42: Fatty Acid Synthesis
BIO EXAM 4
Intro:
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Storage mechanism for excess fuel
Starting material for lipid biosynthesis
Liver is the major site, also in other tissues
FAs synthesized in the cytosol by a different pathway
than FA oxidation (in mito matrix)
NADPH provides reducing equivalents
Acetyl CoA is the starting material
Incremental addition of 2-carbon units
o Leads to predominance of even
numbered FA chains
Glucose Acetyl CoA
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Acetyl CoA is formed in the mito matrix
but cannot cross the inner mito membrane
Acetyl CoA combines with OAA to form
Citrate (this is the first rxn of TCA cycle)
o Citrate is able to cross the
membrane  Acetyl CoA and OAA
released in cytosol
Regeneration of Pyruvate
 Citrate lyase converts citrate back to acetyl CoA and OAA
 OAA is reduced to malate by cytosolic malate dehydrogenase using NADH
 Malate is oxidatively decarboxylated by malic enzyme forming NADPH, pyruvate and CO2.
 Pyruvate reenters the mito matrix and can be reconverted to OAA by pyruvate carboxylase or to acetyl CoA by PDH
 Patients with chronically high carbohydrate intake will need this pathway more and will have higher levels of these
enzymes.
 Importance of production of NADPH!!!
o Only in pentose phosphate shunt (PPP) and this malic enzyme
o NADPH provides the reducing power needed to synthesize hydrocarbon chains for FA synthesis
o PPP deficiency  Malic enzyme can be used to help supply some NADPH
 The key is there is a cytoplasmic “pool” of malate, which can be used by either pathway, depending on insulin (FA
synth) or glucagon (glucose synthesis).
Acetyl CoA  malonyl CoA
 Condensation of the acetyl group is endergonic
 First step in FA synthesis—activation of acetyl CoA by carboxylation to form malonyl CoA (adds 2 C to chain)
 Enzyme is Acetyl CoA carboxylase which uses a Biotin cofactor (adds one carboxyl group)
o Metabolically irreversible and the rate limiting step
 Commits acetyl CoA to FA synthesis and consumes energy
FA Synthase Complex
 Palmitate is synthezised by repeptitive addition of 2 carbon units
o Followed by the reduction of each condensation product by NADPH
 FA Synthase: a single multifunctional protein with 7 different enzyme activities in a single polypeptide chain which
forms a dimer
 ACP: acyl carrier protein- contains phosphopanttheine (similar to CoA) **
o Cofactor for FA synthase
FA Synthesis: 5 Stages
*1) Loading: one acetyl CoA then one malonyl CoA transferred onto ACP (Acyl carrier protein) on the FA synthase forming
acetyl ACP then malonyl ACP
 Acetyl CoA only used at the beginning to start process (initiation), then only malonyl CoA used after that to lengthen
the chain
 Fatty Acyl Synthase has cysteine residue that will form a bond between the acetyl CoA and a second cysteine residue
that binds to the malonyl CoA (now both bound to FA synthase)  conjugating the two together creates a 4 carbon
unit
2) Condensation: acetyl group transfers onto malonyl group, displacing the CO2 but lengthening the chain by 2 carbons
(malonyl comes off of the cysteine residue and attaches to acetyl CoA that is still attached)
 2 groups are condensed into one  Problem is that this creates a beta-keto group (needs to be removed)
o beta-keto group is the site for reduction and dehydration steps
 ACP is the site of FA synthesis
 Carboxyl group activates methylene carbon for new bond with neighbor acetyl group
 Elimination of CO2: respiration energy consumption for anabolism
3)Reduction: the β-keto group is reduced to a β-hydroxyl group by NADPH
 Reduces the double bond
 Ketone carbonyl is reduced to an alcohol group
4) Dehydration: the elements of H20 are removed (H and OH) across the α-β carbon atoms forming an α-β trans double
bond
5) Reduction: the double bond is reduced using NADPH, produces saturated, fully reduced carbon bonds
 The electrons needed to form new C-H bond come from the NADPH hydride, extra proton forms other C-H bond
 Now you have the nacent FA in the form that you can add another 2 carbon units
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Energy content of FA is increasing as it becomes more reduced
Requires energy input: stores the energy for beta oxidation later
Oxygen is needed in high demand in burning and building up fats
By coupling the breakdown of NADPH with FA production we are building up energy within that molecule that will be
alleviated when broken down which is how we produce NADH during the breakdown
The Cycle Repeats..
 This cycle is repeated 7 times forming palmitate (16 carbon chain), uses malonyl ACP each time after initiation to
extend the FA 2 carbons
 The final reaction of FA synthesis is the cleavage of palmitoyl ACP by a thioesterase to release the free fatty acid
 Palmitate is rapidly converted to palmitoyl CoA by acyl CoA Synthetase
o CoA is added once Free FA is removed
 Fatty acid synthesis is energy efficient using only 7 ATP to form 7 malonyl CoA and 2 ATP equivalents to form the acyl
CoA  activated energized FA produced
Elongation of FA
 Elongation and desaturation enzymes are in the cytoplasmic face of the ER
 Elongase: needed to elongate beyond 16C (no longer using FA Synthase)
o Uses fatty acyl CoA as a substrate and extends the chain 2C at a time using malonyl CoA condensation
followed by reduction, dehydration, and reduction using NADPH in analogous fashion to FA synthase
PUFA
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Unsaturated FA that contain one or more double bonds
Fatty acyl CoA desaturase produces the double bond formation in single bonded chains
o requires NADH and O2
o involve an ER ETC which tranfers electrons and protons
o saturated fatty acyl CoA monounsaturated
FA desaturation in humans
 human desaturases can only place db between the carbonyl carbon and C10
o palmitic acid palmitoleic acid or steric acid  oleic acid
 humans cant place db between C10 and the w-carbon (terminal methyl group )
 Problem= Eicosanoid Synthesis
o Eicosanoid- immunological activators that utilize molecules that have db after C10
 Need to have those FA bc we cant produce those dbs
o 20C FA (arachadonic acid)
o key signaling molecules
o Need PUFAs with dbs 3 carbons from the methyl end (w3) and 6 carbons from the methyl end (w6)
Essential FA and PUFA synthesis
 Example: arachidonic acid
 Start with an essential FA: linoleic acid
o FA we can’t make, must consume (plant oils or fish oil)
o We ingest these fats (essential from our diet) that contain those db (like at C12) manipulate that fat to
make the FA we are looking for (linoleic acid arachidonic acid)
 Introduce those double bonds that we can make (1-10) NADH  NAD
 Lengthen the chain to move the bonds to the high number carbon positions- Malonyl CoA (elongation)
 Introduce more double bonds at positions where we can (1-10) NADH NAD
Location
Thioester carrier
Enzymes
Electron carriers
Synthesis
Cytosol
Malonyl CoA
E complex
NADPH
Oxidation
Mitochondria
Acetyl CoA
Individual enzymes
NADH
Protect newly formed FAs *
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Build up of Malonyl CoA in the cytoplasm inhibits
transferase and doesn’t allow fatty acyl transfer into
mitochondria by inhibiting CPT1 (primary player at getting
FA into the mito for oxidation)
Prevents FA from getting immediately oxidized by the beta
oxidation pathway
Regulation of FA synthesis and oxidation**
1) Hormonal control: insulin and glucagon
- same hormones that regulate blood glucose levels also regulate FA metabolism
2) Expression or degradation of enzymes
-Long term regulation involves changing the amount of key enzymes (takes long time, but once adjusted it meets
metabolic needs much better)
3) Phosphorylation or dephosphorylation of cell signal enzymes *
- Short term regulation involves the modulation of the activity of enzymes
-Allosteric activators are inhibitors
-Insulin stimulates FA synthesis by stimulating the dephosphorylation of acetyl CoA carboxylase
-Malonyl CoA allosterically inhibits carnitine acyltransferase I so that acetyl CoA is not translocated into the mito
matrix but remains in the cytosol
-Insulin also causes dephosphorylation of adipocyte hormone sensitive lipase inactivating it and preventing TG
hydrolysis
Acetyl CoA Carboxylase (Regulation)
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Allosteric
o Feed forward and feed back inhibition
Phosphorylation
o Glucagon  inactive
o Insulin  active
Inducible
o Cells can alter the amount of enzyme present
Rate limiting step: committed step
Citrate activates
Palmitoyl CoA inhibits
Synthesis of Triaglycerols and Major Membrane Lipids (43)
Intro:
 Newly synthesized FAs are incorporated into TGs, glycerophospholipids and sphingolipids
o These molecules are constantly turning over: degradation and new synthesis
 Enzymes synthesizing these complex structures are located on the cytoplasmic face of the SER
 All glycerophospholipids and TGs have the same initial pathway to phosphatidic acid:
o TGs and neutral glycerophospholipids proceed via dephosphorylation to 1,2-diacyl glycerol (1,2-DAG): a
glycerol backbone with FAs attached to carbon 1 and 2.
 Synthesis of the final product requires an activated FA or alcohol
 Acidic glycerophospholipid synthesis proceeds via an activated diacylglycerol, CDP-DAG.
Synthesis pathways of TG and phosphatidic acid
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synthesis of phosphatidic acid starts ultimately with glucose,
but locally with dihydroxyacetone phosphate (DHAP)
Liver has glycerol kinase
Liver uses glycerol backbone to form TGs by adding FA-CoAs
DHAP is reduced to glycerol 3-P by G-3-P dehydrogenase.
Preexisiting glycerol can also be phosphorylated in liver to
glycerol 3-P.
Adipose lacks this enzyme to make G-3-P:
o Don’t want adipose tissue to convert glycerol back to
lipid when FA’s are mobilized for energy. Rather, use
the glycerol for glucose synthesis!
Acyl groups are added at C-1 and C-2 by acyltransferases using
acyl CoA, saturated FAs are preferentially added at C-1 and
unsaturated FAs at C-2
TG synthesis and Transport
 C3 of phosphatidic acid is dephosphorylated, forming diacyl glycerol (DAG) ** (beginning of TG synthesis)
 A third FA is attached at C-3 to complete the TG synthesis
 TGs are packaged into VLDLs (TG is major component of VLDL), processed in the Golgi, then secreted into the blood
 Destination is adipose tissue for storage and muscle for energy use.
 Remember: VLDL also as apoCII, which activates LPL (LPL favors muscle use over fat storage)
o Muscle LPL Km is much lower than adipose tissue LPL Km
o Allows muscle to take up FAs from VLDL (and chylomicrons!) even when concentrations of these are low.
o In contrast, adipose tissue will only take up and store FAs from VLDLs and chylomicrons when these are in
high concentrations, like after a meal…
 VLDL assembly (liver) is nearly identical to chylomicron assembly (intestines)
Storage of TG in adipose tissue (fed state)
 TG in blood (in chylomicrons and VLDL) are hydrolyzed to FA and glycerol by lipoprotein lipase (LPL)
o Reassembled once taken in
o LPL is on the outer surface of the plasma membrane of cells lining the capillary tissues.
o LPL is synthesized in adipose cells and secreted into capillaries of adipose tissue in response to insulin
 FAs liberated by LPL are taken up by adipose cells – free glycerol is not utilized by the cells
o Adipose cells don’t have glycerol kinase
o Prevents futile cycle of attaching FAs immediately back onto glycerol 3-P
Degradation of Triaglycerol
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during fasting, adipose TG is broken down by process called
lipolysis
lipases cleave fatty acids from the TG: a hormone sensitive
lipase starts the process.
 signaled by decreasing insulin and increasing glucagon
 cAMP levels rise, protein kinase A is activated
 phosphorylation of hormone-sensitive lipase turns it on
 other lipases cleave remaining FAs from glycerol
backbone
free fatty acids and glycerol are released into the blood
 FAs are oxidized for energy in muscle and kidney
 Free glycerol is used by liver for gluconeogenesis
FAs are carried by albumin in the blood
Recycling of Free FA in adipose tissue
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Not all FA released from adipose cell during
fasting.
Amount regulated by glucagon or epinephrine
Allows fine control of free FA level in blood
Adipose doesn’t have glycerol kinase – must use
DHAP/NADH to make glycerol 3-phosphate.
