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Human serum albumin, systemic inammation, and cirrhosis

Vicente Arroyo1 ,2 , , Rita Garca-Martinez2 , Xavier Salvatella31 Liver Unit, Hospital Clinic, Centre Esther Koplowitz, IDIBAPS, University of Barcelona, Barcelona, Spain; 2 EASL-Cronic Liver Failure Consortium,Fundaci Clinic, Barcelona, Spain; 3 ICREA and BSC-CRG-IRB Research Programme in Computational Biology, IRB Barcelona (IRB), Barcelona, Spain

Summary

Human serum albumin (HSA) is one of the most frequent treat-ments in patients with decompensated cirrhosis. Prevention of paracentesis-induced circulatory dysfunction, prevention of type-1 HRS associated with bacterial infections, and treatmentof type-1 hepatorenal syndrome are the main indications. Inthese indications treatment with HSA is associated with improve-ment in survival. Albumin is a stable and very exible moleculewith a heart shape, 585 residues, and three domains of similarsize, each one containing two sub-domains. Many of the physio-logical functions of HSA rely on its ability to bind an extremelywide range of endogenous and exogenous ligands, to increasetheir solubility in plasma, to transport them to specic tissuesand organs, or to dispose of them when they are toxic. The chem-ical structure of albumin can be altered by some specicprocesses (oxidation, glycation) leading to rapid clearance andcatabolism. An outstanding feature of HSA is its capacity to bindlipopolysaccharide and other bacterial products (lipoteichoicacid and peptidoglycan), reactive oxygen species, nitric oxideand other nitrogen reactive species, and prostaglandins. Bindingto NO and prostaglandins are reversible, so they can be trans-

ferred to other molecules at different sites from their synthesis.Through these functions, HSA modulates the inammatory reac-tion. Decompensated cirrhosis is a disease associated systemicinammation, which plays an important role in the pathogenesisof organ or system dysfunction/failure. Although, the benecialeffects of HAS have been traditionally attributed to plasma

volume expansion, they could also relate to its effects modulatingsystemic and organ inammation.

2014 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Human serum albumin (HSA) and the management of

decompensated cirrhosis: Background and current indications

History

Diuretics, antibiotics, and HSA are the most frequently usedtreatments for the management of patients with cirrhosis.According to the CANONIC study database [1], a prospectiveEuropean investigation in 1348 patients with decompensated cir-rhosis, HSA was indicated in 60% of the patients during hospitaladmission (Table 1).

The history of HSA started in 1940, when a long-term stablesubstitute of blood was required by the US military authoritiesto treat shock on the battleeld during the World War II [2]. Itwas used for rst time in December 1941 in seven severelyburned sailors after the attack on Pearl Harbor. At that period,the association of portal hypertension, hypoalbuminemia, andascites was already known. Not surprisingly, ascites was one of the rst indications of HSA. Three studies published between1946 and 1949 assessing the effect of short- and long-term i.v.infusion of HSA in cirrhotic patients with ascites dened the rstindications of HSA in cirrhosis [35]. Serum albumin concentra-tion and urine volume increased in most patients. Peripheraledema also improved. However, only some patients showedimprovement of ascites. First indication of HSA was, therefore,hypoalbuminemia in patients treated by frequent paracentesis.The introduction of spironolactone and furosemide in the early1960s and the article by Hecker and Sherlock [6] rst describinghepatorenal syndrome (HRS) lead to great changes in the man-agement of ascites. The concept that paracentesis could be fol-lowed by rapid reformation of ascites and renal failureextended rapidly through the medical community and therapeu-tic paracentesis was formally proscribed. Only in 10% of patientsnot responding to diuretics (refractory ascites) was HSA pre-scribed to increase the plasma volume and diuretic effect [7]. Inthe 1970s LeVeen designed the rst peritoneo-venous shunt[8]. It consisted in a multi-perforated intra-peritoneal tube con-nected to a unidirectional valve and to a second tube that subcu-taneously reached the superior vena cava through the internal

