XXIX Congresso SIFO Napoli 12-15 Ottobre 2008...J. H. Henriksen, et al. J. Hepatol. 2001 ; 34 : 53-60. Controls Cirrhotics with ascites Cirrhotics with refractory ascites P < 0.01

$Page 1: XXIX Congresso SIFO Napoli 12-15 Ottobre 2008...J. H. Henriksen, et al. J. Hepatol. 2001 ; 34 : 53-60. Controls Cirrhotics with ascites Cirrhotics with refractory ascites P < 0.01$
Main biological properties of albumin

XXIX Congresso SIFONapoli 12-15 Ottobre 2008

P. AngeliDept. of Clinical and Experimental Medicine University of Padova

HUMAN ALBUMIN

The elipsoid structure of albumin in solution

66.5 Kda

The structure of albumin

• In humans albumin synthesis takes place only in the liver (10-25 g of albumin per day).

• The liver can increase albumin synthesis to only 2-2.7 times normal because most of the liver’s synthetic activity is already devoted to albumin at rest.

• Albumin is not stored by the liver but is secreted into the portal circulation as soon as it is manufactured.

HUMAN ALBUMIN

Synthesis of albumin

DC. Carter, et al. Adv. Protein Chem. 1994 ; 45 : 153-203.

HUMAN ALBUMIN

Albumin distribution in a healthy 70 kg adult with a total albumin pool of 350 g (5g/kg B.W.)

Synthesis (13.3 g/day)(3.8% day)

Intravascular space

147 g (42%)

Extravascular space

203 g (58%)

Degradation (13.3 g /day)(3.8% day)

6.6 g/h

Y. Takeda, et al. J. Lab. Clin. Med. 1963 ; 61 : 183-202.


• Albumin accounts for 75-80% of plasma colloid oncotic pressure

HUMAN ALBUMIN

Albumin and plasma colloid oncotic pressure

High concentration (2/3)High concentration (2/3)

GibbsGibbs--Donnan effect (1/3)Donnan effect (1/3)

NaNa++

HUMAN ALBUMIN

0

5

10

15

20P < 0.005

Overall transvascular transport of albumin in cirrhosis

J. H. Henriksen, et al. J. Hepatol. 2001 ; 34 : 53-60.

Controls Cirrhotics with ascites

Cirrhotics with refractory ascites

P < 0.01

(% IVM • h )-1

ALBUMIN IN CIRRHOSIS

HUMAN ALBUMIN

0

20

40

60

80

100

120

60 120 240

Septic patients Healthy subjects

Changes in the albumin increment (% of initial value) following a bolus of albumin 20%

M.P. Margarson, et al. B.J.A. 2004 ; 92 : 821-826.

P < 0.001P < 0.01P < 0.05

min.

• Effect on endothelial function

HUMAN ALBUMIN


Main properties of albumin

HUMAN ALBUMIN

0

20

40

60

80

100

120

Bovine calf serum Human Albumin Colture medium

Relative protection from apoptosis for cultured human endothelial cells

M.P. Margarson, et al. B.J.A. 2004 ; 92 : 821-826.

P < 0.001

TER (%/hr)

0

2

4

6

8

10

Before Albumin After albumin

P = N.S.

Effect of albumin supplementation on transvascular escape rate (TER) of 125I-labeled albumin in pigs with septic shock.

M. Margason et al. J. Appl. Physiol. 2002 ; 92 : 2139-2145.

HUMAN ALBUMIN

Value in healthy subjects 28%

• Effect on endothelial function • Regulation of acid-base balance

HUMAN ALBUMIN


Main properties of albumin

7.44

HUMAN ALBUMIN

Effect of two boluses of 20% albumin solution (0.5 g/kg B.W.) on acid-base balance

pH

D. Bruegger, et al. Intens. Care. Med. 2005 ; 31 : 1123-1127.

7.40

7.36

0 10 20

* = p <0.05 vs baseline

7.42

7.38

Alb. 20% 0.5/kg B.W.

Alb. 20% 0.5/kg B.W.