Note also precursors that lead to G-3-P and
pathway very similar to gluconeogenesis and 1st
bypass.
Lipids:
Glycerophospholid Synthesis from Phosphatidic Acid *
 Ethanolamine and choline are rapidly phosphorylated upon entering cells and then react with CTP to form CDPethanolamine and CDP-choline, releasing PPi
 Phosphoethanolamine and phosphocholine are transferred from the activated nucleotide derivative to DAG, releasing
CMP
Phospholipid synthesis from phosphatidic acid
-phosphatidylcholine
-phosphaditylethanolamine
-phosphatidyl inositol
-cardiolipin
Interconversion of PLs
 Phosphatidylserine is produced from PE (phosphatidyl ethanotamine)
 Liver can interconvert PE to PC (Phosphatidycholine) by the sequential transfer of three methyl groups from Sadenosyl-methionine (SAM)
 SAM is a methyl group donor, a major player in biochemical reactions.
Phosphatidylinositol and Cardiolipin
 For PI and cardiolipin, activate the DAG itself first
 React the activated DAG with with inositol (PI) or another DAG (cardiolipin).
Synthesis of Spingolipids
 Sphingolipid synthesis can be divided into two parts:
o the formation of ceramide and
o the conversion of ceramide to sphingomyelin and glycolipids
 The precursors to sphingosine are seine and palmitoyl CoA whose condensation forms 3-ketosphinganine with
decarboxylation of the serine
 3-ketosphinganine is converted to a ceramide in three reactions
o the 3-keto group is reduced to a 3-hydroxy group by NADPH
o A Δ4 trans double bond is introduced by oxidation involving FAD
o The amino group is acylated by transferring an FA (usually palmitate) from acyl CoA
Sphingolipids to Glycolipids
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Sphingomyelin is formed by the transfer of
phosphocholine from phosphatidylcholine to
the C-1 hydroxy group of ceramide
Gluco- and galactocerebrosides are formed
by the transfer of the sugar group from UDPglucose or UCP-galactose
Complex glycolipids are formed by adding
additional sugars one at a time
Degradation of Glycerophospholipids and Sphingolipids
 glycerophospholipids are hydrolyzed by phospholipases
o phospholipase A1 releases FA at position 1 on glycerol
o phospholipase A2 releases FA at position 2 on glycerol
o phospholipase C releases the phosphorylated base at position 3
o phopholipase D releases the free base
 sphingolipids are degraded by lysosomal enzymes.
 many diseases are caused by defects in lysosomal enzymes, including those that break down sphingolipids.
Intermediates build up and reach toxic levels.
Cholesterol Metabolism and the Lipoproteins (44 & 45)
Functions of Cholesterol
 Membrane stabilization (plasma membrane)
 Bile salt precursor
 Steroid hormone precursor
 Blood lipoproteins
 Precursors of cholesterol can be used for making ubiquinone, dolichol, and cholecalciferol (active form of VitD)
Sources of Cholesterol
 Synthesized by most cells in the body – primarily the liver and intestine.
o Acetyl CoA is the starting material
 Dietary sources – animal products: eggs, red meat and liver
Cholesterol Synthesis
 All carbons come from Acetyl CoA
o Squalene is built up from 5-C isoprene units
 Cholesterol synthesis can be divided into three stages
o C2 C6 C5  C10  C15  C30 (squalene) C27 (cholesterol)
o Glucose, FA, AA  Acetyl CoA  HMG CoA  Mevalonate isoprene units Squalene  Cholesterol
 Most of the first two stages of synthesis occur in the cytosol but two enzymes (HMG CoA reductase and squalene
synthase) are located on the cytoplasmic face of the ER
 The final stage occurs entirely with ER bound enzymes
 Cytoplasm
o Fed State: want to make FAs and make cholesterol (regulated by insulin)
 Both involve citrate shuttle to get acetyl CoA from the mito matrix  cytosol
 Stage 1: Condensation of acetyl CoA to form mevalonate
o The first two reactions of cholesterol synthesis are the same for ketone body synthesis except that they involve
cytoplasmic isozymes
 Two acetyl CoAs condense to form acetoacetyl CoA
 A third acetyl CoA is added to form 3-hydroxy-3-methylgutaryl CoA (HMG-CoA)
o HMG-CoA reductase reduces HMG-CoA to mevalonate using two NADPH
 Can be inhibited by cholesterol (end product inhibition); so cholesterol inhibits its own synthesis
 First committed step and the rate limiting step of cholesterol synthesis!!!
 It is the site of regulation for cholesterol synthesis
 Located on the cytoplasmic face of the ER, has a cytoplasmic catalytic domain and a regulatory membrane
domain
 Is a target of drug therapy
 Activity is highly regulated.. ***
 Short term regulation is by phosphorylation /
dephosphorylation primarily by the same AMP-activated
kinase that regulates acetyl CoA carboxylase activity.
o When AMP is high (ATP low) HMG-CoA
reductase is phosphorylated, decreasing its
activity
 Glucagon inhibits the phosphatase
o Insulin activates phosphatase to
dephosphorylate the kinase and HMG CoA
reductase active form
 Long term: modify enzyme level in cell (DNA / mRNA)
o Bile salts and cholesterol can inhibit DNA
mRNA (so HMG CoA reductase not produced)
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Stage 2: Conversion of Mevalonate to Squalene:
o Formation of isopentenyl pyrophosphate
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Involves series of cytoplasmic rxn
o 2 phosphorylations using ATP to produce
mevalonate pyrophosphate
o decarboxylation and dehydration with ATP
hydrolysis  isopentyl pyrophosphate
o Isomerase converts isopentyl pyrophosphate to
dimethylallyl pyrophosphate
o 3 ATP enter and CO2 released (decarboxylation)
 5 carbon molecule
Isopentyl pyrophosphate to Squalene
 Geranyl pyrophosphate (C-10) formation involves head to tail condensation of dimethylallyl
pyrophosphate with isopentenyl pyrophosphate and PPi release (2 isoprenes condense)
 Farnesyl pyrophosphate (C-15) is formed by condensation of geranyl PPi with another isopentenyl
PPi with PPi release
 2 farnesyl PPi molecules condense head to head to form squalene and release two PPi
 The reactions of are driven by the rapid hydrolysis of PPi
 The enzyme catalyzing squalene synthesis and all subsequent enzymes are bound to the cytoplasmic
face of the ER
 Takes 6 isoprene units to make the C30 squalene
 Stage 3: Formation of Cholesterol from Squalene
o These reactions all occur on the ER
o Hydroxylation at C-3 and ring closure to form lanosterol
 Squalene binds to a specific sterol carrier protein
 Squalene monooxygenase adds a hydroxyl group at C-3
 Lanosterol cyclase forms the ring structure
o Monooxygenases or mixed function oxygenases consume one molecule of O2 per atom of oxygen introduced
in to a substrate. The other oxygen forms water.
o NADPH provides reducing power for ER monooxygenases
o Lanosterol binds to a second carrier protein and lanosterol undergoes 20 further reactions to form
cholesterol.
o Squalene  Lanosterol  Cholesterol
Cholesterol Esterification
 Cholesterol is a non-polar molecule
 The hydroxyl group (OH) affect solubility in hydrophobic media
 The -OH at C-3 of Cholesterol can be esterified with a fatty acid (Fatty acyl CoA  CoA-SH)  Cholesterol ester
 Greatly increases the hydrophobicity of cholesterol, which increases is solubility in lipoprotein particles an in lipid
droplets in the cytosol of cells.
 Two main enzymes that esterify cholesterol:
o Lecithin:cholesterol acyltransferase (LCAT)
 located in blood
 esterifies cholesterol associated with HDL
 Cholesterol CE which causes Lecithin (PC)  Lysolecithin
o Acyl:cholesterol acyltransferase (ACAT)
 located in cells
 concentrated in cells that need to store cholesterol for steroid synthesis.
 Fatty acyl CoA  CoA-SH which causes cholesterol  CE (more hydrophobic)
 Activated by cholesterol
 Storage as cholesterol esters
Transport of Cholesterol by blood lipoproteins
 Due to the hydrophobic nature of cholesterol, it must be transported by chylomicrons or blood lipoproteins
 Chylomicron cholesterol: from the diet via intestines
o Cholesterol remains associated with chylomicrons during LPL action
o Chylomicron remnants that are taken up by endocytosis into liver cells are digested in lysosomes, which also
processes any cholesterol esters to cholesterol.
o Cholesterol stays intact and enters cholesterol pool in liver
 LDL Cholesterol: from liver to peripheral tissues (makes VLDL)
o Cells take up LDLs, incorporate cholesterol inside
o Intracellular cholesterol signals a decrease in cholesterol synthesis (inhibits HMG CoA reductase)
o Cholesterol down regulates production of LDL receptors
 Less receptors bring less cholesterol into cell
 As receptors are endocytosed, fewer receptors remain on cell surface
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VLDL cholesterol: from the liver
 VLDL IDL LDL
 TGs synthesized in liver are packaged with cholesterol from pool in liver cells into VLDL lipoproteins
 Once in the blood, HDL transfers apoCII and apoE and cholesterol esters to VLDL
 When LPL acts on VLDL, and TG are degraded, IDL is produced
 if TG are further degraded, IDL becomes LDL, OR IDL can be taken up directly by liver by endocytosis
HDL Cholesterol: the “good” cholesterol
 Picks up cholesterol from cells, other lipoproteins (absorbs cholesterol in body)
 Has enzymes to esterify cholesterol, make it more hydrophobic
 Therefore has more cholesterol esters than cholesterols, not much TG
 HDLs return to liver for recycling.
 Sterified cholesterol
Transcriptional control of Cholesterol Synthesis  control HMG CoA reductase activity
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Upregulated by proteins that by steroids
SREBP: sterol regulatory element binding protein
o activates over 30 genes related to fat
metabolism: cholesterol, FA, TG, PL, and
NADPH production proteins
o short half life, must be continually produced
SCAP: SREBP cleavage-activating protein AND S2P: site
2 protease
Senses sterol level and stimulate production of SREBP
Regulation of HMG Reducatase **
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Proteolytic degradation:
o removes the enzyme from the pathway
o Quickly degraded
Reversible covalent modification
o Don’t want to synthesize cholesterol during fasting…
 Glucagon stimulates HMG-CoA reductase
phosphorylation = OFF
o or when ATP is low…
o or when sterol level is high.
 High [AMP] or high [sterol] turns OFF HMGCoA reductase (at the level of the protein)
Other functions of Cholesterol
 Precursor for:
o Bile salts
o Vitamin D
o Steroid hormones (glucocorticoids, mineralcorticoids, androgens, estrogens, progestns)
Synthesis of Bile Salts
 Cholesterol  7a-Hydroxycholesterol  Chenocholic acid or Cholic acid
 bile salts are more polar and hydrophilic than cholesterol.
 A Cytochrome P450 monooxygenase (7α-hydroxylase) incorporates an -OH group first to the 7th carbon
 Note the use of NADPH, typical of oxygenases.
 KEY: rate-limiting step in bile salt synthesis, committed step (committing cholesterol to bile salt synthesis)
 Strongly inhibited by bile salts (don’t want to make too much bile salt)
 Cholic acid is precursor to bile salt
o Conjugation increases the polarity of the molecule, increases the detergent properties of the bile salt  can
interact with both non polar and polar molecules now
o Masks the hydrophobic surface of fats to allow it to interact with the water environment (emulsifying)
o Cholic acid is a primary bile salt (it can be modified into secondary bile salts once released in digestive tract)
Bile Salt Metabolism
 Bile salt metabolism has direct effect on cholesterol metabolism.
 Patients who do not efficiently resorb the bile salts must constantly resynthesize them from cholesterol.
o Pts with issues in malabsorption, storage, gall bladder removal, etc. cant fully absorb the fats ingested
 These patients often have upregulated cholesterol synthesis. (bc they don’t have bile salts stored to inhibit)
o If you are constantly getting rid of bile salts there is no feedback to production
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Steroid Hormones
 Cholesterol  Pregnenolone Progesterone  Aldosterone, Cortisol, Estrone, and Estradiol
Lipoproteins
 TGs, cholesterol and cholesterol esters (CEs) are too hydrophobic to travel in a free form in the blood.
o Uses either chylomicron or lipoprotein
 They are packaged into the hydrophobic core of an aggregate of phospholipid, cholesterol and amphipathic proteins.