Journal of Hepatology 2014 vol. 61 j 396407

Keywords: Cirrhosis; Human serum albumin; Paracentesis-induced circulatorydysfunction; Spontaneous bacterial peritonitis; Hepatorenal syndrome; Systemicinammation; Organ inammation; Oxidative stress; Decompensated cirrhosis;Acute-on-chronic liver failure.Received 26 February 2014; received in revised form 4 April 2014; accepted 6 April 2014 Corresponding author. Address: Centre Esther Koplowitz, Rosell 153, 08036Barcelona, Spain. Tel.: +34 93 2271724; fax: +34 93 3129403.E-mail address: [email protected] (V. Arroyo). Abbreviations: HSA, human serum albumin; HRS, hepatorenal; PICD, paracentesis-induced circulatory dysfunction; SBP, spontaneous bacterial peritonitis; RCT,randomized controlled trial; LPS, lipopolisaccharide; TLR4, Toll-like receptor 4;PAMPs, pathogen-associated molecular paterns; DAMPs, damaged-associatedmolecular paterns; ROS, reactive oxygen species; RNS, reactive nitrogen species;HMA, human-mercapto-albumin; NMA, non-mercapto-albumin; NO, nitric oxide;CRP, c-reactive protein; RAS, renin-angiotensin system; SNS, sympathetic nervoussystem; ADH, antidiuretic hormone; PGs, prostaglandins; BDK, bradikinin; ACLF,acute-on-chronic liver failure; TNFa , tumor necrosis factor a .

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jugular vein. The positive abdominal pressure and the negativeintra-thoracic pressure facilitated the opening of the valve andthe continuous passage of ascites into the circulation. LeVeenshunt was widely used in the management of refractory ascitesfor more than a decade. Following the introduction of LeVeenshunt, HSA disappeared from the therapeutic armamentariumof cirrhosis for more than a decade.

Current indications of albumin

Management of ascitesIn 1988 a Spaniard inter-hospital group [9] demonstrated thatparacentesis, if performed in association to HSA, was an effectiveand safe therapy of ascites. They compared repeated large vol-ume paracentesis (4 liters/day) associated with HSA (8 g per literof ascitic uid removed) vs. diuretics. The incidence of renalimpairment, hyponatremia and encephalopathy was signicantlylower in the paracentesis group. No signicant change in plasmarenin activity was observed indicating no impairment in effectiveblood volume. Survival probability was similar in both groups. Ina second investigation, total paracentesis (complete removal of

ascites in only one tap) associated with HSA was also found tobe safe [10]. Treatment of ascites was therefore considerably sim-plied. Instead of requiring 24 weeks in hospital to compensatea tense ascites with diuretics, patients could be managed by par-acentesis in a single day hospitalization regime [11]. Paracentesisassociated with HSA was subsequently compared to peritoneo-venous shunting in patients with refractory ascites [12]. Perito-neo-venous shunting was superior to paracentesis in the long-termcontrol of ascites. However, due to the high rate of complicationsassociated with the prosthesis, the total time in hospital and theprobability of survival was similar with both treatments. Basedon these data, paracentesis plus HSA was considered the treat-ment of choice for tense ascites.

When paracentesis is performed without HSA or if HSA issubstituted by synthetic plasma expanders, a high proportion of patients develop marked activation of the renin-angiotensin sys-tem, a feature known as paracentesis-induced circulatory dys-function (PICD) [1315]. The prevalence of PICD in patients notreceiving volume expansion is 70%. In patients receiving dextranor polygeline it is also high (37.8%). PICD is due to an accentua-tion of the arterial vasodilation already present in cirrhosis anda lack of appropriated cardiac response [1618] (Fig. 1). PICD,although asymptomatic, is a serious complication. It is not spon-taneously reversible and is associated with shorter time to hospi-tal readmission, higher incidence of renal failure, and shorterprobability of survival [15]. A recent meta-analysis (17 trials,

1,225 patients) comparing HSA vs. alternative treatments (no vol-ume expansion, synthetic plasma expanders or vasoconstrictors)has shown that HSA signicantly reduces the incidence of PICDand mortality [19].

Prevention of type-1 HRS associated with spontaneous bacterial peritonitis (SBP)Despite infection resolution, 2040% of patients with SBP developtype-1 HRS in relation to arterial vasodilation, acute impairmentin cardiac function, and compensatory activation of the renin-angiotensin and sympathetic nervous systems [2023] (Table 2).Type 1 HRS also develops in cirrhotic patients with other type of bacterial infections although the prevalence is lower [2426]. Thereason why SBP is such a frequent precipitating event of type-1HRS is multifactorial. First, an exaggerated inammatoryresponse to sepsis occurs in patients with cirrhosis and asciteswith an increase in plasma levels of cytokines 20-fold greaterthan in individuals without cirrhosis [26]. This feature has alsobeen observed in experimental animals in which doses of bacte-rial endotoxin that do not produce any change in systemic hemo-dynamics in healthy rats, induce arterial hypotension and

increase the plasma levels of cytokines by 100-fold in rats withcirrhosis and ascites [27]. Second, the inammatory response tobacterial infection persists for a longer duration in cirrhosis.Finally, patients with cirrhosis and ascites already have severeimpairment in cardio-circulatory and renal function, which pre-dispose these patients to further deterioration in organ function[28]. In support to this contention, patients with SBP who haveincreased serum creatinine concentration or dilutional hypona-tremia prior to infection, and those with intense inammatoryresponse (high concentration of polymorphonuclear leukocytes,tumor-necrosis-factor alpha and interleukin-6 in plasma, andascitic uid) are at high risk of developing type-1 HRS[23,29,30]. If untreated, type-1 HRS in patients with SBP is asso-ciated with a mortality rate approaching 100% [31].

