1. The metabolic functions of the liver
~
The catabolism of heme
~
The metabolism of iron
Department of Biochemistry, Faculty of Medicine, Masaryk University
2011 (J.D.)
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2. The major metabolic functions of the liver
- the uptake of most nutrients from the gastrointestinal tract (Glc, AA, SCFA)
- intensive intermediary metabolism, conversion of nutrients
- storage of some substances (glycogen, iron, vitamins)
- controlled supply of essential compounds (Glc, VLDL, ketone bodies, plasma proteins etc.)
- ureosynthesis, biotransformation of xenobiotics (detoxification)
- excretion (cholesterol, bilirubin, hydrophobic compounds, some metals)
V. cava inf.
Hepatic veins
Portal vein
2
A. hepatica
3. Metabolism of glucose
• the primary regulation of blood glucose concentration
• glycogen synthesis in the postprandial state
• glycogenolysis and gluconeogenesis in fasting / starvation
• glucose galactose plasma glycoproteins
• glucose glucuronic acid conjugation reactions
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4. Metabolism of lipids
• FA are principal metabolic fuel for the liver
• SCFA go directly from portal blood,
other FA enter liver as chylomicron remnants
• completion and secretion of VLDL and HDL
• production of ketone bodies (alterantive nutrients), they cannot be
utilized in the liver, but they are supplied to other tissues
• secretion of cholesterol and bile acids into the bile is the major way
of cholesterol elimination from the body
The liver cells meet their energy requirements preferentially from fatty acids,
not from glycolysis. Glucose and glycogen are spared for extrahepatic tissues.
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5. Metabolism of nitrogen compounds (AA, purines, bilirubin)
• deamination of amino acids that are in excess of requirements
• intensive proteosynthesis of major plasma proteins and blood-clotting factors
• uptake of ammonium, ureosynthesis
• catabolism of purine bases – uric acid formation
• bilirubin capturing, conjugation, and excretion
Biotransformation of xenobiotics
Detoxification of drugs, toxins, etc. (see the separate lecture)
Vitamins
Hydroxylation of calciols to calcidiols, splitting of -carotene to retinol.
The liver represent a store of lipophilic vitamins and cobalamin (B12).
Iron and copper metabolism
Synthesis of transferrin, ceruloplasmin, ferritin stores, excretion of copper (bile)
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6. Transformation of hormones
• inactivation of steroid hormones – hydrogenation, conjugation
• inactivation of insulin and glucagon (see next page)
• inactivation of catecholamines and iodothyronines - conjugation
• dehydrogenation of cholesterol to 7-dehydrocholesterol and
25-hydroxylation of calciols play an essential role in calcium homeostasis
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7. Insulin, glucagon, and liver
Feature Insulin Glucagon
Formation in beta-cells, pancreas alfa-cells, pancreas
AA / chains / -S-S- bonds 51 / 2 / 3 29 / 1 / 0
Plasma half-life 3 min 5 min
Inactivation in liver, (kidneys) liver
GSH-insulin-transhydrogenase
Inactivation by insulinase
insulinase
During a single pass through liver about 50 % of insulin is removed.
GSH-insulin-transhydrogenase disrupts disulfide bonds:
2 GSH + insulin-disulfide G-S-S-G + insulin-dithiol
Insulinase (insulysin, insulin-specific protease, insulin-glucagon protease)
is cytosolic protease, degrades insulin, glucagon, and other polypetides,
no action on proteins.
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8. The liver parenchymal cell (hepatocyte)
Columns (cords) of hepatocytes are surrounded by sinusoids lined by endothelial
fenestrated layer (without a basement membrane) and Kupffer cells (mononuclear phagocytes)
Plasmatic membrane directed to the space of Disse – the blood pole,
the bile pole – lateral parts of the membrane with gap junctions and parts forming bile capillaries.
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9. Cell types in liver
Hepatocyte ~ 60 % of cells, high metabolic activity
located on sinusoid walls, take up disintegrated
Kupfer cells erythrocytes, macrophages, belong to RES,
high content of lysosomes, catabolism of heme
Stellate (Ito) cells deposits for TAG and lipophilic vitamins
Pit cells lymphocytes (natural killers)
epithelial cells of bile canaliculi,
Cholangiocytes
in membranes: ALP and GMT
of sinusoids make a fenestrated filtr system
Endothelial cells
(no basal membrane)
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10. Hexagonal lobule around central vein is the morphological
description (not the functional unit)
blood
flows through
sinusoids
to central vein
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11. The functional unit is called liver acinus
efferent vessels
at least two terminal
hepatic venules
bile ductules
arterial blood
terminal branches portal (venous) blood
aa. hepaticae from intestine, pancreas,
and spleen
portal field with afferent vessels
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12. The liver receives venous blood v. hepatica v. cava inferior
from the intestine. All the
products of digestion, in addition
to ingested drugs and other
xenobiotics, perfuse the liver
before entering the systemic
circulation.
a. hepatica
The mixed portal and arterial
blood flows through sinusoids v. portae
between columns of hepatocytes.