 Aggregate known as a lipoprotein, part of a class of lipoproteins that circulate in the blood.
 Chylomicron  nacent chylomicron VLDL  IDL  LDL  HDL
o order based on increasing density bc they are giving up their fat content (greater protein content) and
decreasing size
o Each class of lipoproteins is a heterogeneous mixture of particles of varying lipid and apolipoprotein
composition but with specific apolipoproteins in each type.
 Lipoprotein Content:
o Chylomicron—TGs
o VLDL—TGs
o LDL—CE
o HDL—Proteins
o All have an appropriate receptor
Apoproteins**
ApoB-48
Primary tissue source
Intestine
Lipoprotein distribution
Chylomicrons
ApoB-100
Liver
VLDL, IDL, LDL
ApoE
Liver
Chylomicron remnants,
VLDL, IDL, HDL
ApoCII
Liver
Chylomicrons, VLDL, IDL,
HDL
Metabolic function
Assembly and secretion of chylomicrons
from small bowel
VLDL addemly and secretion structured
protein of VLDL, IDL, LDL ligand for LDL
receptor
Ligand for binding several lipoproteins
to the LDL receptor, to the LDL
receptor-related protein (LRP), and
possibly to separate apo-E receptor
Cofactor activator of lipoprotein lipase
(LPL)
Chylomicrons
 carry dietary TGs from the intestine to peripheral tissues, especially muscle and adipose tissue
 deliver cholesterol and phospholipids to the liver.
 Protein contained is:
o apoB-48 (nascent)
o apo E and apo CII added from HDL in blood.
Fate of Chylomicrons
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LPL allows for digestion of these fats
Chylomicron remnants are recycled
into VLDLs in liver
VLDLs
 VLDLs carry endogenously synthesized TGs, cholesterol and cholesterol esters from the liver to tissues, especially TGs
to muscle and adipose tissue
 Smaller than chylomicrons, slightly higher density
 Major difference is the presence of apo B100 rather than apo B48
LDL
 LDL are rich in cholesterol and cholesterol esters
 Delivers cholesterol and cholesterol esters to all peripheral tissues
 Primary cholesterol transport mechanism!!!
The LDL Receptor
 All blood lipoproteins have receptors on cells – best characterized is the LDL receptor
 LDL receptor recognizes apoE and apoB100
o Binds LDL, VLDL, IDL and chylomicron remnants
 Its job is to bind lipoproteins and bring them into the cell by endocytosis
o For LDLs, that means delivering cholesterol and cholesterol esters
Fate of VLDLs and LDLs
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Cholesterol and the LDL receptor
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As VLDLs lose TGs bc they are hydrolyzed by lipases in
the liver, they are converted into IDLs, which are
smaller and have a higher protein content, thus a
higher density.
o Free up FA to adipose and muscle
Some IDL is taken up by the liver, some is converted to
LDL and some give up remaining cholesterol to nascent
HDL
o IDL goes to liver or continues in bloodstream
to give off more FA
o  LDL  liver or can go to peripheral cells
Note role of receptor mediated endocytosis for
importing lipoproteins into the cell…allows for
processing.
LDL binds to the LDL receptor
LDL receptor delivers cholesterol and CE to cell
Cholesterol tightly regulates its own metabolism: inhibits
its own production
o inhibits LDL receptor synthesis
 inhibits DNA mRNA
o controls endogenous cholesterol synthesis
 inhibits HMG CoA reductase, which keeps
the cell from overproducing reductase
Activates storage of excess cholesterol:
o reesterified to form cholesterol ester droplets in
the cytosol (hydrolizes cholesterol)
Endocytosis of LDLs
 LDL particle with ApoB-100 and CE  binds to LDL receptor on surface  receptor mediated endocytosis
 Endosomes fuse with lysosomes  lipoprotein degraded (LDL digested) to basic constituents  cholesterol esters are
hydrolyzed to free cholesterol
o Contents are determines via enzymatic reactions
o Substrate level reactions—knows what is in vesicle
 Some cholesterol moves to ER for storage, most cholesterol is re-esterified to keep free cholesterol level low inside
cell.
 Receptors recycled via Golgi to Plasma Membrane
HDLs (“good” cholesterol)
 Smallest blood lipoprotein with the highest protein content, thus the highest density
 Nascent HDL are synthesized released from the liver and intestinal cells
 HDLs pick up cholesterol from tissues: (absorb cholesterol)
o esterification by LCAT and transport cholesterol esters to IDL transforming IDL  LDL
o OR…carry CEs back to the liver.
 Also pick up free apolipoproteins released from other lipoproteins or act as a reservoir to donate apoproteins to other
lipoproteins.
 Cleans up lipoprotein transport system!!!!!
 Function and fate of HDLs
o Upon release they interact with cells taking up cholesterol
o The cholesterol is esterified by LCAT (lecithin-cholesterol acyl transferase)
o Cholesterol esters are transferred to IDL or transported back to the liver
o HDLs pick up apoC-I,II and III and apoE as they are released from VLDL and chylomicrons
o HDL interacts with nascent chylomicron to give them ApoCII and ApoE  mature chylomicron
o HDL interacts with nascent VLDL to give them ApoCII and ApoE  mature VLDL
o LCAT undergoes production of cholesterol esters producing VLDL from HDL
Atherosclerotic Plaques: Blood Lipoproteins gone bad
 Smooth muscles allow arteries to compress to control blood pressure
 Fat that is distributed will be in different levels of artery
 Minor damage to cells lining vessel wall occurs
 Macrophages (WBC) recruited, uptake LDLs, macrophages get filled with lipid/fat = foam cells
 Foam cells accumulate = fatty streak  separates wall of artery
 Chemical signals induce local growth, platelet aggregation  clot formation and more separation
 More LDL recruited by cells, fat builds up.
 Foam cells trying to chew up fat collect underneath arterial wall  damage  aggregate under wall
 Fibrous plaque
o Fibroblasts excrete fibrous proteins, make a tough cap.
o Cells are trapped and begin to die, leaving debris and fat, more macrophages recruited.
 Process repeats, adding to buildup until vessel is completely blocked
 Advanced lesion
o Dead tissue and lipids accumulate
o Calcification and hemorrhaging occur
 Triple aortic aneurism  bleed out within (silent)  blood pressure will drop but you don’t know whats going on
 Vessel blockage at site of plaque
o Atherosclerotic plaque growing underneath lining injury never gets completely healed grows this
thrombus that is trying to repair blocks off artery bc keeps growing bc of buildup of lipoprotein
SAMPLE QUESTIONS:
1) Which enzyme catalyzes the committed step of cholesterol synthesis? HMG-CoA reductase
2) What is the starting material for cholesterol synthesis? Acetyl CoA
3) The conversion of HMG CoA  mevalonate: is the committed step for cholesterol synthesis
4) Blood lipoproteins are classified by what properties? Function, based on the tissues with which they interact
5) Why does cholesterol regulate the number of LDL receptors on the cell surface? To control the number of LDL particles that
are synthesized by the cell
6) Familial hypercholesterolemia is a disease that involves defective LDL cellular receptors, such that the cells do not take up
LDL at a normal rate. Which of the following would NOT be a result of this disease? Fatty deposits in the coronary arteries
and elsewhere
Protein Nutrition (46)
 Protein supports the maintenance and growth of body tissues
o The primary role of proteins in the body are – structural, enzymes, hormones, transport and immunoproteins.
Plasma proteins that serve transport functions exert the colloid osmotic pressure needed to maintain fluid in
vascular space.
 Protein is essential for life
o Protein digestion begins in the stomach, where some of the protein are split into proteases, peptones, and
large polypeptides.
o MOST protein digestion takes place in the upper portion of the small intestine, but also CONTINUES
throughout the GIT*.
o Contact between chime and the intestinal mucosa ------release of enterokinase --- transforms inactive
pancreatic trypsinogen into active trypsin.
o Proteolytic peptidases located on the brush boarder also act on polypeptides, breaking them down into amino
acids (AA), dipeptides, and tripeptides.
o The end products of protein digestion are absorbed as both amino acids and small peptides.
o peptides and amino acids are transported to the liver via the portal vein for metabolism by the liver.
o Interestingly – the presence of antibodies in the circulation of healthy people suggests that immunologically
significant amounts of large intact peptides escape hydrolysis and enter the portal circulation. How many of
theses become allergens???
 the mechanism as to how a food becomes an allergen are not completely clear –
 foods high in protein-> relatively resistant to complete digestion -> immunoglobulin response
 . Examples: EoE, and EGE
o – only 1% protein is found in the feces.
 AA and protein
o Proteins differ from carbohydrates and lipids in that they contain nitrogen (they are 16% N).
o Plasma proteins that serve transport functions exert the colloid osmotic pressure needed to maintain fluid in
vascular space. (Albumin)
o Protein is our second largest energy store… 4 net Kcal per gram
o Know how to calculate the protein content of a meal- 1oz protein~7gram protein
o 9 AA are essential
 Classification
o Essential: Indispensable amino acids- we must get from diet
o Nonessential: Dispensable amino acids
o Conditionally essential*
 - During growth and in some disease states, the need for several AA increase because
synthesis cannot meet metabolic need.
 Premature infants or severe catabolic stress in adults
 Animal vs. plant protein
o The digestibility of a particular protein (food) – looked at by the WHO and FDA - you will hear it referred to
as the amino acid score. (After correcting for digestibility proteins that provide AA at recommended levels or
more are given a score of 1)
o Digestibility is a major factor affecting protein quality – and is affected by many factors including food
processing. ( True for both animal and plant protein)
o Their AA profile of animal proteins and plant proteins differ---most plants have a limiting AA which must be
complimented to create a “complete protein”… note this compliment does not have to occur exactly at the
same time in the meal.
o In most vegetarian diets, the typical food combinations provide more than adequate amounts of complete
protein. ( note – plant based diets are have many great health advantages)
o For animal proteins- meat preparations like involving acids like vinegars, marinade, and MOIST heat
tenderize tough cuts of meats through denaturation. Acids, salts and heat help the digestibility.
o Vegetable protein is less efficient than animal protein – it is encased in carbohydrate and is less available to
digestive enzymes. Some plants contain enzymes that must be inactivated by heat to allow protein digestion
– (i.e.) soy beans contain tyrpsinase which inactivates trypsin…
 Dietary Reference Intake
o Corn is low in lysine, high in methionine; Beans are low in methionine, high in lysine
o When utilizing plant based foods – the term complimentary protein is often used because the synthesis of
proteins for the body depends on the availability of all necessary amino acids. If an AA in a food is in short
supply it is called “ the limiting AA” – and that source of dietary protein can be combined with another food
which is high In what the original food was short in.
(80% of body need met by ‘recycling’)
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Vegetarian Diets
o Lacto-ovo-vegetarian-- Iron
o Lactovegetarian-- Iron
o Vegans
 Use of complementary proteins
 At risk for calcium, zinc, iron, vitamin B12 deficiency (B12 absorbed in ileum)
o Often more attentive attitude, behaviors to diet, thus, healthy intakes
o Milk makes you not be able to absorb iron
Importance of protein in appropriate amounts
o Times of abundance
 When requirements for AA have been met, excess AA are converted to FA and stored in adipose
tissue
o Times of need
 Atkins diet
 AA Glucose and ATP production (gluconeogenesis)
Need for protein
o Assuming normal health……. Protein needs are influenced by life stage.
o 0.8 g protein/kg/day, healthy adults
o Infants need up to 1.5 gm protein/kg/d
o And children (1 -3 yrs.) 1.1gm prot/kg/d
o 4 to 13 yrs. - 0.95 gm/kg/day
o And from 14 to 18 about 0.85 gm/kg/day
o Pregnancy 1.1gm/kg pre pg. wt. /d
o Lactation 1.3 gm/kg/d
o Protein requirements are increased in certain conditions Surgery
 Burns (High quality protein -- intakes 20-25% of energy intake) due to losses in urine, wounds,
Energy adequacy spares protein
o BUT in the critical patient and/or the chronic patient have frequent inflammation and infection you will need
to –pause and think about protein need, calorie need and catabolism. At this point consider a referral to a
critical care RD.