In 1999 we reported the use of HSA (1.5 g/kg b.wt. at infectiondiagnosis and 1 g/kg b.wt. at the third day) in SBP [31]. HSA pre-vented cardiovascular dysfunction and this was associated with adramatic decrease in the prevalence of type 1 HRS and hospitalmortality (10% in the HSA group and 30% in the control group).A recent meta-analysis has conrmed these ndings [32].

Treatment of type-1 HRThe use of vasopressin analogs and HSA for the treatment of type-1 HRS was based on two features. First, arterial vasodilationin cirrhosis occurs in the splanchnic circulation and vasopressinanalogs act preferentially in this area [28]. Second, studies using

Table 1 . Use of albumin in patients with acute decompensation of cirrhosis in Europe. Results from the CANONIC study database.

Patients with hospitalization follow-up* Patients receiving albumin during hospitalizationPatients (n) 699 425 (60.8%)Bacterial infections (n) 398 (56.9%) 152 (38.2%)

SBP (n) 107 (15.3%) 53 (49.5%)Non-SBP infections (n) 291 (41.6%) 65 (22.3%)

Paracentesis (n) 291 (41.6%) 225 (77.3%)9 L (n) 55 (7.8%) 51 (92.7%)

HRS (n) 179 (25.6%) 75 (41.9%) The CANONIC study was made in 1348 patients. The table only includes patients with hospitalization follow-up. There were patients with more than one complicationrequiring albumin administration.

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head-out water immersion, a procedure that expands the centralblood volume, and vasoconstrictors showed that improvement inrenal function in cirrhosis only occurs when both procedures areapplied simultaneously [33]. In our rst study we used ornipres-sin and HSA [1 g/kg b.wt. on the rst day followed by 20 to 40 g/

day] [34]. A remarkably improvement in circulatory function wasobserved with rapid suppression of the renin-angiotensin andsympathetic nervous systems, progressive normalization inserum creatinine and increase in renal perfusion and GFR (Table 3). Because treatment was associated with ischemic eventsin some patients, the study was repeated using i.v. terlipressin(12 mg/46 h) with identical results but no ischemic complica-tions [35].

Many studies have been subsequently published on the use of terlipressin and albumin for HRS and their main conclusions arethe following: (1) Terlipressin plus HSA reverses type-1 HRS(serum creatinine

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(ClinicalTrials.gov, Identier: NTC01288794) is a RCT in 400patients with ascites comparing standard therapy vs. standardtherapyplusHSA(40 g twiceweeklyin thersttwoweeksandonceweekly for one year). The main end points are mortality and inci-denceof refractoryascites.

Theuseof HSAfor themanagement of non-SBP bacterial infec-tions is an increasing indication in Europe (Table 1). There is datathat these patients also develop type-1 HRS [25,22]. On the otherhand a recent RCT in 110 patients (INFECIR-1 Study) showed thatHSA [1.5 g/kg b.wt. at infection diagnosis and 1 g/kg b.wt. at thethird day] has benecial effects on renal and circulatory function[56].AtrendforbetterprognosisinpatientsreceivingHASwasalsoobserved. A second RCT (INFECIR-2 study, ClinicalTrials.gov, Iden-tier: NTC02034279) has been started by the EASL Chronic LiverFailure Consortium; 512 patients withnonSBPbacterial infectionsand high risk of type-1 HRS and death will be included.

Key Points 1

HSA is an extracellular hearth-shaped three-domainprotein of intermediate size (6.810 4 g/mol) that issynthesized in the liver and is the most abundantprotein in plasma. It is a protein of remarkable solubility(50 mg/ml) owing to its high content (30%) in chargedresidues (39 Asp, 60 Glu, 58 Lys, 23 Arg) and performsa wide range of functions ranging from regulatingoncotic pressure to scavenging reactive oxygenspecies. Both the amount and quality of HSA areimportant for health

The structure of HSA, solved at high resolution in 1992,is stabilized by a remarkably large (17) number ofintra-domain disulfide bonds that render the individualdomains, that are of similar size and structure, verystable. Domains I and III, which are lobes in thestructure, can fold autonomously but domain II, which isthe core of the structure of HSA, cannot. This structuralcoupling provides a framework for the allostericregulation of HSA, whereby the binding of ligands orchemical modifications in one domain generates asignal that can propagate to a different domain

