Protein synthesis in the Liver and the Urea Cycle Dr NC Bird

This lecture will consider the features of how nitrogen is removed from amino acids and converted to urea and the major proteins synthesised by the liver.

glutamine pair are the most common of the donor /acceptor pairs . Serum levels of the aminotransferase enzymes are used clinically as indicators of liver cell damage (these reactions occur in the hepatocytes) – the most clinically useful being Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) –higher levels indicating that more enzyme has leaked from the In this overview we can see how the damaged hepatocyte. amino acid pool is added to from dietary protein and how the intracellular proteins circlulate these free amino acids in a continuous cycle of synthesis and breakdown. Excess amino acids are metabolised (not stored for use as potential energy because this can be done more efficiently using other energy sources). The carbon skeleton, as an α-keto acid, is fed into the citric acid cycle to be incorporated into (a) glucose production whilst the α ketoglutarate acting as an amine ammonia is largely excreted, acceptor giving us L-glutamate and although some is used in the an α keto acid. PLP represents biosynthesis of amine containing pyridoxal phosphate, which is the substances. metabolically active form of Vitamin B6 and is a co-factor in all of these In the liver; input of amine groups reactions. comes from dietary amino acids, (b) alanine from muscle and glutamine from muscle. The α-ketoglutarate / In the bottom reaction alanine is deaminated to pyruvate - part of the glucose alanine cycle.

Glutamate Dehydrogenase reaction

This reaction goes both ways using NAD in the forward reaction and NADP the other way. The forward reaction generates α ketoglutarate which is fed into the citric acid cycle and so hepatocytes are capable of upregulating GDH activity at times of energy depletion (at a cellular level at least). So as the illustration Glutaminase shows, ADP / GDP drive the reaction forward – because they represent In most land animals glutamine is the ‘low energy’ i.e. ATP or GTP carried in the blood to the liver. As is have been ‘used’ by the cell so in the case for glutamate, the amino order to reconstitute them substrate nitrogen is released only within the for the citric acid cycle is generated mitochondria by this enzyme and ATP is replenished. When ATP glutaminase. concentration is high then glutamate is formed and because it is an amino Glutamine synthetase is a cytosolic acid, it is available for incorporation enzyme whereas glutaminase is a into protein. mitochondrial enzyme, so they are located in separate compartments Glutamine Synthetase reaction which ensures that the liver is neither a net consumer or producer of A reaction which produces an amino glutamine. The differences in acid – glutamine - suitable for cellular location of these two incorporation into proteins. Its enzymes allows the liver to principal role appears to be as a scavenge ammonia that hasn’t been circulating ammonia carrier, incorporated into urea and so consequently it is the most abundant ammonia concentration is controlled amino acid found in blood. In this by either incorporation into urea or state it is a neutral, non-toxic glutamine. compound which passes readily through cell membranes. Glucose alanine cycle

In muscle, alanine is the principal ammonia scavenger and transporter. Glutamate collects the ammonia, the enzyme alanine aminotransferase (ALT) transaminates the amino group from glutamate, forming α ketoglutarate, and the amino group gets attached to pyruvate, formed from glycolysis, making alanine. This gets transported in the blood, taken up by the liver where the reverse reaction occurs and the ammonia gets converted to urea. Pyruvate is re- cycled into glucose. The urea cycle.

This is a superb illustration of Discovered by Hans Krebs and Kurt economy of effort in solving two Henseleit in this university in 1932. problems with one cycle. Moving Henseleit was a medical student carbon atoms of pyruvate, as well as here also. excess ammonia, from muscle to liver as alanine. Then in the liver, The essential features of the urea alanine yielding pyruvate – the cycle reactions and their metabolic starting block for gluconeogenesis, regulation are as follows:- and releasing ammonia for conversion into urea. The energetic Arginine either from the diet or burden of gluconeogenesis being protein breakdown, is cleaved by imposed on the liver rather than arginase generating urea and muscle, so that muscle ATP can be ornithine. In subsequent reactions a devoted to muscle contraction. new urea is built on the ornithine (from ammonia and CO2 ) making modules. The common thread to citrulline. This, in turn, is them all is the elevation of ammonia reconfigured into arginine. The levels in the blood. enzymes responsible for this are found partly in the mitochondria and partly in the cytosol (like glutaminase/glutamine synthase).