 Illness causes protein catabolism and affects interpretation of serum protein values!
 There is stress –related protein energy malnutrition
o A ratio of 150 non-protein calories per gram of nitrogen (provided by 6.25g of protein) – Is usually sufficient
to spare protein.
Evaluating protein status
o Acute illness or trauma causes inflammatory stress. Hormones and cell –mediated responses trigger the
break down of lean body mass
o Evaluation of nutrition status of such patients is difficult because NONE of the standard laboratory tests will
truly reflect changes in protein status during the onset of the illness or during refeeding.
o CRP – C-reactive protein--- helps to identify acute hyper metabolic periods
o Hs-CRP – high sensitivity CRP- is a sensitive measure of chronic inflammation .
o Both are NON-specific markers of inflammation
o Creatinine- it is found almost exclusively in the muscle tissue. Used along with Bun it is used to assess kidney
function.
o
When I see a very ill patient, or a long standing bedridden patient with a low Crt… it is a red flag to me to
consider the likelihood of loss of lean muscle
o In metabolically stressed patients, both inadequate and excessive protein can cause problems.
 Excessive animal protein can be suspect in certain cancers
o Even brief periods of protein calorie deprivation can send a patient from anabolism to catabolism – esp. in the
critically ill patient.
 Protein requirements range from 1.25 -2.0 g/kg/d critically ill
 1.2 to 1.5 for healing decubitus ulcers
o Over feeding of protein can also cause problems
 Acidosis and azotemia
 In “dry” patients hypertonic dehydration (tube feeding syndrome) may result from obligate water
losses ( high urea production)
 Other consequences of excessive protein – idiopathic hypercalciuria, osteoporosis, gout, and if >35%
calories – hyperammonemia, hyperaminoacidemia, hyper insulineamia, nausea…
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Additional Guidelines for protein intake
o Acceptable Macronutrient Distribution Ranges
 10-35% of energy (most liberal)
 Assumes kcal intake is sufficient
o DGAs & MyPyramid
 Fat-free or low-fat milk
 2-7 oz. lean meant
 Legumes - 2-3 cups

DGA - Dietary Guidelines for Americans)
Sports nutrition/supplements
o Endurance athletes- 1.2 to 1.4 g/kg/day
o Strength training – 1.2 to 1.7 g/kg/day
o Resistant training - 1.2 to 2 g/kg/d
o Athletes that consume high protein, risk compromising their carbohydrate status– which may impact their
ability to train or compete at their peak levels.
o High protein diets can also contribute to dehydration.
o Note the protein need is only slightly above what is needed for sedentary persons
o The timing of the protein ingested seems to be more important rather than taking in a high quantity of
protein.
Protein Deficiency
o Protein Energy Malnutrition and Protein calorie malnutrition
o Marmasmus- protein and energy deficiency
o Kashiorkor- protein deficiency (children ascites)
Healthcare providers often overlook PEM
Eicosanoids (47)
Arachidonic Acid (AA) Signaling
 AA is the most common FA released by PLA2 following signaling
 PLA2 cleaves the AA from position 2 of the phospholipid to form free Arachidonic Acid… ** OR **
 …DAG lipase can cleave AA from position 2 on DAG after PLC cuts PIP2
 AA enters the eicosanoid pathway
 Produced de novo
 AA acts as a substrate for PG, TX, and LT
 Terminated via degradation
Intro:
 Eicosanoids = class of compounds synthesized from 20 carbon FAs
 consist primarily of prostaglandins (PG), thromboxanes (TX) and leukotrienes (LT)
 elicit a wide variety of effects
o Commonly prostaglandins result in symptoms like pain, inflammation, fever, nausea and vomiting
 normally produced in very small amounts, and they have a very short half-life ***these are among the most potent
signaling compounds we make.
 metabolized to inactive products at the site of synthesis
 Formed in most tissues and act locally (compare to hormones which are produced in specialized tissues and act
system wide.
 Eicosanoid function is diverse
o Synthesis, lifetime and function of eicosanoids is tissue-dependent
o All 3 increase bronchioconstriction
o PG increases vasodialation; TX increases vasoconstriction
PG, LT, and TX
 Prostaglandins are fatty acids:
o 20 carbon atoms
o an internal, saturated 5-carbon ring
o a hydroxyl group at carbon 15,
o a double bond between carbons 13 and 14
o various substituents on the ring
 Thromboxanes structure similar to PG, but they contain a 6-membered ring
o Some TXs have an additional oxygen atom bridging carbons 9 and 11 of the ring.
 Leukotrienes are characterized by three consecutive double bonds (triene)
o Also have an oxygen atom (or two) bound as an OH or as an epoxide
 All synthesized from polyunsaturated fatty acids (PUFAs) containing 20 carbons and 3-5 double bonds (typically
arachidonic acid)
o Essential fats that have to be ingested from our diet that have the double bond after the 10th C
Eicoasanoids are produced in response to cellular signals
 PUFAs usually attached to glycerol backbone of phospholipid.
o Phosphoatidylcholine or phosphatidylinositol.
 PUFA is cleaved from phospholipids by phospholipase A2
o Triggered by stimulus binding to membrane receptors (histamine or cytokines)
o Steroidal anti-inflammatory agents inhibit phospholipase A2 (inhibiting production of Arachidonic Acid)
 Alternatively, phosphatidyl inositol bisphosphate can be cleaved by phospholipase C liberating a 1,2diacylglycerol containing the PUFA, which can be cleaved directly, or in two steps by other lipases.
Three pathways (were only focusing on these 2)
 Once released, PUFA (Arachidonic Acid) is converted to eicosanoids by three major pathways (Tissue dependent)
 You don’t want ppl with asthma taking asprin bc it inhibits cyclooxygenase, which then leads towards production of
leukotrines, which is responsible for bronchial constriction
 Cyclooxygenase (COX): leads to prostaglandins and thromboxanes via PGG2
o COX-1 is a constitutive enzyme in gastric mucosa, platelets, vascular endothelium, and kidney
o COX-2 is inducible and is generated in response to inflammation. Mainly expressed in activated macrophages
but other tissues also
 Lipoxygenase: leads to leukotrienes, HETE and lipoxins via HPETE **
o lipoxygenase is a dioxygenase that inserts a peroxide
PG and TX nomenclature
 PG and TX designate type
 All hydrated carbons
 Functional groups give function of prostaglandin
 All have a capital letter that designates ring substituents
o most common classes are A,E, F
 Some have a subscript, which designates the number of double bonds in the linear portion of the hydrocarbon chain
(but not within the ring) See Figure 35.6 for example.
 compounds in the 2-series are of the greatest significance in humans
 The PGF series has two hydroxyl groups on the ring. A Greek subscript is used to denote the position of the hydroxyl
group at carbon 9
 Thromboxane A2 (TXA2) isthe predominant eicosanoid in platelets…potent vasoconstrictor and stimulates platelet
aggregation. This leads to thrombus formation. Aspirin inhibits COX, and thereby blocks formation of TXA2.
*The Cyclooxegenase Pathway (synthesis of PG and TX from AA)
 Oxygen is added and a 5-carbon ring is formed by a cyclooxygenase that produces the initial prostaglandin, which is
then converted to other classes of PGs or TXs.
 PGH2 may be converted to TXA2
 Next step is tissue specific: function and then degradation
 TX functions
o Produced by platelets and stimulates platelet aggregation; causes vasoconstriction
Diversity of Function of Prostaglandins (depends on tissue type)
 Tightly regulated, made only when needed
 Platlets aggregation
 Smooth muscle vasocontriction
 Vasodialation
 Uterine smooth muscle contraction
 Chemotaxis of WBC
 Osteoclast Bone reabsorption
 Neurons  Fever
 Pain response
Degradation of PG and TX
 PG and TX designed to elicit a short-lived signal: must degrade them to stop signal
 Rapidly inactivated by oxidation of the 15-hydroxyl group critical for activity
o Hydroxyl is oxidized to a ketone
o Half life is seconds to minutes
 Breakdown products are excreted in urine
Steroidal and Nonsteroidal Anti-inflammatory agents **
 NSAIDs block prostaglandin formation by irreversibly inhibiting COX (Cyclooxygenase)
o How asprin gets rid of fever
o Aspirin acylates an active site serine. Other NAISDs bind non-covalently to COX to inhibit them
o Aspirin is the only COX inhibitor that covalently modifies COX
o Aspirin is more potent against COX-1 than COX-2
o There are specific COX-2 inhibitors (more later in Pharmacology)
o Note: NSAIDs don’t block AA formation so AA can form other eicosanoids
 Steroidal anti-inflammatory drugs (hydrocortisone, prednisone) block prostaglandin formation by inhibiting
phospholipase A2
o Blocks release of arachidonic acid from phosphatidyl choline for PG synthesis
Synthesis of Leukotrienes, HETE and Lipoxins: Lipoxygenase pathway **
 Arachadonic acid is starting material
 Lipoxygenase incorporates an oxygen molecule onto a carbon of one of several double bonds : activity of enzyme is
tissue dependent
 Starts with the formation of HPETEs (hydroperoxyeicosatetraenoic acids)
 HPETEs are reduced to the corresponding hydroxyl metabolites (HETEs) or metabolized to form leukotrienes or
lipoxins
Diversity of Function: Leukotrines
 When you inhibit cyclooxygenase you upregulate Leukotrines
 Chemotaxis in neutrophils
 Bronchial constriction
 Edema
 They have multiple effects- don’t want them lingering around or produce them when not needed
Epoxides: the Cyt P450 Pathway
 Note cytochrome P450 pathway. Allows for mono-oxygenase chemistry needed for this reaction
 Produces epoxides, HETEs diHETEs. (similar role as PG and LT)
 Prevalent in ocular, vascular, endocrine and renal systems.
Eicosonoids Mechanism of Action
 Eicosanoids have specific receptors on membranes of the target cell. Some eicosanoids enter nucleus, also.
 Intracellular responses are species-, organ-, and receptor-specific. (different tissues react differently)
 For the most part, eicosanoids act by either cytosolic Ca2+ or cyclic AMP in the cell.
o PGE, PGD, and PGI generally increase intracellular cyclic AMP.
o PGF2a, TXA2 and the leukotrienes elicit increased intracellular Ca 2+ levels, which triggers smooth muscle
contraction.
Clinical AA Metabolite Functions (know these first 5 for test) **
 PGE2- pregnancy, abortion, erection
 PGI2 – bronchial dilation (scleroderma) pulmonary hypertension
 Thromboxane A2- platelet aggregation
 LTB4 acute respiratory distress
 LTD4 asthma, inflammatory bowel disease
 HETE’s release Ca2+ stores and promote cell proliferation
The Alpha and Omega of Fats
 Double bonds can be counted from carboxyl carbon (α) or relative to the terminal (ω) carbon – more common
clinically.
 In humans, ω-3, ω-6 and ω-9 fatty acids predominate
o To get these we have to use essential FA like linoleic to get arachodonic acid
Diet and Inflammation
 Breast milk contains ample arachidonic acid, whereas plant-based formulas do not. Immune boost for breast-fed
babies.
 Docosapenaenoic acid (DPA) is a metabolite of arachidonic acid (other than eicosanoids) that decreases
prostaglandin production
 Similar effect seen for EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid).
o Dietary supplementation with DPA, EPA and DHA can decrease inflammation.
 ω-6 and ω-3 fatty acids are good, because they lead to anti-inflammatory eicosanoids (i.e., Series 1 and 3
prostaglandins) and decrease production of inflammatory prostaglandins (Series 2).
 BUT: ω-6 FAs can lead into arachidonic acid production  Series 2 prostaglandins if they are not completely utilized
by other pathways.
 What you ingest will lead to either proinflammatory or anti-inflammatory eicosanoids
Eicosanoid Pathways
Series 1 and 3 are good,
Series 2 is inflammatory
DHA, EPA and DPA all
inhibit inflammation
Balancing w9, w6, and w3 Fats
 ω-9 FAs are precursors for formation of ω-6 and ω-3 fats, so many diets focus on getting lots of ω-9 and they ignore
ω-6 and ω-3.
