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5 liver
- 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.) 1
- 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 3
- 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. 4
- 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) 5
- 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 6
- 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. 7
- 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. 8
- 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) 9
- 10. Hexagonal lobule around central vein is the morphological description (not the functional unit) blood flows through sinusoids to central vein 10
- 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 11
- 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 12
- 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 13
- 14. Periportal hepatocytes Process Enzyme(s) transamination ALT, AST Citrate cycle succinate DH, malate DH Gluconeogenesis (Cori cycle) LD Ammonia detoxication (urea) carbamoyl-P-synthetase, arginase Deamidation of glutamine glutaminase ROS elimination GSH peroxidase ... Cholesterol synthesis HMG-CoA reductase Gluconeogenesis glucose 6-phosphatase, PEPCK 14
- 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 15
- 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. 16
- 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 20
- 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. 22
- 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 23
- 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 25
- 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) 30
- 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 ↑ ↑ 31
- 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 32
- 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. 33
- 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. 34
- 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 35
- 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 36
- 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) 37
- 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. 38
- 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. 39
- 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 40
- 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. 42