The reactions of one turn of the cycle consume 3 ATP equivalents and a total of 4 high energy nucleotide = PO4 . Urea is the only compound generated by the cycle: all other components are re-cycled. The Neurotoxicity associated with energy consumed by urea Ammonia. production is generated in the production of the cycle Elevated blood ammonia is seen in intermediates. severe liver disease, whether it be as a result of liver failure due to Control of the cycle is via up or down infection, toxicity or substantial regulation of the enzymes surgical resection. This is something responsible for urea formation. So that is seen in the clinical practice with long term changes in the (not uncommonly) and since quantity of dietary protein, ammonia is neurotoxic, is one of upregulation in the order of 20 times those things that staff are conscious has been demonstrated. This can of when a patient with liver disease be due to either increased intake as becomes confused or comatose . with body builders – high protein low fat diets - or in starvation because The mechanism for the increase in muscle proteins are being broken ammonia is basically the same in all down with the amino acid carbon cases. In simple terms, the blood skeletons providing the energy. doesn’t get exposed to enough liver Thus the amount of ammonia that parenchymal cells to have the must be excreted increases. ammonia removed. This can be due either to the fact that there simply Defects in the urea cycle. aren’t enough living, metabolising cells because they’ve been killed off Absence of any of the enzymes by the disease process be it viral or involved in urea synthesis is not toxic. Or because, in cirrhosis for compatible with life. Deficiencies in example, the resistance to blood flow any one of them can occur and are through the liver is so great (because well described in the textbooks, but of fibrosis) that the blood by-passes in terms of their likely impact on your the liver by flowing through large future clinical practice they are collateral veins. It therefore gets insignificant and will probably be delivered to the brain directly. dealt with in your paediatrics Ammonia crosses the blood-brain Albumin leaves the circulation via the barrier readily. Once inside it is interstitium to the lymph system and converted to glutamate via glutamate back to the circulation via the dehydrogenase and so depletes the thoracic duct. Between 4-5% of total brain of α ketoglutarate. As intravascular albumin extravasates ketoglutarate falls, so does per hour. This rate of movement is oxaloacetate and ultimately citric known as the Transcapillary Escape acid cycle activity stops, leading to rate and is determined by :- irreparable cell damage and neural 1.Capillary and interstitial free cell death. albumin concentration. 2.Capillary permeability to albumin 3.Movement of solute/solvent 4.Electrical charges across the capillary wall (albumin has a strongly negative charge)

The biological half-life in the circulation is around 16-18 hours

Functions

1.Binding and transport

There are 4 binding sites on albumin which have varying specificity for different substances. Competitive binding of drugs may occur at either the same site or different sites (causing conformational changes which affect other binding sites). The drugs that are important for *************************************** albumin binding are; warfarin, Major proteins produced by the liver NSAIDS, midazolam, thiopentone. which have significant extra-hepatic roles. 2.Maintenance of colloid osmotic pressure Albumin Colloid osmotic pressure is the term A highly soluble, single polypeptide used to describe the effective protein with a MW of 66000. Around osmotic pressure across blood 9-12 g produced per day with the vessel walls which are permeable to rate of production being controlled by electrolytes but not larger molecules. changes in colloid osmotic pressure It is almost entirely due to plasma and osmolality of the extravascular proteins. liver space. Production can be increased by 2 to 3 fold when necessary. of inhibiting inactivation and therefore prolonging the biological The Starling equation half – lives.