 The “ω-6 oils” are preferentially utilized by eicosanoid pathways that lead away from arachadonic acid production,
which decreases pro-inflammatory series 2 prostaglandin production and favor production of the direct production of
anti-inflammatory series 1 prostaglandins. So want to have these fats available in the diet.
 The “ω-3 oils” are preferentially utilized by eicosanoid pathways that lead to anti-inflammatory series 3
prostaglandins…BUT same enzymes that work on ω-6 fatty acids work on ω-3 fatty acids.
 Too much ω-6 relative to ω-3 will decrease anti-inflammatory effects of ω-3
 AND too much ω-6 will favor formation of arachadonic acid  can be bad for patients with chronic inflammation
issues.
The w6:w3 ratio
 The ratio of ω-3 to ω-6 FAs is critical for balancing pro-inflammatory eicosanoid synthesis from arachidonic acid.
 In times past, we had 6:3 ratios of 1:1 or 2:1, which was great.
 Nowadays the 6:3 ratio is closer to 10:1 to 25:1 due to greatly increased use of plant oil rich in ω-6 FAs.
 More ω-6 means increased likelihood of arachidonic acid production and inflammatory eicosanoids…in pathogenic
conditions.
 So…keep track of the ratio of these FAs in the diet, and work toward striking a balance close to 1:1 for optimum
health.
The Urea Cycle (48)
Nitrogen Metabolism
 In the liver nucleic acid metabolism and AA  Ammonia  Urea cycle Urea out of liver
 Body protein is in a constant state of turnover through the activity of biosynthetic and degradative pathways.
 Waste nitrogen is ammonia: an important metabolic intermediate, but toxic when levels get too high!
o Ammonia plays a pivotal role in nitrogen metabolism.
o Needed for biosynthesis of (also derived from):
 Nonessential amino acids
 Nucleic acids
 Major function of liver is ammonia metabolism.
 Ammonia concentration in liver is 700 mmol/L; >10X higher than plasma
Dietary Proteins: the primary source of nitrogen
 Amino acids (aa) produced from digestion of proteins are absorbed by intestinal epithelial cells and enter blood
 aa enter cellular pools and are used for the synthesis of proteins and
 other nitrogen-containing compounds
 carbon skeletons of aa, after nitrogen removal can be a source of energy
 amino groups of aa are converted to urea via the urea cycle
Ammonia or Ammonium ion
 Ammonia (NH3) is a weak base, and ammonium (NH4+) is its conjugate acid: pKa = 9.3
 Physiological pH is 7.4, so ammonia is nearly 100% protonated…but not quite
 Ratio of ammonium ion, NH4+ to ammonia, NH3 is 100 : 1…more NH4+ means more NH3.
 Ammonia is toxic... control NH4+ level to control NH3 level.
Nitrogen Balance
 “negative” nitrogen balance
o Total nitrogen breakdown at the whole body level exceeds synthesis - excretion exceeds intake losing
nitrogen. (don’t have enough)
 “positive” nitrogen balance
o Total nitrogen incorporation exceeds breakdown at the whole body level – intake exceeds excretion…gaining
nitrogen
 Typical of growing children: lots of amino acid and protein synthesis.
 Nitrogen balance-- Uptake and release of nitrogen are equal.
 High circulating ammonia levels (> 50 µM) are highly neurotoxic.
o Deficiency in liver function  several distinct neurological problems  coma
o Need to clear out the excess waste nitrogen…
Elimination of Nitrogen
 Depends on the availability of water.
o Many aquatic organisms: NH3 diffuses directly across the cell membranes of gill tissue and diluted by the
surrounding water
o Most terrestrial vertebrates: NH3 converted to urea (water soluble, uncharged molecule)  urine
o Birds and terrestrial reptiles NH3 converted to uric acid (insoluble, semisolid slurry)  bird droppings
Nitrogen Metabolism: Big Picture
 Glucose-alanine cycle
o Muscles have the AA that we utilize their backbone for energy
o Produce glutamate to remove the Nitrogen  produces Alanine  released from muscle and taken up from
liver can become glucose and the nitrogen can become urea
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Glutamine Cycle
o A-KG can accept two Nitrogens glutamate glutamine  goes to liver nitrogen comes off glutamine
urea/urine
Deamination of amino acids produces free NH4+ that must be reused (incorporated into muscle as amine group) or
eliminated.
Free NH4+ will lead to free NH3
How is Nitrogen removed from AAs?
 Transamination:
o the major process of removing nitrogen from amino acids (aa)
o nitrogen is transferred as an amino group from an aa to a-ketoglutarate to form glutamate
o catalyzed by transaminases (aminotransferases)
o readily reversible, involved in aa synthesis and degradation
 all aa except lysine and threonine can undergo transamination
 Pyridoxal Phosphate
o Pyridoxal phosphate (a form of Vitamin B6) is the cofactor in transamination
o Vitamin B6 deficiency is problematic for nitrogen metabolism.
Clinical Example: ALT and AST
 Liver cell damage leads to the leakage into blood of cellular enzymes like AST (aspartate tranaminase) and ALT
(alanine transaminase).
 ALT and AST are in high abundance in the liver
 Serum levels of AST and ALT are elevated in chronic alcoholic cirrhosis and acute viral hepatitis.
 Pyruvate  (Glutamate aKG via Aminotransferase) Alanine
Sources of NH4+
 1) Brain and Muscle purine nucleotide cycle
 2) Amino acid deamination’s
 3) Gut urea
Sources of NH4+ for the Urea Cycle**
 1) Glutamate “collects” nitrogen from other aa via transamination
o Oxidative deamination of glutamate by glutamate dehydrogenase (GDH) releases glutamate nitrogen as
ammonium ions  aKG
o Reaction is reversible: note NADH to deaminate, NADPH to reaminate.
o Note regulation! ATP and GTP high? Don’t need αKG in the TCA cycle. ADP and GDP high? Make αKG for TCA
cycle.
 2. Deamination of serine and threonine by serine dehydratase: histidine (histidase) - both PLP-dependent
o serine  pyruvate
 3. Deamination of asparagine (by asparaginase) and glutamine (by glutaminase)
o The amine group is simply removed. No partner molecule, no ketone formed in place where amine was
o “amination” pairs, where the difference in the molecules is the presence or absence of the amine group.
o Asparagine  aspartate
o Glutamine  Glutamate
 4) Purine nucleotide cycle in brain and muscle
 5) Urease-containing bacteria in the intestinal tract convert urea to ammonia and also release ammonia from amino
acids
How to eliminate waste nitrogen?
 The urea cycle: convert free NH4+ to urea
 Three Phases:
o Synthesize carbamoyl phosphate
o Produce arginine
o Cleavage of arginine to produce urea
 Five steps
o Two in mitochondrial matrix and Three in cytosol
 Urea is eliminated from the cell and eliminated from the body via urine.
The Urea Cycle**
1) Step 1
 Mitochondrial matrix: NH4+ and bicarbonate are condensed at the expense of 2 ATPs to form carbamoyl phosphate.
 free ammonium from transamination reaction
 use 2 ATP (1 for conjugation/activates bicarb and 1 donates phosphate)
 Catalyzed by enzyme carbamoyl phosphate synthetase I (CPS I)
2) Step 2
 Carbomoyl phosphate + Ornithine  Citruline via OTC
 Citruline transported in cytoplasm when Ornithine comes into mito (exchange reaction)
3) Step 3
 Aspartate + Citruline  Argininosuccinate (has 2 NH groups) via arginosuccinate synthase
 Aspartate formed by transamination of OAA.
 Requires ATP
4) Step 4
 Arginosuccinate lyase—Argininosuccinate  Arginine and relieves fumarate (can enter TCA)
5) Arginine  ornithine and urea via Arginase
 Ornithine is transported back into mitochondrion in exchange for a citrulline transported out.
--If we have a malfunction in any of these problems it will impact nitrogen metabolism
Urea Cycle and TCA cycle (highly linked)
 Sources of nitrogen for urea synthesis:
o One nitrogen comes from NH4+ one from aspartate.
 The carbon atom comes from CO2 (bicarbonate)
 Fumarate enters the TCA cycle and regenerates OAA
 OAA is transaminated to aspartate
Regulation of Urea Cycle
 N-Acetyl glutamate (NAG): formed when arginine is high, stimulates CPS1 to start moving nitrogen out.
o Glutamate + Acetyl CoA  + Arginine  NAG (positive allosteric regulator of CPS1) activates urea cycle 
carbomyl synthetase
 Substrate availability: urea cycle enzymes have high capacity to move substrate to product, allowing liver to clear
nitrogen quickly and efficiently.
 Induction: under conditions of prolonged high nitrogen load (high protein diet, prolonged fasting), urea cycle enzymes
are produced in greater number to help handle the increased load. (up regulation)
o Note: glucacon leads to increased synthesis of CPS1 to help handle increased nitrogen production during
fasting
Fed State AAs used for protein synthesis, no deamination needed.
Fasted State  AAs used to drive gluconeogenesis during short fast (12 hrs), AA use decreases as fast continues.
 AAs deaminated to provide carbon skeleton for glucose synthesis.
 Nitrogen cleared by cycle
Ammonia Toxicity
 deficiencies in urea cycle enzymes
 damage to liver, impairing urea cycle function
 Overproduction or underproduction of urea cycle intermediates, affected by other pathways
Enzyme Deficiencies**
 (1) CPS I: hyperammonemia. NH4+ can’t be cleared from body
 (2) Ornithine transcabamoylase (OTC): increased blood and urine orotic acid (orotate).
o Most common deficiency involving urea cycle enzymes.
o Increased blood levels of ammonia and orotic acid.
o Carbamoyl phosphate diffuses into cytosol and reacts with aspartate, a step in the pathway of pyrimidine
synthesis. Excess orotate, an intermediate in pyrimidine biosynthesis, is excreted in urine.
o Enzyme: Aspartate transcarbamoylase
 (3) Argininosuccinate synthase: highly elevated blood citrulline
 (4) Argininosuccinate lyase: Argininosuccinate excreted in large amounts, elevated blood citrulline
 (5) Arginase: high blood arginine, low blood urea nitrogen
Liver Dysfunction
 When the capacity of the liver to form urea is reduced, NH4+ accumulation can lead to a variety of problems. For
example:
o Hepatitis (inflammation of the liver) due to parasites, bacteria or viruses
 can cause hepatic necrosis and thereby reduce liver capacity to remove toxic nitrogenous waste.
o Damage to liver by chronic alcohol consumption-metabolism
o Damage to liver by toxic substances, e.g., acetaminophen overdose
 Bacteria in the intestinal tract convert urea back to ammonia and CO 2 using urease enzyme. Fairly normal
process…1/4 of urea released by liver is recycled by bacteria
o Urea is not cleaved by human enzymes.
o Normally, ammonia (NH4+) moves to liver, which reprocesses it
o If liver function is impaired  ammonia toxicity.
Hepatic Encephalopathy: Brain Dysfunction Due to Liver Failure
 Functional and anatomical shunting of nitrogenous metabolites formed in the gut into the systemic circulation,
bypassing their normal metabolism by liver cells
 Brain cells, which depend on the citric acid cycle, become depleted of a-ketoglutarate (and thus ATP) when high
concentrations of ammonia reverses the glutamate dehydrogenase reaction
 Increased synthesis of glutamine causes a deficiency of the neurotransmitter glutamate, which is also precursor of the
neurotransmitter g-aminobutyrate
Treatment Strategies
 Goal is to prevent ammonia toxicity
 Reduce protein intake and potential build-up of ammonia:
o Limit ingestion of aa, replace with equivalent α-keto acids to be transaminated in vivo.
o Causes nitrogen to be used for aa synthesis
 Reduce bacterial source of ammonia in the intestines:
o Treatment with lactulose/levulose, a poorly absorbed synthetic disaccharide which is metabolized by colonic
bacteria to acidic products, causes ammonia to be excreted in feces as NH 4+.
o Antibiotics to kill ammonia-producing bacteria.