Hepatocytes are differentiated
in their functions according to the
decreasing pO2. In a liver acinus,
there are three zones equipped with
different enzymes.
ductus choledochus
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13. Metabolic areas in the acinus
terminal hepatic venule
ZONE 3
ZONE 2 is transition
ZONE 1
terminal
portal venule art. hepatica
bile ductule terminal hepatic venule
Zone 1 – periportal area Zone 3 – perivenous area
high pO2 low pO2
cytogenesis, mitosis high activity of ER (cyt P450, detoxification)
numerous mitochondria pentose phosphate pathway
gluconeogenesis and glycogenolysis hydrolytic enzymes
proteosynthesis glycogen, fat, and pigment stores
ureosynthesis glutamine synthesis
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15. Perivenous hepatocytes
Process Enzyme(s)
Dehydrogenation deamination of Glu GMD
FA synthesis Acetyl-CoA carboxylase
Ethanol catabolism AD
hydroxylations Cytochromes P-450
Ammonia detoxication (glutamine) Glutamine synthetase
Conjugation reactions UDP-glucuronyl transferase
Glycolysis glucokinase
Glycogen synthesis Glycogen synthase
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16. The compositon and functions of bile
Mass concentration (g/l)
Hepatic bile Gall-bladder bile
Inorganic salts 8.4 6.5
Bile acids 7 – 14 32 – 115
Cholesterol 0.8 – 2.1 3.1 – 16.2
Bilirubin glucosiduronates 0.3 – 0.6 1.4
Phospholipids 2.6 – 9.2 5.9
Proteins 1.4 – 2.7 4.5
pH 7.1 – 7.3 6.9 – 7.7
Functions
The bile acids emulsify lipids and fat-soluble vitamins in the intestine. High
concentrations of bile acids and phospholipids stabilize micellar dispersion
of cholesterol in the bile (crystallization of cholesterol → cholesterol gall-stones).
Excretion of cholesterol and bile acids is the major way of removing cholesterol from
the body. Bile also removes hydrophobic metabolites, drugs, toxins and metals
(e.g. copper, zinc, mercury).
Neutralization of the acid chyme in conjunction with HCO3– from pancreatic secretion.
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17. Transport processes in hepatocyte membrane
OCT – organic cation transporter, NTCP – Na/taurocholate cotransporting polypeptide,
OATP – organic anion transporting protein, MRP – multidrug resistance-associated protein
bile duct
hepatocyte
hepatocyte hepatocyte
OCT transcytosis of
org. cations
serum proteins
ATP canalicular membrane
cholesterol
NTCP
bile acids
ATP ATP blood
Na+
bile phospholipids
blood MRP 3
acids bile
OATP acids
bile acids,
org. anions HCO3−, GSH
passive difusion of serum proteins
The bile formation requires active transports from hepatocyte into bile duct 17
18. Three main components of bile (cholesterol, bile acid, lecithin)
create a complicated micellar dispersion system
triangle
diagram
Stable liquid mixture is under yellow curve ABC
Point P: 80 % bile acids, 15 % phosphatidylcholine, 5 % cholesterol 18
19. The catabolism of heme
from:
hemoglobin,
myoglobin,
cytochromes,
other heme enzymes (catalase etc.)
19
20. Heme catabolism: Three oxygen atoms attack protoporphyrin
HO O O OH
NH N
3 O2 H 3C CH3
NH NH
N HN heme oxygenase
B A (NADPH:cyt P450 oxidoreductase)
NH NH
H 3C
CH2
O O
O CH3
OO H2 C
CO
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21. Degradation of heme to bilirubin – bile pigments
Erythrocytes are taken up by the reticuloendothelial cells (cells of the spleen,
bone marrow, and Kupffer cells in the liver) by phagocytosis.
