xxix congresso sifo napoli 12-15 ottobre 2008...j. h. henriksen, et al. j. hepatol. 2001 ; 34 :...
TRANSCRIPT
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Main biological properties of albumin
XXIX Congresso SIFONapoli 12-15 Ottobre 2008
P. AngeliDept. of Clinical and Experimental Medicine University of Padova
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HUMAN ALBUMIN
The elipsoid structure of albumin in solution
66.5 Kda
The structure of albumin
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• 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.
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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.
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Main biological properties of albumin
• Albumin accounts for 75-80% of plasma colloid oncotic pressure
HUMAN ALBUMIN
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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
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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
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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.
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• Effect on endothelial function
HUMAN ALBUMIN
• Albumin accounts for 75-80% of plasma colloid oncotic pressure
Main properties of albumin
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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
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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%
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• Effect on endothelial function • Regulation of acid-base balance
HUMAN ALBUMIN
• Albumin accounts for 75-80% of plasma colloid oncotic pressure
Main properties of albumin
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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.
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(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
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• Effect on endothelial function • Regulation of acid-base balance• Ligand-binding
HUMAN ALBUMIN
• Albumin accounts for 75-80% of plasma colloid oncotic pressure
Main biological properties of albumin
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SH
NH2
DRUGSNSAIDsWarfarinBenzodiazepinesFurosemide
Exogenous moleculesBenzeneAflatossina G
Endogenous moleculesBilirubinBiliary saltsLCFA
HUMAN ALBUMIN
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• Effect on endothelial function • Regulation of acid-base balance• Ligand-binding• ROS/RNS scavennging and antioxidant function
HUMAN ALBUMIN
• Albumin accounts for 75-80% of plasma colloid oncotic pressure
Main biological properties of albumin
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Oxidative stress
↑ Production of ROS
↓ Reduction of Antioxidant
ALBUMIN AND OXIDATIVE STRESS
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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
ALBUMIN AND OXIDATIVE STRESS
Oxidative stress due to cellular surface modifications
Inflammation, endotoxins
Reduced tissue perfusion
Endotoxins, exogenous toxins
Induction
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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
ALBUMIN AND OXIDATIVE STRESS
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1. INTRACELLULARSODCatalasePeroxidaseGlutathioneSelenium
2. MEMBRANEVitamin Eß CaroteneUbiquinone
3. EXTRACELLULAR (PLASMA)Metal-Binding Proteins Caeruloplasmin, TransferrinAlbuminUric acidVitamin EVitamin C
Biological anti-oxidant systemsALBUMIN AND OXIDATIVE STRESS
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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)
ALBUMIN AND OXIDATIVE STRESS
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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
ALBUMIN AND OXIDATIVE STRESS
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SH
NH2
ROS/RNSH2O2ONOO–
Lipid hydroperoxide
ALBUMIN AND OXIDATIVE STRESS
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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)
ALBUMIN AND OXIDATIVE STRESS
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Oxidative stress
ALBUMIN AND OXIDATIVE STRESS
- Effects of ROS on albumin structure and functions- Effects of albumin infusion on:
• ROS production • ROS-related organ dysfunction
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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
ALBUMIN AND OXIDATIVE STRESS
ER. Stadman, et al. Amino Acids 2003 ; 25 : 207-218.
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ALBUMIN AND OXIDATIVE STRESS
Albumin dimers induced by peroxides in human plasma
Y. Ogasawara, et al. Biochim. Byoph. Res. Com. 2006 ; 340 : 353-358.
Exposure to OOHNormal
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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
*
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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)
ALBUMIN AND OXIDATIVE STRESS
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Oxidative stress
ALBUMIN AND OXIDATIVE STRESS
- Effects of ROS on albumin structure and functions- Effects of albumin infusion on:
• ROS production • ROS-related organ dysfunction
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time
ROS
Neutrophils ‘oxidative burst’
T-cells
ALBUMIN AND OXIDATIVE STRESS
Cellular sources of ROS in oxidative stress due to sepsis
•- Induction of iNOS and overproduction of nitric oxide
(NO) and reactive nitrogen species (RNS)
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ALBUMIN AND OXIDATIVE STRESS
Role of NF-kB in oxidative stress
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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
ALBUMIN AND OXIDATIVE STRESS
Mechanism of action of NF-kB
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ALBUMIN AND OXIDATIVE STRESS
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
{
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NAD(P)H•
p67
p47
gp91
rac
•O2-
O2 H+
NAD(P)+
p22phox
• Endothelial cells
• Vascular smooth muscle cells
• Cardiac myocytes
NADH/NADPH Oxidase
ALBUMIN AND OXIDATIVE STRESS
Cellular sources of ROS in oxidative stress due to sepsis
• TNF-α
• Thrombin
• Angiotensin II
• Platelet-derived growth factor
+
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ALBUMIN AND OXIDATIVE STRESS
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
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ALBUMIN IN OXIDATIVE STRESS
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
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Decreased BH4 reduces NO bioavailability in endothelial cells by causing eNOS uncoupling
eNOSL-arginine + O 2 L-citruline + NO .