 Remove excess aa:
o administer agents that covalently bind aa and are excreted in urine.
o Ex: Benzoate binds glycine to form benzoylglycine (hippurate). Phenylacetate (unpalatable, given as sodium
phenylbutyrate) binds glutamine to form phenylacetylglutamine
 Covalently bind AA and gets rid of Nitrogen excess
Practice question**
I- CPSI
II- Argininosuccinate synthase
III- Arginase
IV- OTC
V-Argininosuccinase
Amino Acid Generation and Degradation (49 & 50)
Essential AA: PVT TIM HALL
PKU treatment: restrict Phe in the diet—since tyrosine is synthesized from Phe, tyrosine becomes essential from dietary
intake
AA Metabolism
1) for the most part, we get amino acids from our diet
2) This “spares” us from having to invest a lot of energy in making amino acids
-we do have the pathways available to synthesize amino acids if the need arises (except for the essential ones)
3) Nonessential amino acids for mammals are usually derived from intermediates of glycolysis or the TCA
Synthesis of AA
•
•
•
•
•
•
some are glycolytic
intermediates,
some are TCA cycle
intermediates.
Ser is the source for Cys and Gly
Glutamate is the source for Pro
and Arg
Note the transamination pairs:
OAA  Aspartate and
α-KG  Glutamate.
Note: Asn can be made from Asp
by adding an amine from
glutamine same for Gln from
glutamate
AA synthesized from Glycolitic intermediates (from glucose)
 Glycine
 Serine (intermediate)
 Cysteine
 Alanine
 Note: Serine is an intermediate for both glycine and cysteine

Their carbons can be reconverted to glucose in the liver (gluconeogenesis)
Serine Biosynthesis
 3-phosphoserine  serine via 3-phosphoserine phosphatase
 3 phosphoglycerate is the metabolic intermediate for serine
o 3-phosphoglycerate dehydrogenase
 induced when serine levels are low
 repressed when serine levels are high (end product inhibition/feedback)
Glycine Biosynthesis
 Serine is the intermediate metabolite for glycine
 Serine + THF  (PLP, remove water)  Glycine (reversible)
Cysteine Biosynthesis
-Carbon skeleton and nitrogen for cysteine derived from
serine
-Methionine- sulfur donor
-Cysteine becomes essential in diet if dietary met is low
-Example of end product inhibition/neg feedback
Cystathionuria
-intermediate cystathionine accumulates in urine
-seen in premature infants  as they mature
cystathionase lyase increases so conc of cystathionine
decreases
-in adults it is caused by enzyme deficiency or dietary
deficiency of PLP (vit B6) which is needed by both
enzymes
If there is not an adequate nutritional supply of Serine or Glycine, the body must make these AA in order to provide starting
materials for all of their derivatives ( such as, Chlorophyll, Heme, and Cobalmin from Glycine)
Alanine-Glucose Cycle
 Alanine pyruvate (transamination cycle that is reversible; uses PLP and ALT)
 Alanine is the major gluconeogeneic AA bc it transfers/eliminates Nitrogen to the liver
Aspartate Biosynthesis
 Oxaloacetate (OAA)  tranamination with PLP and AST Aspartate
 OAA is the amino group acceptor
 High in liver cells
o If liver cells are damaged AST shows up in the blood (KEY MARKER); example: hepatitis and chirrhosis
Asparagine Biosynthesis
 Aspartate Asparagine via Asparagine synthetase
 Glutamine provides the nitrogen formation
 Aspariginase degrades asparagine has been used as an anti-tumor agent for treatment of leukemia
Glutamine Biosynthesis
 Intermediate is Glutamate
 Glutamine synthetase is one of only three enzymes that can fix free amide (glutamate glutamine)
 Reverse rxn by glutaminase is important in kidney (NH3 NH4 excreted; dec. H+ and inc. urine pH)
Proline and Arginine Biosynthesis
 Glutamate 5-semialddehyde
 Transamination urea cycle reactions  Arginine (during growth arginine is essential for children)
 Spontaneously cyclizes  reduced Proline
Tyrosine Biosynthesis**
 Phenylalanine  Tyrosine Acetoacetate (TCA)
o both are glucogenic and ketogenic
 Phenylalanine hydroxylase requires BH4 which reduces to BH2 to form Tyrosine
 Phenylketonuria (PKU) results from deficiency of phenylalanine hydroxylase: (PAH)
o Excess Phe and decreases in tryosine---remove Phe from diet and add tyrosine sources
o Phe excess causes neuro disorders (PKU)
o Can cause deficiency in thyroid hormones and nuerotransmitters (L-Dopa, Dopamine, Norepinephrine, etc)
o Infants appear normal at birth
o if untreated in first month of life
 gradual irreversible mental retardation
 delayed psychomotor maturation
 other neurological symptoms.
 Irreversible if you don’t treat with dietary
o characteristic musty or “mousy” odor in urine from the presence of phenylalanine metabolites
o Accumulation of metabolites: decreases pH and inhibits enzymes and transporters
 Malignant hyperphenylalaninemia
o Patients have normal PAH activity but are deficient in dihydropteridine reductase (DHPR).
 Secondary defect—cant regenerate BH4 which is needed for PAH
 Despite dietary restriction of Phe, have progressive neurological symptoms and die within first 2
years of life
 BH4 also a cofactor in the hydroxylation of tryptophan (serotonin synthesis) and of tyrosine
(dopamine, epinephrine and norepinephrine synthesis).
 Resulting neurotransmitter deficits cause neurological manifestations and death.
 - Both from the primary and secondary defects
 treatment includes administration of precursors of serotonin and norepinephrine
Catabolism of AA**
 Glucogenic—supply the gluconeogenesis pathway via pyruvate and TCA intermediates
 Ketogenic—converted to acetyl CoA or acetoacetate
Glucogenic
Glycine
Serine
Valine
Histidine
Arginine
Cysteine
Proline
Hydroxyproline
Alanine
Glutamate
Glutamine
Aspartate
Asparagine
Methionine
Ketogeneic
Leucine
Lysine
Both
Threonine
Isoleucine
Phenylalanine
Tyrosine
Tryptophan
Reverse Transamination
 Reentry into pathways from which carbon skeletons arose by reverse transamination
Alanine pyruvate
Aspartate oxaloacetate
Glutamate a-ketoglutarate
 Glutamine and asparagine are first hydrolyzed to glutamate and aspartate
 Then enter TCA Cycle at α-ketoglutarate
Arginine, Histidine, and Proline
 All can create Glutamine for the TCA cycle via a-KG
Histidine Catabolism: Histidenemia
 Rare autosomal recessive
 Deficiency of histadase
 Relatively begnin neonatal disease
 Increase blood, urine, and CSF levels of histamine
 Decrease of urocaninc acid (cant produce glutamate or aKG)
Serine Degradation
 In humans, serine is generally degraded by transamination to hydroxypyruvate, then reduced and phosphorylated to
2-phosphoglycerate, an intermediate of glycolysis
 This forms phosphoenolpyruvate (PEP) and then pyruvate
 In many mammals, serine is degraded by serine dehydratase to pyruvate.
Glycine Degradation
 1) To serine by a reversible reaction.
 2) To CO2 and NH4 after transferring carbon 2 to TH4 (FH4).
 3) To glyoxylate (oxidation) and then to oxalate or to CO2 and H2O.
 Oxalate produced from glycine or obtained from the diet forms precipitates with calcium Kidney stones (renal calculi)
are often composed of calcium oxalate.
Threonine Degradation
1. Converted to acetyl CoA and glycine
2. Cleaved by a dehydratase and then decarboxylated to form propionyl CoA
Branched Chain AAs
 Degraded by related pathways
 A BCAA transaminase catalyzes the first step forming an α-ketoacid
 An dehydrogenase then oxidatively decarboxylates the α-ketoacid
 The BCAA are universal fuels and catabolism of these AA occurs at low levels in the mitochondrial of most tissues
 Muscle carries out the highest level of oxidation of these amino acids
 Maple Syrup Urine Disease
o deficiency of first enzyme leads to accumulation of the α-ketoacids and unable to produce CoA product
o cant produce glucogenic or ketogenic derivatives downstream
o This is a branched –chain ketonuria
o Deficiency of α-ketoacid dehydrogenase that oxidatively decarboxylates the branched-chain α-ketoacids.
o Results in accumulation of branched-chain amino acids and their corresponding α-keto acids in blood and
urine (urine has odor of maple syrup or burnt sugar).
o Evident at the end of first week after birth. Infant difficult to feed, may vomit and may be lethargic. Without
treatment death by 1st year (extensive brain damage occurs in surviving children)
o Rx: restrict intake of BCAA to the only the amount required for protein synthesis (nutritional intervention)
Cysteine Catabolism
 When cysteine is degraded
o Nitrogen is converted to urea
o Carbons to pyruvate
o Sulfur to sulfate (excreted in urine)
 Cystinosis is a rare but serious disorder in which the cysteine transporter of lysosomal membranes is defective.
Cysteine cannot be transported out of lysosomes, forms crystals in many tissues and impairs cellular function.
 Renal failure occurs by age 6-12 yrs
 Makes them candidate for kidney transplant
Methionine
Homocystinuria Type I **
 vitamin B6 deficiency causes elevated blood homocysteine levels, and this can contribute to cardiovascular disease
 Cystathionine synthase deficiency
 Plasma methionine & homocysteine elevated. **note Met is elevated also**
 Rx:- High dose Vitamin B6, restrict dietary methionine
 Mental retardation is frequently the 1st indication of this deficiency:
o Dislocation of the lens of the eye
o Osteoporosis
o Vessel thrombosis
 Oxidation of homocysteine to homocystine
 Renal tubular reabsorption of methionine is efficient, so it may not appear in urine. But, homocystine less efficiently
reabsorbed, thus large amounts excreted in urine daily in homocystinuria.



Type II
o deficiency in the synthesis of methyl
cobalamin,=CH3-B12 (cofactor)
Type III
o deficiency in the synthesis of N5-methyltetrahydrofolate =5-CH3-FH4 (cofactor)
BOTH associated with elevated plasma homocysteine but not
methionine
 Methionine can’t be generated **
Albinism



Defect in tyrosinase
o Works in two places and causes both phemetain and
eumotains
complete or partial absence of pigment in skin
vision defects: photophobia, nystagmus and astigmatism
Alcaptinuria
 Error in Tyrosine metabolism
 Defect in homogentisate oxidase
 Homogentisate accumulates, autoxidizes forming a dark pigment, discolors urine (black!!) and stains diapers
 Later in life, chronic accumulation of the pigment may cause arthritic joint pain
Tyrosinemia
 Frequent in newborn infants, especially premature, “benign” form…plasma Tyrosine returns to normal by dietary
restriction of protein
 Type II: deficiency in tyrosine aminotransferase (TAT),
o eye and skin lesions, neurological symptoms.
o Rx: restrict Tyr and Phe dietary intake
 Type I: (Tyrosinosis)
o Deficiency in fumaryl acetoacetate hydrolase
o Acute form associated with liver failure, cabbage-like odor
o Without Rx death within first year of life.
Degradation of Tryptophan
 One ring carbon produces formate
 Non-ring portion forms alanine
 Kynurenine intermediate
o can be converted to urinary excretion products eg. xanthurenic acid (particularly in vitamin B6 deficiency)
o or degraded to CO2 and acetyl CoA
o or converted to the nicotinamide moiety of NAD and NADP
Degradation of Lysine
 Lysine cannot be directly transaminated at either of its amino groups
 Lysine is catabolized by a complex pathway ultimately forming acetyl-CoA. It is strictly ketogenic.
 During it’s degradation NADH and FADH2 are generated as energy sources
Methylmalonic Acidemia
 The final product of methylmalonyl-CoA mutase enzymatic reaction s uccinyl-CoA.
o excess methylmalonyl-CoA build-up causes [propionyl-CoA] to increase above normal Results in propanoic
acidemia
 Propanoic acidemia +methylmalonic acidemia
o -depletes ATP levels by oxidative stress
o -decreases biosynthesis of myelin, urea and glucose
 Methylmalonic acidemia causes oxidative stress on the mitochondrial enzymes involved in the urea cycle
(ammonia-dependent-carbamol phosphate synthase or CPS1) and inhibits its mechanism of action. The combination
of inhibited urea cycle, poor protein metabolism, low TCA Intermediatessymptoms of methylmalonic acidemia.
