydrolase in the nitricoxide-generating systems of rats using monoclonal antibodyrdquoArchives of Biochemistry and Biophysics vol 300 no 2 pp 657ndash662 1993

[7] R J Nijveldt T Teerlink M P C Siroen A A Van LambalgenJ A Rauwerda and P A M Van Leeuwen ldquoThe liver is animportant organ in the metabolism of asymmetrical dimethy-larginine (ADMA)rdquo Clinical Nutrition vol 22 no 1 pp 17ndash222003

[8] F PalmM LOnozato Z Luo andC SWilcox ldquoDimethylargi-nine dimethylaminohydrolase (DDAH) expression regulationand function in the cardiovascular and renal systemsrdquo TheAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 293 no 6 pp H3227ndashH3245 2007

[9] Y Hattori K Kasai and S S Gross ldquoCationic amino acidtransporter gene expression in cultured vascular smoothmusclecells and in ratsrdquo The American Journal of PhysiologymdashHeartand Circulatory Physiology vol 276 no 6 pp H2020ndashH20281999

[10] J P Cooke ldquoAsymmetrical dimethylarginine the ubermarkerrdquoCirculation vol 109 no 15 pp 1813ndash1819 2004

[11] HHe SWang X Li et al ldquoAnovelmetabolic balancemodel fordescribing themetabolic disruption of and interactions between


cardiovascular-relatedmarkers during acute myocardial infarc-tionrdquoMetabolism Clinical and Experimental vol 62 no 10 pp1357ndash1366 2013

[12] J T Kielstein and C Zoccali ldquoAsymmetric dimethylarginine acardiovascular risk factor and a uremic toxin coming of agerdquoTheAmerican Journal of Kidney Diseases vol 46 no 2 pp 186ndash202 2005

[13] R PMookerjee R NDaltonN ADavies et al ldquoInflammationis an important determinant of levels of the endogenousnitric oxide synthase inhibitor asymmetric dimethylarginine(ADMA) in acute liver failurerdquo Liver Transplantation vol 13no 3 pp 400ndash405 2007

[14] P Vallance A Leone A Calver J Collier and S MoncadaldquoAccumulation of an endogenous inhibitor of nitric oxidesynthesis in chronic renal failurerdquoThe Lancet vol 339 no 8793pp 572ndash575 1992

[15] A Surdacki ldquoL-arginine analogsmdashinactive markers or activeagents in atherogenesisrdquo Cardiovascular and HematologicalAgents in Medicinal Chemistry vol 6 no 4 pp 302ndash311 2008

[16] R Schnabel S Blankenberg E Lubos et al ldquoAsymmetricdimethylarginine and the risk of cardiovascular events anddeath in patients with coronary artery disease results from theAtheroGene Studyrdquo Circulation Research vol 97 no 5 pp e53ndashe59 2005

[17] M Trocha A Merwid-Ląd E Chlebda-Sieragowska et alldquoAge-related changes in adma-ddah-no pathway in rat liversubjected to partial ischemia followed by global reperfusionrdquoExperimental Gerontology vol 50 pp 45ndash51 2014

[18] G P Yagnik Y Takahashi G Tsoulfas K Reid N Murase andD A Geller ldquoBlockade of the L-arginineNO synthase pathwayworsens hepatic apoptosis and liver transplant preservationinjuryrdquo Hepatology vol 36 no 3 pp 573ndash581 2002

[19] P Martın-Sanz L Olmedilla E Dulin et al ldquoPresence ofmethylated arginine derivatives in orthotopic human livertransplantation relevance for liver functionrdquo Liver Transplan-tation vol 9 no 1 pp 40ndash48 2003

[20] W Laleman A Omasta M van de Casteele et al ldquoA role forasymmetric dimethylarginine in the pathophysiology of portalhypertension in rats with biliary cirrhosisrdquo Hepatology vol 42no 6 pp 1382ndash1390 2005

[21] G Palladini A Ferrigno V Rizzo et al ldquoLobe-specificheterogeneity and matrix metalloproteinase activation afterischemiareperfusion injury in rat liversrdquo Toxicologic Pathologyvol 40 no 5 pp 722ndash730 2012