At the molecular level the most striking feature of HSAis its ability to bind to a wide range of ligands, whichis intimately linked with one of its physiological roles,to transport relatively hydrophobic species thatwould otherwise not be soluble in plasma. Thisincludes small molecules such as hormones, fattyacids, bilirubin and heme as well as metals such asCu, Fe and Hg but also more complex substancessuch as lipopolysaccharide. In addition to endogenousligands HSA can also bind to exogenous ligands suchas drugs, greatly contributing to their bioavailability

HSA can be modified irreversibly by non-enzymaticglycosylation of Lys side chains, mainly Lys 525 , as wellas by oxidation, mainly of the only free Cys residuein the protein, Cys 34 . The former occurs in diabeticpatients whereas the latter is frequent in acute onchronic liver failure. Although the mechanisms bywhich these chemical modifications alter the structuralproperties of HSA are not known there is substantialevidence that they alter the physiological functions ofthe protein, presumably by collective changes in itsstructure

Chemistry and physiology of HSA

Molecular structure and chemical properties

HSA is a multi-domain protein stabilized by 17 disulde bondsthat confer a remarkable stability to its structure [57]. In additionto the 34 cysteine residues involved in disulde bonds HSA alsopossesses a free cysteine (Cys-34) that is relatively solvent-exposed (Fig. 2) and plays an important role in its antioxidantactivity [58,59]. The structure of HSA at low resolution (6 )was rst solved in 1989 [60] and a high resolution atomic struc-ture (2.8 ) was reported in 1992 [57]. These revealed this pro-tein to be composed of three domains of similar size (residues1197, 198381, and 381585) arranged in a heart shape andformed by eight a -helices. Owing to their structural similaritythe domains can be divided in sub-domains (IA, IB, IIA, IIB, IIIA,and IIIB) that have functional relevance as they dene the bindingsites of many HSA ligands (Fig. 2) [61].

Due to the presence of a substantial number of intra-domaindisulde bonds the domains of HSA have signicant stability.Domains I and III are particularly stable and a construct contain-

ing domains I and II [1381] as well as a construct containingdomains II and III [198585] fold autonomously by adoptingthe same structure that they have in the full-length protein[62,63]. In spite of its large size and owing to its remarkable sta-bility and its relatively simple structure, where residues that areconnected by disuldes or in contact are not particularly distantin sequence, HSA can be refolded from the fully reduced stateunassisted by molecular chaperones [64,65]. This greatly facili-tates its synthesis in the laboratory and the study of its structuraland biophysical properties [62].

Again owing to its relatively simple structure and modularnature HSA appears to be a very exible protein. According to

N-ter

Sudlowssite I

Sudlowssite II

Cys34

C-ter

IA

IB

IIIBIIIA

IIB

IIA

Fig. 2. Molecular structure of human serum albumin. Crystal structure (pdbcode 1e7i) of HSA with an indication of the its subdomains (IA, IB, IIA, IIB, IIIA anIIIB), of the N and C termini, of Sudlows sites I and II and of the seven LCFAbinding sites (FA1 to FA7). The heavy atoms of the side chain of residue Cys-34are shown as purple spheres. LCFA binding sites also bind prostaglandins. Cys-34binds ROS and RNS, including NO [61].



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various experimental approaches the heart shape that the proteinadopts in the crystal is likely to be only one in the range of inter-domain orientations that HSA samples show in solution. Experi-ments monitoring the rate of exchange between exchangeablehydrogens and water have shown these to be particularly labile,indicating that in addition to inter-domain motions HSA under-goes local unfolding events where the hydrogen bonds that stabi-lize its secondary structure are transiently disrupted [66].

Many of the physiological functions of HSA rely on its abilityto reversibly bind to an extremely wide range of ligands toincrease their solubility in plasma, to transport them to specictissues or organs or to dispose of them when they are toxic.The structures of HSA bound to various of these substrates havebeen determined by X-ray crystallography, revealing that onmany occasions the protein must experience substantial struc-tural changes to accommodate the ligand and therefore stronglysuggesting a functional role for its high exibility. Although manybinding sites for ligands have been reported in HSA the mostimportant sites are commonly known as Sudlows sites I and II(Fig. 2) which are found in subdomains IIA and IIIA, respectively.

Long chain fatty acids (LCFAs) are intermediates in lipidmetabolism that circulate in plasma both in soluble form as wellas associated with HSA. According to crystallographic studiesthey can bind up to seven sites in HSA (FA1 to FA7) includingboth Sudlows sites, with site I corresponding to FA7 and site IIcorresponding to FA3 and FA4 [67]. It is however likely that manyof these sites are secondary and therefore do not play an impor-tant role in LCFA binding in physiological conditions, where it isthought that up to two LCFA molecules, bound to Sudlows site I,are bound to HSA at a given time.