ALBUMIN AND OXIDATIVE STRESS
-
BH 4
O 2-
H 2O 2ONOO
+ BH 4
BH 2
+
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eNOS
↑ ROS ↑ iNOS
ALBUMIN AND OXIDATIVE STRESS
Effect of ROS and iNOS on eNOS in endothelial cells
⊗ ⊗
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Microvascular dysfunction
Organ failure
Multiorgan failure (MOF)
• Reduced microcirculatory flow rate
• Increased flow heterogenity
• Decrease capillary density
ALBUMIN AND OXIDATIVE STRESS
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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.
ALBUMIN AND OXIDATIVE STRESS
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• Scavenging of ROS and RNS• Reduced ROS and RNS production• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction
ALBUMIN AND OXIDATIVE STRESS
Potential effects of albumin in sepsis
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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.
ALBUMIN AND OXIDATIVE STRESS
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• Scavenging of ROS and RNS• Reduced ROS and RNS production• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction
ALBUMIN AND OXIDATIVE STRESS
Potential effects of albumin in sepsis
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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)
ALBUMIN AND OXIDATIVE STRESS
*
F. Meziani, et al. Am. J. Pathol. 2007 ; 171 : 1753-1761.
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• Reduced ROS and RNS production and/or availability
• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction
ALBUMIN AND OXIDATIVE STRESS
Potential effects of albumin in sepsis
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Response to acethylcholine (Ach) in mesenteric arteriola of septic mice
F. Meziani, et al. Am. J. Pathol. 2007 ; 171 : 1753-1761.
ALBUMIN AND OXIDATIVE STRESS
O = LPS▲ = LPS plus
saline
= LPS plus albumin
● = Control
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Effects of albumin on expression of inducible NOS (iNOS) in aorta of septic mice
Quantification for NO synhesis
F. Meziani, et al. Am. J. Pathol. 2007 ; 171 : 1753-1761.
Immunohistochemical staining for iNOS
Control LPS Albumin + LPS
ALBUMIN AND OXIDATIVE STRESS
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• Reduced ROS and RNS production and/or availability
• Improvement of microvascular dysfunction• Improvement of cardiac dysfunction
ALBUMIN AND OXIDATIVE STRESS
Potential effects of albumin in sepsis
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• 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
ALBUMIN AND OXIDATIVE STRESS
Mechanisms of sepsis-induced cardiac dysfunction
A. Rudiger et al. Crit. Care Med. 2007 ; 35 : 1599-1608.
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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)
ALBUMIN IN OXIDATIVE STRESS
Effect of albumin resuscitation on myocardic oxygenation in endoxemic rats
*
*
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• 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
ALBUMIN AND OXIDATIVE STRESS
Mechanisms of sepsis-induced cardiac dysfunction
A. Rudiger et al. Crit. Care Med. 2007 ; 35 : 1599-1608.
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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
F. Meziani, et al. Am. J. Pathol. 2007 ; 171 : 1753-1761.
ALBUMIN AND OXIDATIVE STRESS
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Albumin β-adgrenergic signaling in cardiac tissueALBUMIN AND OXIDATIVE STRESS
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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)
ALBUMIN AND OXIDATIVE STRESS
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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 AND OXIDATIVE STRESS
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Albumin β-adgrenergic signaling in cardiac tissueALBUMIN AND OXIDATIVE STRESS
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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
P. Angeli et al. AALSD 2007
ALBUMIN AND OXIDATIVE STRESS
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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
ALBUMIN AND OXIDATIVE STRESS
G. Ceolotto, et al. Hepatology 2008 (in press).
Rats with cirrhosis plus albumin
0
1
2
3
4
P. Angeli et al. AALSD 2007
*
P < 0.01 vs control