(pH)

0,000

0,020

0,040

0,060

0,080

Albumin Saline

P < 0.05

Mean difference in pH between baseline value and mean value during albumin or saline resuscitation in critically ill patients

R. Bellomo et al. Crit. Care Med. 2006 ; 34 : 2891-2897.

HUMAN ALBUMIN

• Effect on endothelial function • Regulation of acid-base balance• Ligand-binding

HUMAN ALBUMIN



SH

NH2

DRUGSNSAIDsWarfarinBenzodiazepinesFurosemide

Exogenous moleculesBenzeneAflatossina G

Endogenous moleculesBilirubinBiliary saltsLCFA

HUMAN ALBUMIN

• Effect on endothelial function • Regulation of acid-base balance• Ligand-binding• ROS/RNS scavennging and antioxidant function

HUMAN ALBUMIN



Oxidative stress

↑ Production of ROS

↓ Reduction of Antioxidant

ALBUMIN AND OXIDATIVE STRESS

Oxidative stess due to metabolic or drug induction

Oxidative stress due to change in intracellular pO2

Oxidative stress due to inefficiency of respiratory chain

Cytochrome P450Cytochrome b5

Xanthine oxidase

NADPH oxidases NADH dehydrogenaseCytochrome oxyidase

Cellular sources of ROS in oxidative stress


Oxidative stress due to cellular surface modifications

Inflammation, endotoxins

Reduced tissue perfusion

Endotoxins, exogenous toxins

Induction

Reactive oxygen species (ROS)

• Molecules like hydrogen peroxide • Radicals like the hydroxyl radical: the most reactive • The superoxide anion which is both ion and radical

O O

Oxygen O2

O O

H

H

Hydrogen Peroxide

O

H

Hydroxyl Radical

O O

Superoxide anion


1. INTRACELLULARSODCatalasePeroxidaseGlutathioneSelenium

2. MEMBRANEVitamin Eß CaroteneUbiquinone

3. EXTRACELLULAR (PLASMA)Metal-Binding Proteins Caeruloplasmin, TransferrinAlbuminUric acidVitamin EVitamin C

Biological anti-oxidant systemsALBUMIN AND OXIDATIVE STRESS

600

Plot of the total antioxidant capacity of human serum as a function of the albumin concentration

Ant

ioxi

dant

cap

acity

(µm

ol/)l

H. Ihara, et al. J. Clin. Lab. Anal. 2004 ; 18 : 45-49.

400

200

200 400 600 800

r = 0.992, p <0.001

Albumin concentration (µmol/l)


0

20

40

60

80

100

120

Ant

ioxi

dant

Lev

el (%

)

Control Cirrhotic Cirrhotic + Albumin

*= p < 0.01 vs other groups

Effect of albumin on the plasma antioxidant level in cirrhotic rats level

*

Angeli P. et al. AALSD 2007


SH

NH2

ROS/RNSH2O2ONOO–

Lipid hydroperoxide


0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

healthy subjects patients with heart failure

Plasma levels of oxidized proteins in patients with heart failure

C. Banfi, et al. Eur. J. Heart Failure 2008 ; 10 : 244-251.

P < 0.01

(nmoles/mg protein)


Oxidative stress


- Effects of ROS on albumin structure and functions- Effects of albumin infusion on:

• ROS production • ROS-related organ dysfunction

Consequences of oxidation of proteins

Oxidation of side-chains - some of which can be used as damage markers

Backbone fragmentation

Formation of new reactive species

(peroxides, DOPA)

Release of further radicals and occurrence

of chain reactions

Dimerization or aggregation

Unfolding or conformational changes

Loss of structural or functional activity

Alterations in cellular handling / turnover

X•

or non-radical oxidant

Protein


ER. Stadman, et al. Amino Acids 2003 ; 25 : 207-218.


Albumin dimers induced by peroxides in human plasma

Y. Ogasawara, et al. Biochim. Byoph. Res. Com. 2006 ; 340 : 353-358.

Exposure to OOHNormal

0

50000

100000

150000

200000

Noramal serum Chronic liverfaiolure

Acute on chronicliver failure

K. Oetti, et al. Biocim. Biophys. Acta. 2008 (in press)

* = P < 0.05 vs native HSA

(luminescence counts)

ALBUMIN IN OXIDATIVE STRESS

Carbonyl groups in albumin of serum from patients with liver failure

*

0

0

0

0

0

0

0

healthy subjects patients with fatty liver

Cobalt binding capacity of albumin in patients with fatty liver

GJ. Amirtharaj, et al. Biovhim Biophys. Acta 2008 (in press).