Drugs such as warfarin, indomethacin, ibuprofen, diazepamare another heavily studied class of HSA ligands. Similarly towhat is the case for LCFAs these molecules can bind to varioussites in HSA and, although they tend to mainly interact with Sud-lows sites I and II, this can vary depending on the relative con-centration of competing ligands and on the allosteric effects

discussed below [68]. That these and other drugs can competefor HSA binding sites for their transport in plasma emphasizesthe importance of understanding the physicochemical basis of their interaction with HSA [69].

Given that HSA is a exible protein that can bind to varioussubstrates in different sites there have been suggestions that itis an allosteric protein, i.e., that its afnity for specic ligandscan depend on whether a second ligand is occupying a differentbinding site. Specic examples of such a behavior, among others,include an allosteric coupling between the binding sites of hemethat binds to FA1 in sub-domain IB, and the drug warfarin, thatbinds to Sudlows site I [70]. The structural, dynamical, and ther-modynamic coupling between domains occurring in HSA ishighly suggestive of this possibility as these are sufcient condi-tions to give rise to allosteric binding [71].

Given its high stability HSA has a long half-life in plasma, of about 19 days [2]. During this relatively long time its chemicalstructure can be altered by oxidation as well as by non-enzymaticglycosylation, among other irreversible modications. The accu-mulation of chemically altered HSA has been linked to specicpathologies such as cirrhosis [72] and diabetes [73]. The highlyexible nature of this protein and the growing body of evidence,suggesting that the various binding sites that contribute to HSAfunction are strongly coupled, suggests that such chemicalalterations can have functional consequences. In this scenario

Key Points 2

The total extracellular pool of albumin of a 70 kg b.wt.healthy man is 360 g which represents 3.2 x 10 21 molecules of albumin. The weight of the extracellularpool of albumin is similar to the total liver proteinweight. 66% of the extracellular albumin is in the

interstitial space and 33% in the intravascular space.The transfer of albumin from the intravascular tothe interstitial space (transcapillary escape rate ofalbumin) is 4-5% per hour and approximately thesame percentage is returned to the intravascularcompartment throughout the lymphatic system; 80% ofthe extravascular pool is in the muscle and skin

Albumin is synthesized only by the hepatocytes andit is not stored, rather the protein is rapidly secreted(synthesis occurs within 2-3 minutes and secretionwithin 25 minutes). Each hepatocyte synthesizesapproximately 15 x 10 6 albumin molecules per minutewhich represents a total hepatic synthesis of 13-14g (3.5% of the total albumin pool) per day of newlysynthesized albumin entering the intravascular space.

An equivalent mass of albumin is catabolized. Thehalf life of albumin is 19 days. An average molecule,therefore, leaves the circulation every 23 h, whichmeans approximately 28 trips from and back to thecirculation during its lifetime, and makes around 15,000trips around the circulation

The plasma concentration of albumin (35-45 g/L) and

the total albumin pool are regulated by changes inalbumin gene transcription and in albumin catabolism.The most important physiological factor regulatingsynthesis is the plasma concentration of albumin.Hypoalbuminemia stimulates albumin synthesis.Conversely, albumin infusion depresses albuminsynthesis. The administration of other macromolecules(dextrans) has similar effects pointed to the colloidosmotic pressure as the function on which thisregulation is based. Albumin catabolism is regulatedby the total albumin pool. A decrease in albumin poolleads to a reduced albumin catabolism. Changes inalbumin synthesis are less pronounced than changesin catabolism

In patients with decompensated cirrhosis, albuminmetabolism is much more complex than in healthysubjects, since there are additional mechanismsaffecting synthesis and catabolism. For example,systemic inflammation is associated to a markedfall in albumin synthesis, a process that is mediatedby cytokines (IL-6 and TNF). On the other hand,structural changes of the albumin molecule relatedto specific binding of ligands (i.e., glucose, reactiveoxygen or nitrogen species) are associated to rapiddegradation and catabolism of the modified molecules.Since these processes are present in patientswith decompensated cirrhosis or ACLF, albuminreplacement requires high doses in these patients. Infact, the amount of albumin given to a patient of 70 kgb.wt. with SBP (1.5 g/kg b.wt. at infection diagnosis and1 g/kg b.wt. at the third day) represents 48.6% of thetotal extracellular pool of albumin

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the oxidation or glycosylation of specic positions in the surfaceof HSA would cause structural changes that could propagate todifferent regions of structure either by concerted conformationalchanges or through local changes in the stability known to giverise to long range effects in simpler systems.