3 H2O
hemoglobin CO
3 O2
heme oxygenase verdoglobin
(NADPH:cyt P450 oxidoreductase)
Fe3+
globin
NADPH
biliverdin reductase biliverdin
bilirubin
In blood plasma, hydrophobic bilirubin molecules (unconjugated bilirubin) are
transported in the form of complexes bilirubin-albumin. 21
22. Real conformation of bilirubin
4
15
Theoretical polarity of the two carboxyl groups of unconjugated bilirubin is masked
by the formation of six intramolecular hydrogen bonds between the carboxyl groups
and electronegative atoms within the opposite halves of bilirubin molecule.
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23. The hepatic uptake, conjugation, and excretion of bilirubin
UNCONJUGATED bilirubin (bilirubin-albumin complex) in hepatic sinusoids
albumin
the amount that can leak from excretion
bilirubin receptor
(bilitranslocase) plasma membrane of hepatocytes
ligandin
(protein Y)
UDP-glucuronate
UDP
glucosyluronate
transferase terminal bile
on ER membranes ductule
CONJUGATED bilirubin
is polar, water-soluble
active transport (ABC transporter)
into bile capillaries
bilirubin bisglucosiduronate
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24. The formation of urobilinoids by the intestinal microflora
Conjugated bilirubin is secreted into the bile.
In the conjugated form, it cannot be absorbed
in the small intestine.
In the large intestine,
bacterial reductases and -glucuronidases
catalyze deconjugation and hydrogenation
4 H (vinyl → ethyl)
of free bilirubin to mesobilirubin and urobilinogens: GlcUA
A part of urobilinogens is split to mesobilirubin
dipyrromethenes, which can condense 4 H (reduction of bridges)
to give intensively coloured bilifuscins.
Urobilinogens are partly
– absorbed (mostly removed by
the liver), a small part appears 2H
i-urobilinogen
in the urine, urobilin
– partly excreted in the feces; 4H
on the air, they are oxidized 2H
to dark brown faecal urobilins. stercobilinogen
stercobilin 24
25. Overview of bilirubin metabolism in healthy individuals
Plasma: unconjugated bilirubin-albumin < 20 mol/l
bilirubin ← hemoglobin
uptake of bilirubin,
conjugation, excretion
V. lienalis
(the blood from the spleen
flows into the portal vein)
most of urobilinogens
are removed in liver small amounts of urobilinogens
(oxidation ?) not removed by the liver
conjugated bilirubin
portal
urobilinogens
A. renalis
urobilinogens
and dipyrromethenes
Urine:
urobilinogens < 5 mg/d
Feces:
urobilinoids and bilifuscins ~ 200 mg/d
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26. In the absence of intestinal microflora, bilirubin remains in stool
(before colonization in newborns or during treatment with broad-spectrum antibiotics)
Plasma: normal bilirubin concentration
Uptake of bilirubin
and its conjugation
Excretion into the bile
Conjugated bilirubin
Intestinal flora is lacking
or inefficient (speedy
passage in diarrhoea)
Urine:
urobilinogens
Feces: BILIRUBIN (golden-yellow colour are absent
that turns green on the air),
urobilins and bilifuscins are absent 26
27. Major types of hyperbilirubinemias
Hyperbilirubinemia – serum bilirubin > 20 mol/l
Icterus (jaundice) – yellowish colouring of scleras and skin,
(serum bilirubin usually more than 40 mol/l)
The causes of hyperbilirubinemias are conventionally classified as
prehepatic (hemolytic) – increased production of bilirubin,
hepatocellular due to inflammatory disease (infectious
hepatitis), hepatotoxic compounds (e.g. ethanol,
acetaminophen), or autoimmune disease; chronic
hepatitis can result in liver cirrhosis – fibrosis of
hepatic lobules,
posthepatic (obstructive) – insufficient drainage of intrahepatic
or extrahepatic bile ducts (cholestasis). 27
28. Hemolytic hyperbilirubinemia in excessive erythrocyte breakdown
Blood serum:
unconjugated bilirubin elevated
intensive uptake, conjugation,
and excretion into the bile
increased urobilinogens
are not removed sufficiently
high supply of urobilinogens
(bilirubin-albumin complexes
high portal conj. bilirubin cannot pass the glomerular filter)
urobilinogens
Urine: increased
urobilinogens
Feces: polycholic (high amounts (no bilirubinuria)
of urobilinoids and bilifuscins) 28
29. Hepatocellular hyperbilirubinemias may have different etiology
The results of biochemical test depend on whether an impairment of hepatic
uptake, conjugation, or excretion of bilirubin predominates.