P < 0.05

(Co bound/g albumin)


Oxidative stress


- Effects of ROS on albumin structure and functions- Effects of albumin infusion on:

• ROS production • ROS-related organ dysfunction

time

ROS

Neutrophils ‘oxidative burst’

T-cells


Cellular sources of ROS in oxidative stress due to sepsis

•- Induction of iNOS and overproduction of nitric oxide

(NO) and reactive nitrogen species (RNS)


Role of NF-kB in oxidative stress

Activating Stimuli(ROS)

IkB kinase/NEMO/IKAP

IκBα

p65

p50 IκBα

p65p50

PIκBα

Degradation

p65p50

p65p50

NUCLEUS

mRNA

Inflammatory/Immune Proteins

Translocation

Transcription

Target genes


Mechanism of action of NF-kB


Role of NF-kB in oxidative stress due to sepsis

NF-κB

Infection/endotoxin Reactive oxygen species

• Adesion molecules • Chemokins and • Cytokines• iNos and Cox2

{

Recruitment of otherleukocytes

Involvement of other types of cells

{

NAD(P)H•

p67

p47

gp91

rac

•O2-

O2 H+

NAD(P)+

p22phox

• Endothelial cells

• Vascular smooth muscle cells

• Cardiac myocytes

NADH/NADPH Oxidase


Cellular sources of ROS in oxidative stress due to sepsis

• TNF-α

• Thrombin

• Angiotensin II

• Platelet-derived growth factor

+


Effect of endotoxin and cytokines on iNSOS in endothelial cells

F. Wu, et al. Amirtharaj, et al. J. Cell. Physiol. 2008 (in press).

NADH inhinitors

Endothelial cells knock out for NADH


0

20

40

60

80

100

Control H2O2 with BH4supplementation

BM. Boulden, et al. Free Rad. Biol. Med. 2006 ; 41 : 810-817.

(pm

ol/m

g pr

otei

nn)

The role ROS in the pathogenesis of endothelial dysfunction in sepsis

Endothelial availability of BH4

Decreased BH4 reduces NO bioavailability in endothelial cells by causing eNOS uncoupling

eNOSL-arginine + O 2 L-citruline + NO .


-

BH 4

O 2-

H 2O 2ONOO

+ BH 4

BH 2

+

eNOS

↑ ROS ↑ iNOS


Effect of ROS and iNOS on eNOS in endothelial cells

⊗ ⊗

Microvascular dysfunction

Organ failure

Multiorgan failure (MOF)

• Reduced microcirculatory flow rate

• Increased flow heterogenity

• Decrease capillary density


Effects of topical acethylcholine administration in patients with sepsis

Patients with sepsis Control subjects

Parameter Baseline Acethylcholine

Total n° of vessels (n/mm) 4.9 (4.1-5.7) 6.0 (4.7-6.4) 5.4 (5.4-6.3)

Proportion of vessels perfused (%)

83 (77-96) 99 (98-100) 98 (97-99)

Proportion of capillaries perfused (%)

44 (24-60) 94 (77-96) 94 (92-95)

Absent flow (capillaries) (%)

29 (8-44) 1 (0-3) 3 (2-5)

Internittent flow (capillaries) (%)

24 (19-38) 8 (3-19) 5 (3-6)

D. De Backer, et al. Am. J. Resp. Crit. Care Med. 2002 ; 166 : 98-104.


• Scavenging of ROS and RNS• Reduced ROS and RNS production• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction


Potential effects of albumin in sepsis

Mean level, µM

Free nitric oxide 0.0034 ± 0.00058

S-nitrosothiol 7.19 ± 5.73

S-nitrosoprotein 7.92 ± 5.45

Mean plasma levels of nitric oxide and S-nitrosothiols in humans

J. S. Stamler, et al. Proc. Natl. Acad. Sci 1992 ; 89 : 7674-7677.


• Scavenging of ROS and RNS• Reduced ROS and RNS production• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction



0

5

10

15

20

25

sham LPS LPS + HSA

Staining of the p65 subunit of NF-κB in aorta in septic mice

* = P < 0.01 vs others

(arbitrary units)


*

F. Meziani, et al. Am. J. Pathol. 2007 ; 171 : 1753-1761.

• Reduced ROS and RNS production and/or availability

• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction



Response to acethylcholine (Ach) in mesenteric arteriola of septic mice



O = LPS▲ = LPS plus

saline

= LPS plus albumin

● = Control

Effects of albumin on expression of inducible NOS (iNOS) in aorta of septic mice

Quantification for NO synhesis


Immunohistochemical staining for iNOS

Control LPS Albumin + LPS


• Reduced ROS and RNS production and/or availability

• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction



• Microvascular dysfunction• Depressed postreceptor signaling pathway• Impaired calcium liberation from the sarcoplasmatic

reticulum through over-expression of S100A8 and S100A9

• Impaired electromechanical coupling at the myofibrillar level

• Cell death


Mechanisms of sepsis-induced cardiac dysfunction

A. Rudiger et al. Crit. Care Med. 2007 ; 35 : 1599-1608.

0

2

4

6

8

10

12

Control LPS LPS plus albumin

C. Tokunaga, et al. J. Exp. Crit. Care Med. 2007 ; 35 : 1341-1347.

* = P < 0.05 versus othe groups

(Tissue PO2 mm Hg)


Effect of albumin resuscitation on myocardic oxygenation in endoxemic rats

*

*

• Microvascular dysfunction• Depressed postreceptor signaling pathway• Impaired calcium liberation from the sarcoplasmatic

reticulum through over-expression of S100A8 and S100A9

• Impaired electromechanical coupling at the myofibrillar level

• Cell death


Mechanisms of sepsis-induced cardiac dysfunction

A. Rudiger et al. Crit. Care Med. 2007 ; 35 : 1599-1608.

Effects of albumin on expression of inducible NOS (iNOS) in aorta of septic mice

Albumin reduces iNos expression in heart

Albumin reduces NO overproduction in heart

Albumin reduces NO overproduction in lung



Albumin β-adgrenergic signaling in cardiac tissueALBUMIN AND OXIDATIVE STRESS

0

40

80

120

Dose-responses to isoproterenol in isolated left ventricular papillary muscles from bile duct ligated- (BLD) rats and sham-

operated rats

H. Liu et al. Gastroenterology 2000 ; 118 : 937-944.

(Con

trac

tility

% o

f bas

al)

10 10 10 10- 8 - 7 - 6 - 5

BLD

Sham

BLD + L-Name

Isoproterenol (M)


Effects of albumin on cardiac contractility in cirrhotic rats

P. Angeli et al. AALSD 2007

-10.0 -9.5 -9.0 -8.5 -8.0

0

5

10

15

20

25

∆L

VD

P (m

m H

g)

Control

Cirrhotic + albumin

Cirrhotic

Log . Isoproterenol

* = P < 0.01

**


Albumin β-adgrenergic signaling in cardiac tissueALBUMIN AND OXIDATIVE STRESS

β2- AR β1- ARACGαs Gαs

Gαi

cAMP

PKA

Troponin I

RGS2

PDE2a

Ca2+

Myofibril

L-type C a2+

Ca 2+

SR

PLN

(-) (-)

(+)

(+)

(+)

(-)

(+)

(+)

β-adgrenergic signaling in cardiac tissue



0

1

2

3

4

Gen

e E

xpre

ssio

n (∆∆C

t)

RGS2 PDE2A Gαi2 Adcy3

Control ratsRats with cirrhosis

*

*

*

*

* = p < 0.01 vs controll

Effects of albumin on β-adgrenergic signaling in cardiac tissue


G. Ceolotto, et al. Hepatology 2008 (in press).

Rats with cirrhosis plus albumin

0

1

2

3

4


*

P < 0.01 vs control

Documents

XXIX Congresso SIFO Napoli 12-15 Ottobre 2008...J. H. Henriksen, et al. J. Hepatol. 2001 ; 34 : 53-60. Controls Cirrhotics with ascites Cirrhotics with refractory ascites P < 0.01