Special AA Products Derived from AA (51)
Glutathione
 Huge antioxidant role in cells
 Gamma-peptide bond, which is not attacked by peptidases
 Substrate for γ-glutamyltransferase, GSH-peroxidase
 Reduces dehydro-ascorbate which is produced by the reaction of ROS + ascorbate
 GSH is synthesized by a specific enzyme pathway in the cytosol of nucleated cells and is transported into
mitochondria through a transporter in the inner mitochondria
 Present in all mammalian cells (many non-mammalian cells too) except in neurons
 Deficiency of GSH causes death in new born (rats) and cataract & mitochondrial swelling in adults
 Glutathione Synthase deficiency very rare disorder
 Glutathione levels can be low due to dietary or environmental factors (chronic free radical stress)
Creatinine
 Functions
o Reservoir of high energy phosphate.  Creatine travels from liver to other tissues (particularly muscle &
brain) where it reacts with ATP to form creatine phosphate. Regenerates ATP (CPK reaction reversible).
o Transfer of high energy phosphate.  Creatine phosphate shuttle of heart and skeletal muscle allows rapid
transport of high energy phosphate (ATP formed in oxidative phosphorylation) from mitochondrial matrix to
cytosol for muscle contraction.
 Brain and muscle contain large amounts of creatine kinase (CK).
 Serum CK is elevated in patients who had a stroke or heart attack.
 Constant amount of creatinine excreted in the urine daily & reflects muscle mass
 Urine Creatinines
o Creatinine cannot be further metabolized
o Amount of creatinine produced depends on muscle mass
o Amount of creatinine excreted daily is constant when renal function is normal
o Concentration of a compound in a given urine specimen varies with time of day and water intake
o Better indication of the true rate of excretion of a compound
 amount of compound / amount of creatinine in the urine specimen
 Serum Creatinine
o Indicator of Glomerular Filtration Rate (GFR)
o Extent of rise in serum creatinine indicates severity of glomerular dysfunction in the kidney
Heme**
 Allows for oxygen binding and storage
 Produced in all mammalian tissues, highest in liver (cytochromes) and bone marrow (hemoglobin). Muscle
(myoglobin)
 Consists of one ferrous ion and a tetrapyrrole ring. Four pyrrole rings are joined together by methenyl (-CH=) bridges
to form a porphyrin ring
 Synthesis
o From Succinyl CoA and Glycine
o PLP is coenzyme: Vitamin B6 deficiency associated with microcytic, hypochromic anemia
o Step 1) rate-limiting step: catalyzed by d-ALA (d-aminolevulinate) synthase**
o Regulation of heme synthesis Heme represses synthesis of d-ALA synthase as well as directly inhibits it.  no
transcription
o
o
o
Final reaction catalyzed by ferrochelatase (heme synthase): iron as (ferrous) is incorporated to form
heme. (a number of conjugations to build up porphorin ring with iron in center)
**Lead Poisoning: Inactivation of d- ALA dehydratase (contains zinc) and ferrochelatase.
 g-ALA and protoporphyrin accumulate.
 Reduced heme production resulting in:
 - anemia (lack of hemoglobin)
 - reduced energy production (lack of cytochromes for e- transport chain)




Porphyrias
o rare inherited disorders caused by deficiencies of enzymes in the heme biosynthesis pathway  metabolite
o increase in blood & urine of porphyrins & their precursors
o Neurological effects: Accumulated intermediates have toxic effects
o Photosensitive skin lesions: Oxidation of accumulated porphyrinogens to porphyrins by light. Porphyrins
react with O2 to form oxygen radicals which may damage skin
Source of Iron:
o Diet. In meats, as heme it is readily absorbed. (main source)
o Non-heme in plants not readily absorbed-chelated to oxalates, tannins, phytates.
o Vitamin C increases uptake of non-heme iron in GI tract
o Iron transport: carried in blood as transferrin (complexed with the protein apotransferrin)
o Iron storage: In all cells especially liver, spleen and bone marrow. Stored as ferritin- iron complexed with the
iron storage protein apoferritin. Small amounts of ferritin enter blood and can be used to determine
adequacy of the body’s iron stores.
o Blood ferritin levels: a sensitive indicator of amount of iron in body’s stores.
Hemosiderin: a form of ferritin complexed with additional iron.
o This is important bc if you have a person that is tired, like a female on her period, if there is low iron she is
anemic
Iron Metabolism
o Dietary iron ingested absorbed via intestinal epithelium
o Transferritin which is iron plus ferritin is taken up by all tissues
o The byroduct of iron is hemocydurin
o Hemoglobin, myoglobin, and cytochrome use iron as their functional purposes
Degradation of Heme
o Hemoglobin in RBC  Heme  Bilirubin-albumin  Bilirubin diglucuronide
o RBC have high amount of Hemoglobin
o At end of life cycle (20days) its spits out heme  bilirubin which is attached to albumin to flow through
blood allows bilirubin to be broken down
o Liver takes up bilirubin and goes through P450 system which will conjugate it to make it more hydrophilic to
leave body
o In the reticuloendothelial system
 Heme is oxidized to biliverdin  biliverdin is reduced to bilirubin
 Meshwork within the liver that allows for the blood to interact directly with hepatocytes
 Junction between blood and hepatocytes
 Heme bound to albumin is taken up by reticuloendothelial system in liver, is oxidized to
bilivirdinbilirubin
o In the liver: Bilirubin is converted to a more water-soluble compound bilirubin diglucoronide
 This conjugated form of bilirubin is excreted in bile
o
o
o
o
o
o
Plasma Bilirubin:
 **Conjugated bilirubin (clinical) is expressed as direct bilirubin (couples readily with diazonium
salts to form azo dyes)
 **Unconjugated bilirubin (indirect) bound to albumin must be released by an organic solvent
before it will react with diazonium salts
 Looking for conjugated vs. unconjugated  will tell you if your liver is working
 If build up is conjugated the issue is not at the liver
 If it is unconjugated then the liver isnt doing its job bc its not conjugating the bilirubin for excretion
out of body
In the Intestine
 Bacteria deconjugate bilirubin diglucoronide and convert bilirubin to urobilinogens
 Some urobilinogen is absorbed into the blood and excreted in urine
 Most of the urobilinogen is oxidized to urobilins such as stercobilin. These pigments give feces its
brown color
 That’s why we don’t look in feces bc its all unconjugated
Hyperbilirubinemia and Jaundice (deposition of bilirubin pigment in tissues) can occur:
 When bilirubin is presented to the liver in amounts that exceed its capacity to conjugate (e.g. severe
hemolysis), elevated serum bilirubin (unconjugated).
 Conjugated < 20 % of total
 -too much heme
 In hepatocyte dysfunction (e.g. viral hepatitis) elevated serum bilirubin (conjugated & unconjugated)
 Conjugated 20-50 % of total
 As a result of hepatic obstruction of the drainage ducts from liver to the intestine (e.g. gallstones,
cancer-head of the pancreas), elevated serum bilirubin (conjugated)
 Conjugated > 50% of total
Urine Bilirubin
 increases only when serum conjugated (direct) bilirubin is elevated.
 Unconjugated bilirubin (bound to albumin) does not pass into urine
Neonatal Jaundice
 occurs frequently due to an immature system for conjugating and excreting bilirubin.
 Toxicity from build up of bilirubin
 Treatment: Phototherapy of the infant causes photoxidation of bilirubin to a water-soluble
compound, thereby facilitating its excretion
We don’t want bilirubin building up—we want it excreted
Nitric Oxide (NO)
 Gas that can diffuse rapidly into cells
 Breakdown of Arginine NO  gas to be to produced diffuses through cells  NO activates guanylyl cyclase 
increases cGMP  relaxes smooth muscles
 Biological messenger involved in vasodialation, neurotransmission, bacterial and tumoricidal activity
 Activates guanylyl cyclase (increases cGMP synthesis)
 Nitroglycerin is converted to NO and dilates coronary arteries in treating angina apectoris (chest pain caused by
narrowing of coronary arteries)
 Synthesis of NO
o Arginine  Citruline but NO is produced via NO synthase
o NO lasts 100 msecs in blood & few seconds in tissues
o NO combines with oxygen to form nitrite
o Nitrite is converted to nitrate and excreted in the urine
 Synthesis of hormones/neurotransmitters
o Via decarboxylation of Amino Acids
o Increases threshold (inhibits ability of neurons to activate)
o Glutamate  GABA:
 an inhibitory neurotransmitter is synthesized by the decarboxylation of glutamate
o Histidine is decarboxylated  Histamine (causes vasodilation, bronchoconstriction in the lungs and
stimulates HCl secretion in the stomach)
 Tryptophan Serotonin  Melotonin (Also, Tryptophan  NAD)
o Serotonin, 5-hydroxytrptamine (5-HT) (neurotransmitter, vasoconstrictor) produced by hydroxylation and
decarboxylation of tryptophan
o Melatonin (sleep-wake cycles, reproductive function) is synthesized by acetylation and methylation of
serotonin



Catecholamines: Dopamine, norepinephrine, and epinephrine are synthesized from Phe and Tyr
o Manipulation of phenylalanine  dopamine  norepinephrine  epinephrine (fight or flight)
Melanins
o in melanocytes of the skin, eyes and hair
o 3,4-dihydroxyphenylalanine (DOPA) is oxidized to quinones which polymerize to form melanin pigments
o Tyrosine is hydroxylated by a copper containing isozyme (tyrosinase) which differs from the one that
converts Tyr to catecholamines in other cell types
o Phenylalanine is derivatized  tyrosine  Dopa Melanin
Albinism
o Hypomelanosis due to heritable defects in the Cu-dependent tyrosine hydroxylase (tyrosinase), or other
enzymes that convert Tyr to melanin.
o Just understand if you don’t have enzyme tyrosinase in order to manipulate tyrosine you wont get melanin
o Tyrosine substrate and enzyme  Melanin
o Lack of melanin pigment in the skin, hair & eyes.
Single Carbon Transfer (52)
 Pyruvate  OAA
 The most oxidized form of carbon (carbon dioxide) is transferred by biotin (e.g. pyruvate carboxylation to form
oxaloacetate) (biotin is the only one involved in CO2 transfer)
 One carbon groups at lower levels of oxidation (-CHO, =CH-, -CH2, -CH3) are transferred by reactions involving:
o Tetrahydrofolate (FH4)
o Vitamin B12 (cobalamin)
o S-adenosylmethioine (SAM)
Tetrahydrofolate (FH4)
 Tetrahydrofolate (FH4 , THF) is produced from the vitamin folate. (folic acid)

Folate is synthesized by bacteria and plants from the bicyclic pteridine ring, p-aminobenzoic acid (PABA) and
glutamate.
o Certain antibiotics will mimic PABA and inhibit bacterial ability to replicate via the mimicing of PABA

Additional glutamate residues can be joined to folate by amide linkages to the glutamate side-chain.
 Absorption/modification of folate
o Dihydrofolate reductase- folate FH2  FH4 (functional form) [utilizes NADPH]
o Enzymes of the intestinal brush border remove all but one glutamate residue before folate is absorbed
o Folate is reduced to FH2 and FH4
o When FH4 enters cells, 4 or 5 additional glutamate residues are added
o Reduced polyglutamate derivative participates as a cofactor
 One carbon groups transferred by FH4 are attached to N5 or N10 or form a bridge between the two.
o The carbon group may be oxidized or reduced on FH4 prior to transfer…but not methyl groups since they
cannot be readily oxidized.
o Formyl Methyl
o Once you are reduced to methyl it is stuck at methyl
 One Carbon
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THF can transfer 1 C in a number of forms
except for CO2 to its products
Serine hydroxymethyl transferase
methylene which can be oxidized or reduced
Serine is the major source of one carbon
units. Because serine is synthesized from
glucose, dietary carbohydrate is the major
source of one carbon units.
o
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Purines
 The C transfer in THF is involved in building purine backbone
 Carbons donated from serine to THF in formation of glycine
 Those carbons now on THF are transferred to purine backbone
 Purine precursors to form purines: C2 and C8 of purines obtained from the one carbon pool
 Glycine to form serine (readily reversible reaction)
Other recipients
 Thymidylate synthase is an important enzyme conversion of dUMP to dTMP
 important role of folic acid in birth
 methyl group being transferred is from THF
 FdUMP formed from 5FU (5-fluorouracil) (a pyrimidine analog) inhibits thymidylate synthase**
 5-FU causes thymineless death especially of rapidly-dividing tumor cells. 5-FU used in the treatment
of colorectal carcinoma.
 inhibits production of TMP from UNP
THF can give off all these C at different reduced states
Gives B12 cobalmin its methyl group  B12 makes methionine from homocysteine
Folate Deficiency
o Causes accumulation of formiminoglutamate (FIGLU, produced during His degradation)
o Histidine load test is used to detect folate deficiency.