Blood serum: unconj. bilirubin is elevated,
when its uptake or conjugation is impaired
conj. bilirubin is elevated,
impairment in when its excretion or drainage is impaired
uptake, conjugation,
or excretion ALT (and AST) catalytic concentrations increased
portal urobilinogens
are not removed efficiently
urobilinogens and conjugated
bilirubin pass into the urine
(not unconj. bilirubin-albumin complexes)
conj. bilirubin
(unless its excretion is impaired)
Urine: increased
urobilinogens
(unless bilirubin excretion is impaired)
Feces: normal contents bilirubinuria
(unless excretion is impaired) 29
(when plasma conj. bilirubin increases)
30. Obstructive hyperbilirubinaemia
Blood serum:
leakage of conj. conjugated bilirubin elevated
bilirubin from the cells bile acids concentration increased
into blood plasma uptake of bilirubin catalytic concentration of ALP increased
and its conjugation
conjugated bilirubin passes into the urine
ubg
low conj. bilirubin
(if obstruction is not complete)
Urine: urobilinogens
are lowered or absent
Feces: urobilinoids and bilifuscins decreased bilirubinuria
or absent (grey, acholic feces)
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31. Laboratory findings in hyperbilirubinemia
Bilirubin Ubg ALT ALP
Type
serum urine stool urine serum serum
Hemolytic ↑↑unconj. - polycholic ↑ N N
Hepatic ↑↑both ↑ N or ↑↑ ↑ ↑
Obstructive ↑↑conj. ↑ acholic - N or ↑ ↑
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32. Laboratory tests for detecting an impairment of liver functions
• Plasma markers of hepatocyte membrane integrity
Catalytic concentrations of intracellular enzymes in blood serum increase:
An assay for alanine aminotransferase (ALT) activity is the most sensitive one.
In severe impairments, the activities of
aspartate aminotransferase (AST) and
glutamate dehydrogenase (GD) also increase.
Increase of catalytic concentrations:
moderate injury
ALT AST GD
severe damage
cytoplasm mitochondria
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33. • Tests for decrease in liver proteosynthesis
Serum concentration of albumin (biological half-time about 20 days),
transthyretin (prealbumin, biological half-time 2 days) and transferrin,
blood coagulation factors (prothrombin time increases),
activity of serum non-specific choline esterase (CHE, CHS).
• Tests for the excretory function and cholestasis
Serum bilirubin concentration
Serum catalytic concentration of alkaline phosphatase (ALP)
and -glutamyl transferase (GMT)
Test for urobilinogens and bilirubin in urine
Estimation of the excretion rate of bromosulphophthalein (BSP test) is
applied to convalescents after acute liver diseases.
• Tests of major metabolic functions are not very decisive:
Saccharide metabolism: low glucose tolerance (in oGT test)
Lipid metabolism: increase in VLDL (triacylglycerols) and LDL (cholesterol)
Protein catabolism: decreased urea, ammonium increase (in the final stage of liver failure, hepatic coma)
• Special tests to specific disorders: serological tests to viral hepatitis,
serum -fetoprotein (liver carcinoma), porphyrins in porphyrias, etc.
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34. Metabolism of iron
The body contains 4.0 – 4.5 g Fe:
hemoglobin 2.5 – 3.0 g Fe,
tissue ferritin stores up to 1.0 g Fe in men (0.3 – 0.5 g in women),
myoglobin and other hemoproteins 0.3 g Fe,
circulating transferrin 3 – 4 mg Fe.
The daily supply of iron in mixed diet is about 10 – 20 mg.
From that amount, not more than only l – 2 mg are absorbed.
Iron metabolism is regulated by control of uptake, which have to replace the
daily loss in iron and prevent an uptake of excess iron.
A healthy adult individual loses on average 1 – 2 mg Fe per day in desquamated cells
(intestinal mucosa, epidermis) or blood (small bleeding, so that women are more at
risk because of net iron loss in menstruation and pregnancy).
There is no natural mechanism for eliminating excess iron from the body.
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35. Linguistic note:
How to express two redox states of iron
Fe2+ Fe3+
Infix -o -i
hemiglobin = methemoglobin
Biochemical examples hemoglobin
ferritin, transferrin
Chemical example Latin ferrosi chloridum ferri chloridum
Old English ferrous chloride FeCl2 ferric chloride FeCl3
New English iron(II) chloride iron(III) chloride
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36. Food sources and absorption of iron
Animal Plant
• blood, blood products • broccoli
• red meat, liver • green leafy vegetables
• bioavailability ~ 20 % • bioavailability ~ 10 %
General availability heme-Fe2+ > Fe2+ > Fe3+
gastroferrin, ascorbate, citrate, sugars,
Supporting factors
amino acids, low pH, iron deficit
phytates (cereals), oxalates (spinach),
Inhibiting factors tanins (tea, red wine),
intestinal adsorbents, antacids, iron excess
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37. 10 – 20 mg Fe Absorption of iron in duodenum and jejunum
Phosphates, oxalate, and phytate (inositolhexakisphosphate),
present in vegetable food
form insoluble Fe3+ complexes and disable absorption.