[22] V Rizzo A Anesi L Montalbetti G Bellantoni R Trotti andG VMelzi DrsquoEril ldquoReference values of neuroactive amino acidsin the cerebrospinal fluid by high-performance liquid chro-matography with electrochemical and fluorescence detectionrdquoJournal of Chromatography A vol 729 no 1-2 pp 181ndash188 1996

[23] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[24] Y Tain and C Baylis ldquoDetermination of dimethylargininedimethylaminohydrolase activity in the kidneyrdquo Kidney Inter-national vol 72 no 7 pp 886ndash889 2007

[25] P Chomczynski and K Mackey ldquoSubstitution of chloroformby bromo-chloropropane in the single-step method of RNAisolationrdquo Analytical Biochemistry vol 225 no 1 pp 163ndash1641995

[26] H Esterbauer and K H Cheeseman ldquoDetermination ofaldehydic lipid peroxidation products malonaldehyde and 4-hydroxynonenalrdquoMethods in Enzymology vol 186 pp 407ndash4211990

[27] M Trocha A Merwid-Lad A Szuba et al ldquoEffect of simvas-tatin on nitric oxide synthases (eNOS iNOS) and arginine andits derivatives (ADMA SDMA) in ischemiareperfusion injuryin rat liverrdquo Pharmacological Reports vol 62 no 2 pp 343ndash3512010

[28] P Lluch B Torondel P Medina et al ldquoPlasma concentrationsof nitric oxide and asymmetric dimethylarginine in humanalcoholic cirrhosisrdquo Journal of Hepatology vol 41 no 1 pp 55ndash59 2004

[29] R P Mookerjee M Malaki N A Davies et al ldquoIncreasingdimethylarginine levels are associated with adverse clinicaloutcome in severe alcoholic hepatitisrdquo Hepatology vol 45 no1 pp 62ndash71 2007

[30] A Cziraki Z Ajtay A Nemeth et al ldquoEffects of coronaryrevascularization with or without cardiopulmonary bypasson plasma levels of asymmetric dimethylargininerdquo CoronaryArtery Disease vol 22 no 4 pp 245ndash252 2011

[31] M P C SiroenM CWarle T Teerlink et al ldquoThe transplantedliver graft is capable of clearing asymmetric dimethylargininerdquoLiver Transplantation vol 10 no 12 pp 1524ndash1530 2004

[32] T Brenner T H Fleming C Rosenhagen et al ldquoL-arginine andasymmetric dimethylarginine are early predictors for survivalin septic patients with acute liver failurerdquo Mediators of Inflam-mation vol 2012 Article ID 210454 11 pages 2012

[33] Y Tain Y Kao C Hsieh et al ldquoMelatonin blocks oxidativestress-induced increased asymmetric dimethylargininerdquo FreeRadical Biology andMedicine vol 49 no 6 pp 1088ndash1098 2010

[34] M Fukai T Hayashi R Yokota et al ldquoLipid peroxidationduring ischemia depends on ischemia time in warm ischemiaand reperfusion of rat liverrdquo Free Radical Biology and Medicinevol 38 no 10 pp 1372ndash1381 2005

[35] X Hu D Atzler X Xu et al ldquoDimethylarginine dimethylam-inohydrolase-1 is the critical enzyme for degrading the car-diovascular risk factor asymmetrical dimethylargininerdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 31 no 7 pp1540ndash1546 2011

[36] D Wang P S Gill T Chabrashvili et al ldquoIsoform-specificregulation by NGNG-dimethylarginine dimethylaminohydro-lase of rat serum asymmetric dimethylarginine and vascularendothelium-derived relaxing factorNOrdquoCirculation Researchvol 101 no 6 pp 627ndash635 2007

[37] K Hasegawa S Wakino S Tatematsu et al ldquoRole of asym-metric dimethylarginine in vascular injury in transgenic miceoverexpressing dimethylarginie dimethylaminohydrolase 2rdquoCirculation Research vol 101 pp e2ndashe10 2007

[38] M Davids M C Richir M Visser et al ldquoRole of dimethylargi-nine dimethylaminohydrolase activity in regulation of tissueand plasma concentrations of asymmetric dimethylargininein an animal model of prolonged critical illnessrdquo MetabolismClinical and Experimental vol 61 no 4 pp 482ndash490 2012

