Protein Synthesis In The Liver And The Urea Cycle

Protein synthesis in theLiver and the Urea CycleDr NC BirdThis lecture will consider the featuresof how nitrogen is removed fromamino acids and converted to ureaand the major proteins synthesisedby the liver.In this overview we can see how theamino acid pool is added to fromdietary protein and how theintracellular proteins circlulate thesefree amino acids in a continuouscycle of synthesis and breakdown.Excess amino acids are metabolised(not stored for use as potentialenergy because this can be donemore efficiently using other energysources). The carbon skeleton, asan α-keto acid, is fed into the citricacid cycle to be incorporated intoglucose production whilst theammonia is largely excreted,although some is used in thebiosynthesis of amine containingsubstances.In the liver; input of amine groupscomes from dietary amino acids,alanine from muscle and glutaminefrom muscle. The α-ketoglutarate /glutamine pair are the most commonof the donor /acceptor pairs . Serumlevels of the aminotransferaseenzymes are used clinically asindicators of liver cell damage (thesereactions occur in the hepatocytes) –the most clinically useful beingAspartate aminotransferase (AST)and alanine aminotransferase (ALT)–higher levels indicating that moreenzyme has leaked from thedamaged hepatocyte.(a)α ketoglutarate acting as an amineacceptor giving us L-glutamate andan α keto acid. PLP representspyridoxal phosphate, which is themetabolically active form of VitaminB6 and is a co-factor in all of thesereactions.(b)

In the bottom reaction alanine isdeaminated to pyruvate - part of theglucose alanine cycle.Glutamate Dehydrogenase reactionThis reaction goes both ways usingNAD in the forward reaction andNADP the other way. The forwardreaction generates α ketoglutaratewhich is fed into the citric acid cycleand so hepatocytes are capable ofupregulating GDH activity at times ofenergy depletion (at a cellular levelat least). So as the illustrationshows, ADP / GDP drive the reactionforward – because they representthe ‘low energy’ i.e. ATP or GTPhave been ‘used’ by the cell so inorder to reconstitute them substratefor the citric acid cycle is generatedand ATP is replenished. When ATPconcentration is high then glutamateis formed and because it is an aminoacid, it is available for incorporationinto protein.Glutamine Synthetase reactionA reaction which produces an aminoacid – glutamine - suitable forincorporation into proteins. Itsprincipal role appears to be as acirculating ammonia carrier,consequently it is the most abundantamino acid found in blood. In thisstate it is a neutral, non-toxiccompound which passes readilythrough cell membranes.GlutaminaseIn most land animals glutamine iscarried in the blood to the liver. As isthe case for glutamate, the aminonitrogen is released only within themitochondria by this enzymeglutaminase.Glutamine synthetase is a cytosolicenzyme whereas glutaminase is amitochondrial enzyme, so they arelocated in separate compartmentswhich ensures that the liver is neithera net consumer or producer ofglutamine. The differences incellular location of these twoenzymes allows the liver toscavenge ammonia that hasn’t beenincorporated into urea and soammonia concentration is controlledby either incorporation into urea orglutamine.

Glucose alanine cycleIn muscle, alanine is the principalammonia scavenger and transporter.Glutamate collects the ammonia,the enzyme alanineaminotransferase (ALT)transaminates the amino group fromglutamate, forming α ketoglutarate,and the amino group gets attachedto pyruvate, formed from glycolysis,making alanine. This getstransported in the blood, taken up bythe liver where the reverse reactionoccurs and the ammonia getsconverted to urea. Pyruvate is recycled into glucose.This is a superb illustration ofeconomy of effort in solving twoproblems with one cycle. Movingcarbon atoms of pyruvate, as well asexcess ammonia, from muscle toliver as alanine. Then in the liver,alanine yielding pyruvate – thestarting block for gluconeogenesis,and releasing ammonia forconversion into urea. The energeticburden of gluconeogenesis beingimposed on the liver rather thanmuscle, so that muscle ATP can bedevoted to muscle contraction.The urea cycle.Discovered by Hans Krebs and KurtHenseleit in this university in 1932.Henseleit was a medical studenthere also.The essential features of the ureacycle reactions and their metabolicregulation are as follows:Arginine either from the diet orprotein breakdown, is cleaved byarginase generating urea andornithine. In subsequent reactions anew urea is built on the ornithine