The best studied irreversible alteration of the structure of HSAis the oxidation by reactive oxygen species of Cys-34 from mer-capto-albumin, which has the native thiol group, to non-mer-capto-albumin, where the Cys side chain is involved in adisulde bond with either glutathion or another HSA molecule[58,59]. The reaction can proceed further to the sulnic and sul-fonic acid derivatives of the side chain. An obvious consequenceof this reaction is a decrease in the anti-oxidant properties of HSA, that rely on the thiol form of Cys-34, but there is also evi-dence that it can indirectly lead to decreases in other functionsof HAS, such as its ability to bind drugs in Sudlows sites [72,74].

Physiology

Synthesis and metabolismAlbumin is synthesized by the liver and rapidly released to the

intravascular compartment [2]. The total amount of albumin inhumans is approximately 360 g, 120 being in the intravascularand 240 in the extravascular compartment. Intravascular albu-min is constantly being exchanged (45%/h) through the endo-thelium with the extravascular pool. In organs having sinusoidsor capillaries with fenestrated endothelium albumin can passthrough the large capillary gaps. In the remaining capillaries withcontinuous endothelium, albumin is transported by an activetranscytotic mechanism mediated by the gp60 receptor (albon-din) [7578]. There is a second group of receptors (gp18 andgp30) expressed in many tissues that governs degradation of albumin [7881]. These surface cell receptors shows 1000-foldhigher afnity for chemically modied albumin (i.e., oxidizedalbumin). Once internalized, this modied albumin is degraded.Finally, a third type of albumin receptor (FcRn) that rescues albu-min from lysosomal degradation contributes to extend the albu-min half life [78,8284].

PhysiologyThe effects of HSA rely in the following features: an appropriatedmolecular mass and negative charge generate high oncotic pres-sure; a high solubility in water and density of binding sites makethe protein an ideal vehicle to transport water-insoluble sub-stances; the effects of active exogenous and endogenous sub-stances are modulated following binding to HSA; the highextracellular concentration of HSA magnied its biologicalactions.

Among the substances transported, fatty acids stand up. HSAis the main fatty acid binding protein in the extracellular space.

It carries fatty acids from the intestines to the liver and fromthe liver to muscle and to and from adipose tissues [85,86].Transportation of unconjugated bilirubin from the spleen or bonemarrow to the liver is the best example of albumin transportationof a water insoluble endogenous metabolite to its elimination site[87]. Other endogenous substances that bind to albumin are theeicosanoids, bile acids, steroids, hematin, tiroxin, vitamin D, andfolate [2,88]. Among the exogenous molecules transported byalbumin there is a wide variety of drugs [2,69,89]. In additionto drug solubility, albumin binding decreases toxicity andincreases drug half-life [69]. Drug binding to HSA can be affected

by other drugs that compete with specic sites in the molecule.As a consequence, an increase in the free fraction may lead tochanges in pharmacokinetics and pharmacodynamics. Drug-albumin binding may be also altered in diseased states, includingliver and renal diseases [58,90,91], due to the increase in endog-enous substances that compete for binding sites, but pharmaco-kinetic consequences of this does rarely cause important effects[92,93].

An outstanding feature of HSA is its capacity to bind pro-inammatory substances and mediators of inammation. Lipo-polysaccharide (LPS), lipoteichoic acid, and peptidoglycan aresurface components of gram-negative and gram-positive bacteriathat activate the innate immune system through Toll-like recep-tor 4 (TLR4) and induce inammation. HSA binds these moleculesby electrostatic and hydrofobic forces [9497]. Cell activation byLPS requires an ordered interaction with several host proteinsincluding LPS-binding protein, CD14 and co-receptor MD-2.Recent studies showed that albumin may also participate in LPSpresentation to TLR4 facilitating disaggregation of LPS-Lipid Apolymers and donation of monomers to CD14 [98100]. This sug-gests that albumin promotes the inammatory response. In con-trast, LPS-albumin complexes are much less effective in immuneactivation than endotoxin-CD14 complexes [100]. Giving theabundance of albumin and the lower relative cell activationcapacity of the LPS-albumin complex, albumin could play a rolein moderating the inammatory response to bacterial infections.Therefore, albumin could play a dual role either stimulating ormoderating immune cells activation depending on pathophysio-logical conditions [98,100].