[39] R Lanteri R Acquaviva C Di Giacomo et al ldquoRutin in rat liverischemiareperfusion injury effect on DDAHNOS pathwayrdquoMicrosurgery vol 27 no 4 pp 245ndash251 2007

[40] G L Volti V Sorrenti R Acquaviva et al ldquoEffect of ischemia-reperfusion on renal expression and activity of NG-NG-Dimethylarginine dimethylaminohydrolasesrdquo Anesthesiologyvol 109 no 6 pp 1054ndash1062 2008


[41] T Hu M Chouinard A L Cox et al ldquoFarnesoid X receptoragonist reduces serum asymmetric dimethylarginine levelsthrough hepatic dimethylarginine dimethylaminohydrolase-1gene regulationrdquo Journal of Biological Chemistry vol 281 no52 pp 39831ndash39838 2006

[42] T Fujino K Murakami I Ozawa et al ldquoHypoxia downregu-lates farnesoid X receptor via a hypoxia-inducible factor-inde-pendent but p38 mitogen-activated protein kinase-dependentpathwayrdquo FEBS Journal vol 276 no 5 pp 1319ndash1332 2009

[43] Z Wang Y Liu Y Han et al ldquoProtective effects of aliskiren onischemia-reperfusion-induced renal injury in ratsrdquo EuropeanJournal of Pharmacology vol 718 pp 160ndash166 2013

[44] M Trocha A Merwid-Lad T Sozanski et al ldquoInfluence ofezetimibe on ADMA-DDAH-NO pathway in rat liver subjectedto partial ischemia followed by global reperfusionrdquo Pharmaco-logical Reports vol 65 no 1 pp 122ndash133 2013

[45] T Teerlink Z Luo F Palm and C S Wilcox ldquoCellular ADMAregulation and actionrdquo Pharmacological Research vol 60 no 6pp 448ndash460 2009

[46] B Betz K Moller-Ehrlich T Kress et al ldquoIncreased symmet-rical dimethylarginine in ischemic acute kidney injury as acausative factor of renal L-arginine deficiencyrdquo TranslationalResearch vol 162 no 2 pp 67ndash76 2013

[47] T A Koeppel J C Thies P Schemmer et al ldquoInhibition ofnitric oxide synthesis in ischemiareperfusion of the rat liver isfollowed by impairment of hepatic microvascular blood flowrdquoJournal of Hepatology vol 27 no 1 pp 163ndash169 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


DDAH emerged as critical regulators of NO bioavailability[8] Studies of gene silencing or deletion in rodents led tothe conclusion that plasma levels of ADMA are regulatedby DDAH-1 whereas the significance of DDAH-2 lies inpreserving the endothelial function [8]

ADMA is also able to interfere with NO synthesis bycompeting with arginine and SDMA for cellular transportacross cationic amino acid transporters (CATs) Interestinglythe liver expresses CATs abundantly especially CAT-2A andCAT-2B suggesting a higher uptake of ADMA in this organas compared with the heart lungs and kidneys [9]

ADMA has been shown to correlate with cardiovascularrisk factors [10 11] and is considered a predictor of cardio-vascular events [12] Moreover ADMAplasma concentrationincreases in patients suffering from hepatic dysfunction[13] and end-stage kidney disease [14] and in situations ofendothelial dysfunction and increased atherosclerotic risk[15 16] Furthermore in ischemiareperfusion (IR) injuryADMA which is increased by reducing DDAH activity maywell influence NO production by competing with argininefor the binding site in the active NOS centre [17] Indeedevidence for the protective effects of NO synthesis was seenwhen NOS inhibitors dramatically worsened liver necro-sis and apoptosis [18] Correlation between methylargininederivatives and liver function and survival after liver trans-plantation was also observed [19] However this does notexhaust the relationship between ADMA and hepatic IRinjury Indeed the molecular mechanisms involved in IRinjury are not completely understood and only a few workshave reported changes induced by hepatic IR injury on theADMADDAH pathway which needs to be considered as apoint of interest potentially capable of reducing the effects ofIR In addition a previous studymerely reported cirrhosis bybile duct ligature (BDL) which induced an increase in plasmaADMA levels this did not happen with thioacetamide-(TAA-) induced cirrhosis [20] Yet no explanation aboutthis event has been reported Understanding themechanismsinvolved in ADMA elimination increases the possibility ofunderstanding its modulation which particularly crucial inseveral pathological conditions [10ndash14]