Fe2+ is absorbed much easier than Fe3+. Reductants such as
ascorbate or fructose promote absorption, as well as Cu2+.
8 – 19 mg Fe Gastroferrin, a component of gastric secretion, is a glycoprotein
that binds Fe3+ maintaining it soluble
elimination of insoluble transferrin–2 Fe3+
Fe salts in feces
ENTEROCYTE
bile pigments
DUODENUM
heme transporter
heme (Fe2+)
Fe3+ ferritin
soluble Fe2+ hephestin
(ferrooxidase)
DMT1
divalent metal transporter
Fe2+ Fe2+
soluble Fe3+ ferric reductase
(gastroferrin)
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38. Transferrin (Trf)
is a plasma glycoprotein (a major component of 1-globulin fraction), Mr 79 600.
Plasma (serum) transferrin concentration 2.5 – 4 g / l (30 – 50 mol/l)
Transferrin molecules have two binding sites for Fe ions,
total iron binding capacity (TIBC) for Fe ions is higher than 60 mol/l.
Serum Fe3+ (i.e. transferrin-Fe3+) concentration is about 10 – 20 mol/l,
14 – 26 mol/l ♂
11 – 22 mol/l ♀
Circadian rhythm exists, the morning concentrations are
higher by 10 - 30 % than those at night..
Saturation of transferrin with Fe3+ equals usually about 1/3.
Because the biosynthesis of transferrin is stimulated during iron deficiency (and
plasma iron concentration decreases), the decrease in saturation of transferrin
is observed.
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39. Iron is taken up by the cells through
a specific receptor-mediated endocytosis
• • transferrin-2Fe3+
• •
transferrin
receptor
Some receptors are released from the plasmatic membranes. Increase in serum
concentration of those soluble transferrin receptors is the earliest marker
of iron deficiency.
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40. Transport and distribution of iron
LIVER CELLS
ENTEROCYTES
biosynthesis of transferrin
food iron Fe3+ transferrin–Fe3+ Fe3+ ferritin
ferritin
SPLEEN
Fe3+ ferritin
hemoglobin hemoglobin
breakdown
BONE MARROW
hemoglobin
synthesis loss of blood
ferritin
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41. Ferritin occurs in most tissues
(liver, spleen, bone-marrow, enterocytes)
The protein apoferritin is a ball-shaped
homopolymer of 24 subunits that iron(III)
hydroxide hydrate
surrounds the core of hydrated Fe(OH)3. core
One molecule can bind few thousands
of Fe3+ ions, which make up to 23 % of
apoferritin ferritin
the weight of ferritin. (colourless) (brown)
Minute amounts of ferritin are released into the blood plasma from the extinct
cells. Plasma ferritin concentration 25 – 300 g/l is proportional to the ferritin
stored in the cells, unless the liver is impaired (increased ferritin release from the
hepatocytes).
If the loading of ferritin is excessive, ferritin aggregate into its degraded form,
hemosiderin, in which the mass fraction of Fe3+ can reach 35 %.
Ferritin was discovered by V. Laufberger, Professor at Masaryk university, Brno, in 1934. 41
42. Hepcidin
is a polypeptide (25 AA, 8 Cys), discovered as the liver-expressed antimicrobial
peptide, LEAP-1, in 2000. It is produced by the liver (to some extent in myocard
and pancreas, too) as a hormone that limits the accessibility of iron and also
exhibits certain antimicrobial and antifungal activity.
The biosynthesis of hepcidin is stimulated in iron overload and
in inflammations (hepcidin belongs to acute phase proteins type 2),
and is supressed during iron deficiency .
Notice the fact that the same two factors stimulating hepcidin synthesis
inhibit the biosynthesis of transferrin.
Effects of hepcidin: It – reduces Fe2+ absorption in the duodenum,
– prevents the release of recyclable Fe from macrophages,
– inhibits Fe transport across the placenta,
– diminishes the accessibility of Fe for invading pathogens.
Hepcidin is filtered in renal glomeruli and not reabsorbed in the renal tubules,
the amount of hepcidin excreted into the urine corresponds with the amount
synthesized in the body. There is a positive correlation between this amount of
hepcidin and the concentration of ferritin in blood plasma.
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