(from ammonia and CO2 ) makingcitrulline. This, in turn, isreconfigured into arginine. Theenzymes responsible for this arefound partly in the mitochondria andpartly in the cytosol (likeglutaminase/glutamine synthase).The reactions of one turn of the cycleconsume 3 ATP equivalents and atotal of 4 high energy nucleotidePO4 . Urea is the only compoundgenerated by the cycle: all othercomponents are re-cycled. Theenergy consumed by ureaproduction is generated in theproduction of the cycleintermediates.Control of the cycle is via up or downregulation of the enzymesresponsible for urea formation. Sowith long term changes in thequantity of dietary protein,upregulation in the order of 20 timeshas been demonstrated. This canbe due to either increased intake aswith body builders – high protein lowfat diets - or in starvation becausemuscle proteins are being brokendown with the amino acid carbonskeletons providing the energy.Thus the amount of ammonia thatmust be excreted increases.Defects in the urea cycle.Absence of any of the enzymesinvolved in urea synthesis is notcompatible with life. Deficiencies inany one of them can occur and arewell described in the textbooks, butin terms of their likely impact on yourfuture clinical practice they areinsignificant and will probably bedealt with in your paediatricsmodules. The common thread tothem all is the elevation of ammonialevels in the blood.Neurotoxicity associated withAmmonia.Elevated blood ammonia is seen insevere liver disease, whether it be asa result of liver failure due toinfection, toxicity or substantialsurgical resection. This is somethingthat is seen in the clinical practice(not uncommonly) and sinceammonia is neurotoxic, is one ofthose things that staff are consciousof when a patient with liver diseasebecomes confused or comatose .The mechanism for the increase inammonia is basically the same in allcases. In simple terms, the blooddoesn’t get exposed to enough liverparenchymal cells to have theammonia removed. This can be dueeither to the fact that there simplyaren’t enough living, metabolisingcells because they’ve been killed offby the disease process be it viral ortoxic. Or because, in cirrhosis forexample, the resistance to blood flowthrough the liver is so great (becauseof fibrosis) that the blood by-passesthe liver by flowing through largecollateral veins. It therefore getsdelivered to the brain directly.

Ammonia crosses the blood-brainbarrier readily. Once inside it isconverted to glutamate via glutamatedehydrogenase and so depletes thebrain of α ketoglutarate. Asketoglutarate falls, so doesoxaloacetate and ultimately citricacid cycle activity stops, leading toirreparable cell damage and neuralcell death.Albumin leaves the circulation via theinterstitium to the lymph system andback to the circulation via thethoracic duct. Between 4-5% of totalintravascular albumin extravasatesper hour. This rate of movement isknown as the Transcapillary Escaperate and is determined by :1.Capillary and interstitial freealbumin concentration.2.Capillary permeability to albumin3.Movement of solute/solvent4.Electrical charges across thecapillary wall (albumin has a stronglynegative charge)The biological half-life in thecirculation is around 16-18 hoursFunctions1.Binding and jor proteins produced by the liverwhich have significant extra-hepaticroles.AlbuminA highly soluble, single polypeptideprotein with a MW of 66000. Around9-12 g produced per day with therate of production being controlled bychanges in colloid osmotic pressureand osmolality of the extravascularliver space. Production can beincreased by 2 to 3 fold whennecessary.There are 4 binding sites on albuminwhich have varying specificity fordifferent substances. Competitivebinding of drugs may occur at eitherthe same site or different sites(causing conformational changeswhich affect other binding sites).The drugs that are important foralbumin binding are; warfarin,NSAIDS, midazolam, thiopentone.2.Maintenance of colloid osmoticpressureColloid osmotic pressure is the termused to describe the effectiveosmotic pressure across bloodvessel walls which are permeable toelectrolytes but not larger molecules.It is almost entirely due to plasmaproteins.

The Starling equationNet Driving Pressure Kf x [(Pc – Pi)– rc(pc – pi)]Kf is the filtration coefficientPc – hydrostatic pressure in thecapillaryPi – hydrostatic pressure in theinterstitiumrc – the reflection coefficient is acorrection factor used to correct themagnitude of the measured gradientto take account of the ‘effectiveoncotic pressure’ (ie in systemswhere protein concs are low e.g.CSFthe rc will be close to 1 whereas inthe liver lymph the conc of protein ishigh and the value is close to 0pc – oncotic pressure in the capillarypi- oncotic pressure in theinterstitium.So net fluid flux is proportional tothis driving pressure. Also, thecapillary hydrostatic pressure fallsalong the capillary from the arteriolarto the venous end and so the drivingpressure decreases.3.Free RadicalsAlbumin has a large number ofsulphydryrl groups. These thiols areable to scavenge free radicals –nitrogen and oxygen species. Thismay be particularly important insepsis.4.Anticoagulant effects.Albumin has both anticoagulant andantithrombotic effects, both of whichare poorly understood. They may berelated to its binding of nitric oxideradicals which would have the effectof inhibiting inactivation andtherefore prolonging the biologicalhalf – lives.What causes albumin to decrease?1.Decreased synthesis.In liver disease or in largeresections, the functional mass ofthe liver is reduced and therefore itsability to synthesise proteins islikewise reduced.2.Increased catabolismVery slow decline in levels becauseit is synthesised at such a rate andsynthesis can be upregulatedseveral fold.3.Increased loss* Nephrotic syndrome – where thereis increased glomerular permeabilitywhich allows proteins to filter throughand so loss of up to several grams ofprotein per day can occur* Exudative loss in burns. Extensivetissue damage with concomittentdamage to the capillaries andtherefore loss of protein through thewall.* Haemorrhage* Gut lossA rare syndrome – protein losingenteropathy in which the wall of thegut is unusually permeable to largemolecules. More common howeveris ulcerative colitis where the site ofulceration is the site of increasedpermeability