The present study was carried out to clarify whether(1) ADMA clearance also occurs through the bile and (2)hepatic IR induces changes in ADMA levels by quantifyingits content in hepatic tissue serum and bile samples Inaddition enzymatic activity and protein expression ofDDAHand mRNA expression of DDAH-1 and PRMT-1 and CAT-2protein at the end of reperfusion were examined

2 Materials and Methods

21 Animals The use of animals in this experimental studywas approved by the National Institute for Research and theanimals were cared for in accordancewith its guidelinesMaleWistar rats (200ndash250 gr 2-3months old Charles River CalcoLecco Italy) with free access to water and food were used

22Materials All reagentswere of the highest grade of purityavailable and were purchased from Sigma (Milan Italy)

23 IschemiaReperfusion (IR) Procedure The rats wereanesthetized with sodium pentobarbital (40mgkg ip) theabdomenwas opened via amidline incision and the bile ductwas cannulated (PE-50) Ischemia to the left andmedian lobewas induced by clamping the branch of the portal vein andthe branch of the proper hepatic artery after bifurcation tothe right lobe for 30 or 60min with microvascular clips [21]with the abdomen temporarily closed with a suture After30 or 60min of ischemia (119899 = 8 each group) the abdomenwas reopened the clips were removed the abdomen wasclosed again and the liverwas allowed to reperfuse for 60min(Figure 1) By using partial rather than total hepatic ischemiaportal vein congestion and subsequent bacterial translocationinto the portal venous blood were avoided A set of 60minischemia experiments followed by 60min reperfusion wasperformed after 5 hours of BDL (119899 = 6) (Figure 1) All theanimals were maintained on warm support to prevent heatloss rectal temperature was maintained at 37 plusmn 01∘C Toprevent postsurgical dehydration and hypotension 1mL ofsaline was injected in the inferior vena cava Sham-operatedcontrol animals (119899 = 8 each group) underwent similarmanipulation of the liver hilum without vascular occlusionor BDL and were kept under anesthesia for an equal length oftime

24 Bile Serum and Tissue Sampling Bile duct was cannu-lated as follows bile duct was closed with a 40 silk sutureligation placed in distal position This causes the bile ductto swell upstream becoming more discernible Then a cutwas opened in the bile duct with spring scissors and aslant-shaped polyethylene tubing was inserted When thetubing was completely filled with bile it was fixed with40 silk suture ligation This operation was conducted usingmagnifying spectacles Bile was collected in darkened vialsduring the reperfusion period Blood and tissue samples werealso collected after 60min reperfusion Blood was drawnfrom the vena cava and centrifuged at 3000 g for 10min at4∘C Hepatic biopsies were quickly removed from the centrallobe and immediately frozen in liquid nitrogen as were bileand serum samples until they were analyzed

25 Biochemical Assays Liver injury was assessed by serumrelease of alanine transaminase (ALT) aspartate transami-nase (AST) and alkaline phosphatase (ALP) by an automatedHitachi 747 analyzer (RocheHitachi Indianapolis IN USA)

ADMA levels in serum bile and tissue were evalu-ated by direct ELISA kit according to the manufactur-ing procedure (Immundiagnostik AG Germany) Quanti-tative analysis of ADMA was also performed in depro-teinized rat bile by reversed-phase high-performance liquidchromatography (RP-HPLC) with o-phthalaldehydebeta-mercaptoethanol (OPAbetaME) and fluorescence detectionas previously described with some modifications [22] Thechromatography was carried out on the HPLCHT400Esystem (ESSECI-Group Como Italy) equipment routinelyused for amino acid analysis that allows automatic onlinemixing of all reagents for OPA derivatizationThe derivativeswere separated on a Teknokroma Mediterranean sea C18


Group 1

Group 2

Group 3




Ischemia (60min)

Ischemia (60min)

Ischemia (30min)