Net Driving Pressure = Kf x [(Pc – Pi) What causes albumin to decrease? – rc(pc – pi)] Kf is the filtration coefficient 1.Decreased synthesis. Pc – hydrostatic pressure in the capillary In liver disease or in large Pi – hydrostatic pressure in the resections, the functional mass of interstitium the liver is reduced and therefore its rc – the reflection coefficient is a ability to synthesise proteins is correction factor used to correct the likewise reduced. magnitude of the measured gradient to take account of the ‘effective 2.Increased catabolism oncotic pressure’ (ie in systems where protein concs are low e.g.CSF Very slow decline in levels because the rc will be close to 1 whereas in it is synthesised at such a rate and the liver lymph the conc of protein is synthesis can be upregulated high and the value is close to 0 several fold. pc – oncotic pressure in the capillary pi- oncotic pressure in the 3.Increased loss interstitium. So net fluid flux is proportional to * Nephrotic syndrome – where there this driving pressure. Also, the is increased glomerular permeability capillary hydrostatic pressure falls which allows proteins to filter through along the capillary from the arteriolar and so loss of up to several grams of to the venous end and so the driving protein per day can occur pressure decreases. * Exudative loss in burns. Extensive tissue damage with concomittent 3. Free Radicals damage to the capillaries and therefore loss of protein through the Albumin has a large number of wall. sulphydryrl groups. These thiols are * Haemorrhage able to scavenge free radicals – * Gut loss nitrogen and oxygen species. This A rare syndrome – protein losing may be particularly important in enteropathy in which the wall of the sepsis. gut is unusually permeable to large molecules. More common however 4. Anticoagulant effects. is ulcerative colitis where the site of ulceration is the site of increased Albumin has both anticoagulant and permeability antithrombotic effects, both of which are poorly understood. They may be related to its binding of nitric oxide radicals which would have the effect Consequences of decreased serum Pre-eclampsia – the association of albumin hypertension, proteinuria and oedema in pregnancy- there is a 1.Decreased colloid oncotic pressure paradoxical decrease in plasma volume and capillary leak syndrome. Decreased colloid oncotic pressure and oedema formation (oedema Burns / Trauma being the accumulation of fluid in the interstitial space). The formation of Loss through leaky capillaries oedema is determined by the rate of although burns patients can also fluid flux and the clearance of fluid by develop a protein losing enteropathy the lymphatics. Sepsis In critical illness there is increased leakage of albumin and decreased Increased capillary permeability – synthesis. There is a stronger possibly due to bacterial endotoxins correlation between colloid oncotic and cytotoxic T cells. There is also a pressure and total protein because profound reduction in plasma the decreased albumin synthesis is albumin associated with marked fluid compensated for by increased shifts. synthesis of acute phase proteins. There is controversy about the use 2.Decreased ligand binding of albumin in clinical practice. Previously studies have shown a Drug kinetics are altered, similarly correlation between low serum hormone transport can be affected. albumin and mortality. Therefore the obvious thing to do, it would seem, would be to raise serum albumin Disease processes associated with levels by albumin infusion. However, low serum albumin these ‘normalisation ‘ regimes have not proved to be effective – in fact Malnutrition – a diet with a high one meta- analysis has suggested a proportion of low-grade cereal higher mortality rate in critically ill proteins (such as maize) can lead to patients treated with albumin. It is a deficiency in the essential amino still used by some – others adopt the acid lysine which in turn leads to a ‘’treat the reason for the capillary decrease in protein synthesis. In leakage and the patient will get contrast starvation does not lead to better’ strategy. low albumin levels – protein from muscle is used as the energy Clotting proteins produced by the source. liver Liver disease Renal diseases – albumin loss With the exception of von Willebrand through the glomerulus and to a Factor (VIII) the liver is responsible small extent during dialysis. for the production of all of the coagulation proteins. Many are serine proteases which require activation

Clotting cascade

In this example Factor 9a (a serine protease itself) activates Factor 10 by cleavage of the heavy chain.

Many have a γ carboxyglutamic acid residue which requires Vitamin K (also stored in the liver) for conversion.