Systemic inammatory response can be triggered by antigensderived from bacteria (Pathogens-Associated Molecular Patterns,PAMPs) or by intrinsic factors released into the circulation as aresult of trauma or cell injury (Damaged Associated MolecularPatterns, DAMPs). Specialized receptors of the innate immunesystem recognized these factors and release inammatory medi-ators among which cytokines, and reactive oxygen (ROS) and

nitrogen species (RNS) are the most important [101,102]. Gener-ation of ROS and RNS (superoxide, nitric oxide, hydrogen perox-ide, hydroxyl radical, peroxynitrite, hypochlorous acid, nitrogendioxide radical, hydroperoxide radical, and peroxyl radical) byneutrophils, macrophages, and endothelial cells, which representthe innate immune system, is the rst line of defense against sep-sis [103105]. The aim of the modication of the redox state inplasma and extracellular uid is to destroy the bacteria by energydepletion and oxidative damage of lipids, proteins, and DNA. Thisextracellular oxidative burst however may be transferred intomitochondria leading to cell dysfunction and organ failure[103]. Extracellular uid and the mitochondria dispose of antiox-idant systems to prevent excessive oxidative damage [103105].HSA is the main extracellular defense against oxidative stress. Itprovides 80% of extracellular thiols, which are potent scavengersof ROS and RNS [60,106,107]. The glutathione system is the mostabundant mitochondrial antioxidant. Intracellular albumin catab-olism represents a source of sulfur-containing amino acids for thesynthesis of glutathione. Albumin therefore modulates the intra-cellular levels of this antioxidant system [108].

Two mechanisms account for the antioxidant effect of HSA.The rst is related to its capacity to bind and inactivate free met-als such as cooper and iron, which catalyse the formation of aggressive ROS [109,110]. HSA-bound bilirubin inhibits lipid per-oxidation and represents an indirect antioxidant effect [111]. The



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second and most important mechanism is related to the capacityof HSA to trap free radicals. Two-thirds of the HSA moleculesexist in a reduced form with a free thiol group in the Cys-34 res-idue. Working as a free radical scavenger, the Cys-34 residue isable to trap multiple ROS and RNS [60,107]. As indicated, in phys-iological conditions the Cys-34 residue exists in two differentforms, namely human-mercapto-albumin (HMA; reduced) inwhich the thiol group is in the free state and human non-mer-capto albumin (NMA; oxidized) [60,107,112]. In this later formthe thiol group may be present as a disulde that is formedreversibly with Cys or glutathione or as sulnic or sulfonic acidthat is formed irreversibly. Irreversible albumin oxidation is asso-ciated with a loss of function and rapid degradation. Cys-34 is agood indicator for evaluating oxidative stress in the systemic cir-culation [60,107]. HSA contains six methionine residues whichcan also be oxidized [112,113].

Under nitrosative stress by nitric oxide (NO) or other nitrosy-lating agents, mercaptalbumin can be converted in nitroso-HSA.This is a reversible reaction, so that NO can be transferred toother molecules [114,115]. HSA also binds arachidonic acid, pros-taglandins, thomboxanes, and leukotrienes [116120]. HSA hastwo-sided effects on eicosanoids. In some cases the protein,which has intrinsic enzymatic activity, catalyzes their synthesisor degradation. In others (i.e., PGI2, thromboxanes, and leukotri-enes) it stabilizes the molecule by delaying hydrolysis. HSA,therefore, functions as storage, carrier, and supplier of NO andeicosanoids to target sites to initiate physiological actions (vaso-dilation and inhibition of platelet aggregation) and as protectoragents to diminish harmful biological effects if produced in toolarge amounts. By these mechanisms HSA modulates endothelialfunction and inammation.

Decompensated cirrhosis is associated with systemicinammation

Three major features characterize decompensated cirrhosis. Therst is multi-organ dysfunction. The second is a systemic inam-matory reaction with increased plasma and ascitic uid concen-tration of cytokines and C-reactive protein (CRP) [121129].Finally, the third is an increased systemic oxidative stress witha high levels of oxidized HSA and of other markers of oxidativestress [58,72]. Systemic inammation, oxidative stress, and organdysfunction are moderate in patients with decompensated cir-rhosis and severe in patients with acute-on-chronic liver failure(ACLF) [1,130] (Fig. 3). Translocation of bacterial products (i.e.,lipopolysaccharide, bacterial DNA) or of viable organisms fromthe intestinal lumen to the circulation due to quantitative andqualitative changes in gut microbiota, impairment in intestinalmucosal barrier, increased epithelial permeability, and impaired

intestinal immunity, are important mechanisms of systemicinammation in cirrhosis [1,23,26,130,131]. However, systemicinammation may also occur in response to acute liver injury(i.e., acute alcoholic hepatitis) or other mechanisms [1].