3 Results








0

01

02

03

04

05

Bilia

ry A

DM

A (n

mol

mL)

30 60(min)

ShamIR

lowastP le 0002

lowastP le 003

N = 8

(a)

0

02

04

06

08

1

12

14

Seru

m A

DM

A (n

mol

mL)

30 60(min)

ShamIR

BDL IR

lowastP le 005

N = 6ndash8

(b)

0

3

6

9

12

15

18

30 60

Hep

atic

AD

MA

(pm

olm

g pr

ot)

(min)

ShamIR

BDL IR

lowastP le 005

lowastP le 003

N = 6ndash8

(c)



Gene Sequence


















4 Discussion





0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile



0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Group 1

Group 2

Group 3




Ischemia (60min)

Ischemia (60min)

Ischemia (30min)










3 Results








0

01

02

03

04

05

Bilia

ry A

DM

A (n

mol

mL)

30 60(min)

ShamIR

lowastP le 0002

lowastP le 003

N = 8

(a)

0

02

04

06

08

1

12

14

Seru

m A

DM

A (n

mol

mL)

30 60(min)

ShamIR

BDL IR

lowastP le 005

N = 6ndash8

(b)

0

3

6

9

12

15

18

30 60

Hep

atic

AD

MA

(pm

olm

g pr

ot)

(min)

ShamIR

BDL IR

lowastP le 005

lowastP le 003

N = 6ndash8

(c)



Gene Sequence


















4 Discussion





0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile



0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

01

02

03

04

05

Bilia

ry A

DM

A (n

mol

mL)

30 60(min)

ShamIR

lowastP le 0002

lowastP le 003

N = 8

(a)

0

02

04

06

08

1

12

14

Seru

m A

DM

A (n

mol

mL)

30 60(min)

ShamIR

BDL IR

lowastP le 005

N = 6ndash8

(b)

0

3

6

9

12

15

18

30 60

Hep

atic

AD

MA

(pm

olm

g pr

ot)

(min)

ShamIR

BDL IR

lowastP le 005

lowastP le 003

N = 6ndash8

(c)



Gene Sequence


















4 Discussion





0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile



0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4 Discussion





0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile



0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

AST ALT ALP

(UL

)

Sham

0

1

2

3

4

5

6

7

8

AST ALT ALP

(UL

)

00

01

02

03

04

05

06

30 60

Dire

ct b

iliru

bin

(UL

)

0

30

60

90

120

150

180

AST ALT ALP

(UL

)

(UL

)

20

50

80

110

140

170

200

AST ALT ALP0

2

4

6

8

10

12

Dire

ct b

iliru

bin

(UL

)

(min)

minus10

30 60(min)

lowastP le 0001

lowastP le 0005

lowastP le 0006

lowastP le 005

lowastP le 004

lowastP le 0001

lowastP le 0001

lowastP le 0005

lowastP le 0005

lowastP le 002

lowastP le 002

lowastP le 002

lowastP le 001

lowastP le 00004

lowastP le 004

IR 30min IR 60min

Sham

ShamIR

ShamIR 30min

ShamIR 60min Sham

Serum

Serum

Serum

Bile

IR

Bile

Bile



0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

006

012

018

DD

AH

activ

ity

(nm

ol ci

trul

line

min

mg

prot

ein)

30 60(min)

ShamIR

lowastP le 004

lowastP le 003

N = 8

(a)

0

20

40

60

80

100

120

140

Sham IR

DD

AH

-1 p

rote

in (

ver

sus c

ontro

l)

lowastP le 002

N = 8

(b)

0

02

04

06

08

1

12

Sham IR

DD

AH

-1 m

RNA

(mRN

A ex

pres

sion) lowast

P le 004

N = 8

(c)








0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

14

16PM

RT-1

(au

)

ShamIR

lowastP le 001

N = 8





0

02

04

06

08

1

12

14

CAT-

2 (n

gm

g pr

otei

n)

ShamIR

N = 8











Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Abbreviations




Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Changes in ADMA/DDAH Pathway after ...downloads.hindawi.com/journals/bmri/2014/627434.pdf · citrulline as follows. Solution A (diacetyl monoxime mM, thiosemicarbazide