Closed interactions exist between bacterial translocation,local inammation, and cardiovascular dysfunction in decompen-sated cirrhosis (Fig. 3). Activation of the intestinal immune sys-tem by bacterial translocation causes local release of NO andother vasodilators leading to the characteristic hyperdynamic cir-culation of cirrhosis and in more advanced stages, effective hyp-ovolemia, activation of the renin-angiotensin system (RAS),

sympathetic nervous systems (SNS) and antidiuretic hormone(ADH), and ascites formation [28,130134]. On the other hand,the activated sympathetic nervous system induces changes inthe gut microbiota and impairs intestinal immunity, thus produc-ing a vicious circle promoting the progression cardiovascular dys-function [131]. A slow but progressive impairment of leftventricular function and cardiac output also develops in decom-pensated cirrhosis and contributes to circulatory dysfunction[135,136]. Recent data suggest that impairment in cardiac func-tion in experimental cirrhosis is related to inammation,tumor-necrosis factor a (TNFa )-related activation of inducibleNO-synthase and oxidative stress in the cardiac tissue [137].

ACLF is characterized by acute development of organ failure(s)(liver, renal, brain, coagulation, circulation, respiration) inpatients with compensated or decompensated cirrhosis [1]. ACLFdevelops in the setting of severe systemic inammatory reactiondue to bacterial infection, acute alcoholic hepatitis or other pre-cipitating events [1,23]. The number of organ failures correlatesdirectly with the degree of systemic inammation (Fig. 4) [1].Therefore, whereas systemic inammation is chronic and moder-ate in decompensated cirrhosis, it is acute and severe in ACLF(Fig. 4). The mechanism of organ failure in ACLF is complex(Fig. 3). Acute impairment in cardiovascular function leading tointense organ hypoperfusion is a major feature [21]. Howeverrecent studies in sepsis suggest that extension of systemicinammation to organs leading to abnormal distribution of bloodow within the microcirculation and cell dysfunction related tomitochondrial oxidative stress are also important mechanisms[138,139].

Bacterial infection is a frequent precipitating event of hepaticencephalopathy. Peripheral inammation may affect cerebralfunction through afferent vagal nerves activated by cytokines atthe site of inammation, by lipopolysaccharide or cytokines thatinteract to brain in areas lacking the blood-brain-barrier or bydiffusion to the brain of endothelial mediators [140144]. Activa-tion of microglia and synthesis of pro-inammatory cytokines in

the brain have also been demonstrated in experimental models of liver failure [141]. Circulatory dysfunction in patients with sys-temic inammation reduces brain perfusion [145]. Finally, sys-temic inammation increases the inhibitory effect of ammoniain brain function [146,147]. There are marked differences inhepatic encephalopathy between patients with decompensatedcirrhosis and patients with ACLF [148,149]. In the rst grouphepatic encephalopathy is of low grade and diuretics the mostfrequent precipitating event. In contrast, hepatic encephalopathyin ACLF is severe and bacterial infections or acute liver injury themain precipitating factor. Organ dysfunction in cirrhosis there-fore varies according to the mechanism and degree of systemicinammation.

HSA may have therapeutic effects unrelated to volumeexpansion

Considering the potential role of systemic inammation in cir-rhosis and the effect of HSA modulating the innate immuneresponse and oxidative stress, it is rational to suggest that someeffects of HSA (i.e., prevention and treatment of type-1 HRS)might be related to these features. Indeed, there are many stepsin the process of acute decompensation of cirrhosis and ACLF thatcould be inuenced by HSA (Fig. 3). There are three studies sup-porting this contention.

Review


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Two studies compared the systemic hemodynamic changes inuncomplicated patients with SBP treated with albumin orhydroxyethyl starch [150,151]. Circulatory function onlyimproved in patients receiving albumin. An increase in left ven-tricular function, cardiac output, and peripheral vascular resis-tance was observed, indicating a simultaneous effect of HSA inthe heart and in the peripheral circulation. An effect of HSA mod-ulating the cardiac and endothelial effect NO was proposed toexplain the different circulatory response on the basis of distinctchanges in the plasma levels of NO metabolites and von Wille-brand Factor between groups [152154].

The third investigation was performed in cirrhotic rats withascites [135]. Plasma volume expansion with HSA but not withhydroxyethyl starch improved left ventricular function ex vivo.This effect was related to a normalization of activated proteinexpression of TNFa and inducible NO synthase, MAD(P)H-oxidaseactivity and nuclear translocation of NF-j B in cardiac tissue, indi-cating a decrease of heart inammation and oxidative stress.

Conict of interest

VA has a grant from Grifols. RG and XS declared that they do nothave anything to disclose regarding funding or conict of interestwith respect to this manuscript. The EASL Chronic Liver FailureConsortium has an unrestricted grant from Grifols.

Acknowledgements

We are grateful to the Esther Koplowitz Foundation for itssupport.

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Leukocyte count C-reactive protein

N o A C L

F

A C L F

- 1

A C L F

- 2

A C L F

- 3

N o A C L

F

A C L F

- 1

A C L F

- 2

A C L F

- 3

1412

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8

642

0

7060

50

40

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0

x 1 0 9

c e

l l s

****

***

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/ L

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Review

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