 Feed His and measure urine FIGLU
o Results in increased dUMP/dTMP ratio
 Increased incorporation of uracil into DNA
o Inhibition of DNA repair
 due to lack of dTTP
 Leads to DNA fragmentation
o Macrocytic, megaloblastic anemia: Inability of hematopoietic (and other) cells to synthesize DNA and to
divide….insufficient RBCs
o Impaired DNA synthesis & repair leads to large cells with abundant cytoplasm as cells persistently try but are
unable to divide.
Sulfa Drugs
o Inhibit thymidine synthase in bacteria
o Sulfa drugs are used in the treatment of certain bacterial infections
o Folate synthesized in bacteria from p-aminobenzoic acid (PABA)
o Sulfa drugs (eg. sulfanilamide) compete with PABA
o PABA is required for bacterial growth, folate cannot be absorbed by bacteria
o Humans can obtain folate from dietary sources: fresh green vegetables (foliage), liver,
Methotrexate
o Cometes with FH2 for binding of the second dihydrofolate reductase **(strong comp. inhibitor and mimics
THF)
o Inhibits formation of dTMP from dUMP
o Used in the treatment of leukemia
Vitamin B12: Cobalmin
 Complex structure, corrin ring coordinated with cobalt (similar to heme-porphyrin ring coordinated with iron)
 Transfers methyl groups via the methyl cobalmin
 Intrinsic factor
o Vitamin B12 cannot be synthesized by higher plants or animals, only by bacteria
o sources: dietary meat, eggs, dairy products, poultry, seafood, intestinal flora (minor)
o absorption of B12 in ileum requires intrinsic factor, a glycoprotein secreted by gastric parietal cells
o lack of intrinsic factor results in pernicious anemia.
 Inability to carry oxygen in blood
o Common problem particularly in the elderly.
o Treatment: Vitamin B12 given by injection, not oral.
 Functions
o 1) Transfer of methyl group from FH4 to homocysteine to form methionine:
 A one carbon group transfers from serine  FH4 (reduction to methyl)  B12,  homocysteine
(forms methionine)  SAM (S-adenosyl methionine), then to other molecules
 B12 deficiency  folate deficiency bc it is trapped in methyl form
 Methyl trap theory: trapping FH4 as methyl FH4
o 2) Coenzyme in the conversion of methylmalonyl CoA to succinyl CoA catalyzed by methylmalonyl CoA
mutase.
 This reaction is part of the metabolic route for the conversion of carbons from valine, leucine,
isoleucine, thymine, and the last 3 carbons of odd chain fatty acids to the TCA cycle intermediate
succinyl CoA (anaplerotic reaction)
 If no B12 you cant make BCAA which then cant form glucogenic precursors
 Cant produce energy
 Clinical manifestations of B12 deficiency
o Hematopoetic - caused by adverse effects on folate metabolism.
 Megaloblastic anemia (similar to folate deficiency).
 Lack of B12 causes folate deficiency by trapping FH4 as methyl-FH4.
 No free FH4 available for dTMP synthesis.
o Neurological – progressive demyelination caused by inability to convert methylmalonyl CoA to succinyl CoA
in the brain.
 Methylmalonyl CoA accumulation interferes with myelin formation.
SAM
 SAM (methyl group donor) participates in the synthesis of compounds that contain methyl groups
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Synthesis
o Synthesized from met and ATP
o Starts with methylated THF gives methyl group to B12  methyl cobalmin homocysteine methionine
o After transferring the methyl group SAH is formed which is cleaved to homocysteine and adenosine
o Homocysteine can accept a methyl group from methyl-B12 to regenerate Met
o Requires methionine!! (obtained from the diet or from homocysteine)
 BUT…Methionine also needed for cysteine synthesis. (unique rel. btwn. Met and Cys)
 Homocysteine provides the sulfur for Met & for Cys
 When Cys levels are high, its synthesis via cystathionine is inhibited, favoring Met synthesis from
homocysteine
 Thus adequate Cys in the diet “spares” the need for methionine…adequate SAM levels
 By having cysteine in diet it will inhibit the breakdown of homocysteine
 If you don’t have cysteine that methionine is shuttle to make more cysteine
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Nucleic acid degradation: (53)
Purine Ring Synthesis
 Synthesized de novo using AA as precursors—lots of ATP used!
 Occurs in liver
 Origin of ring carbons and nitrogens**
o Glycine gives backbone CCN
o Aspartate, Glutamine, Formate, and CO2
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o
PRPP is the source of the ribose moiety
IMP is central intermediate and initial product (major goal of purine synthesis)
o Hypoxanthine is the base for IMP
o Inosine is modified to make the major purines
First step:
o PRPP  5’-phosphoribosyl-1-amine catalyzed by PRPP synthetase (negatively regulated by presence of
IMP, AMP, GMP)
o Highly regulated but not the committed step
Committed Step:
o PRPP PRA (5 phospho-b-d-ribosylamine) by Glutamine PRPP amidotransferase
o PRA Glycine GAR
IMP can go either to AMP or GMP (both inhibit their own synthesis from IMP—feedback inhibition)
IF you have a defect in IMP you don’t get AMP or GMP
GMP is the substrate for AMP synthesis and AMP is the substrate for GMP synthesis  reciprocal substrate effect
and balances synthesis of both molecules
For DNA synthesis ribose is reduced to deoxyribose by ribonulcotide reductase
Reduction of deoxyribose occurs at the nucleotide diphosphate (NDP) level
Feedback inhibition  multilevel regulation by synthesis product **
o
Degradation of Purines**
o occurs mainly in the liver.
o pathways for the degradation of AMP and GMP merge at the point where xanthine is formed from Guanine
and Hypoxanthine
o Xanthine oxidase: Hypoxanthine  Xanthine (H2O2 is released)
o Note where allopurinol affects xanthine production=> GOUT
 Allopurinol inhibits xanthine oxidase
Xanthine is converted by xanthine oxidase to uric acid, which is not very soluble
Sodium urate is relatively insoluble and can crystallize in tissues (eg. the synovial lining of joints, particularly
that of the big toe)
o Gout is caused from:
 (1) overproduction uric acid
 (2) inadequate (more common) excretion of uric acid
o Overproduction uric acid due to:
 Over activity of PRPP synthetase
 Deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
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---a purine salvage enzyme
o Treat Gout with Allopurinol!!!
 converted in cells to oxypurinol, an inhibitor of xanthine oxidase (Product inhibition)
 prevents high levels of uric acid
 degraded purine is spread over 3 products (urate, hypoxanthine, xanthine)  none of these three
exceeds its solubility constant
 precipitation does not occur, the symptoms of gout subside
Purine Salvage Pathway**
o Require much less high energy phosphate than the de novo pathway
o Capture and reuse purines
o Two enzyme used
 HGPRT- hypoxanthine-guanine phosphoribosyltransferase (IMP/GMP competitive inhibitors of
HGPRT)
 Adenine phosphoribosyltransferase (ARPT) (inhibited by AMP)
 Can salvage adenine and make AMP IMP (skips steps so saves energy)
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o
Synthesis of Pyrimidine Nucleotides
 Precursors of ring are Glutamine, Aspartate, and HCO3 The immediate precursor is carbamoyl phosphate
 Six step pathway for de novo synthesis of UMP
o 1) analogous to first step of urea cycle except glutamine is source of N
o 2) Catalyzed by carbamoyl phosphate synthetase II (CPSII)- uses CO2 and glutamine  CAP
 Commited step!!!!! (A CAP)
 ACTase
o 3) Ring closure- loss of H2O  Pyrimidine
o Dihydrooratic acid OMP  UMP
o Defective ezymes: orotate phosphoribosyl transferase and orotidylate decarboxylase
 Hereditary Orotic aciduria
 Pyrimidines cannot be synthesized, growth cannot occur. Individuals treated with uridine
that can be phosphorylated to UMP, this bypasses the block where enzyme deficient
o UMP UTP (uses 2 ATP to add additional PO4 groups)  CTP (uses low GTP to activate glutamine)
 Regulation
o Carbamoyl phosphate synthetase II is:
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negatively regulated by UTP (end product inhibition)
 positively regulated (activated )by ATP, PRPP
o OMP decarboxylase is down-regulated by UMP (and to a lesser extent CMP)
o CTP synthetase is down-regulated by CTP itself (end product inhibition)
 Reduction of Ribonucleotides to deoxynuclotides
o Ribonucleotide reductase requires thioredoxin
 ADP  dADP
 CDP dCDP
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Deoxythymidylate (dTMP) (required for DNA synthesis) is formed by methylation of dUMP by the enzyme
thymidylate synthase
o 5-fluorouracil (5-FU) forms FdUMP which inhibits thymidylate synthase.
o 5-FU is used in the treatment of colon cancer (because they cant make dTMP)
Pyrimidine Degradation
o Pyrimidine nucleotides are hydrolyzed to the nucleosides (base+ sugar) and P i
o Then thymine, uracil, cytosine and (deoxy) ribose phosphate are produced
o Catabolism of the pyrimidine bases do not cause problems, in contrast to purine bases (where urate can
precipitate leading to gout)
Pyrimidine Salvage**
o Pyrimidine phosphoribosyl transferase can act on orotic acid, uracil, or thymine (NOT cytosine) ---uses
much less energy than de novo synthesis
o 5-FU
o
Catabolism
o Surplus nucleotides degraded to uracil or thymine
o Products of degradation
 Beta amino acids (indicators of DNA turnover)
 CO2 (excreted in lungs)
 NH4+ (excreted in lungs)
o Cytosine is deaminated to uracil, which is further degraded to CO2, NH3 and b-alanine
Summary  Bases can be recycles via salvage reactions which resynthesize the bases or go to catabolism
Disease/Condition
Hereditary orotic aciduria
Enzyme Deficiency
OMP decarboxylase
Orotate phosphoribosyl transferase
Action/Presentation
Severe anemia; retarded growth (bc
cells cant divide properly)
Rx: feed cytidine/uridine
Purine nucleoside phosphorylase
deficiency
Results in increased [purine nucleotide],
decreased uric acid formation
Impaired T cell function
Severe Combined Immune Deficiency
(SCID)
Adenosine deaminase (ADA)
Increase purine synthesis
hypernuricemia, neurologic
symptoms (mental; self-mutilation); Xlinked in males
Rx: allopurinol but no effect on
neurologic symptoms
Gout (least severe but painful)
PRPP or partial HGPRT
Na+urate cyrstals in joints
Inflammtion of joints, kidney stones
Rx: allopurinol  oxypurinol
Cancer drug treatment:
1)Hydroxyurea
2)Aminopterin/Methotrexate
3) Fluorodeoxyuridylate
1) nucleoside diphosphate reductase
2) dihydrofolate reductase
3) thymidylate synthetase
1)NTPS  dNTPs blocked
2) Dihydrofolate tetrahydrofolate
3) dUMPdTMP
Inter-tissue metabolism of AAs (54)
Know which tissues use/contribute AA to blood and how they use them once they are taken out of blood.
Alanine and glutamine are used as shuttles to move nitrogen through blood (because free nitrogen in blood= bad news)
Brain, kidneys, gut, liver, and muscle
Maintenance of AA pools in blood
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Inter-organ AA exchange during an overnight fast
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Pool of AA used for synthesis of new proteins
and other molecules needed
The body maintains a large pool of free amino
acids, even in the absence of intake of dietary
protein.
Ensures continuous availability of individual
amino acids for tissues for synthesis of proteins,
neurotransmitters and other nitrogen
containing-compounds.
Provides complete pool of specific amino acids
that can be used as oxidizable substrates
We can synthesize new glucose or store it as
glycogen
We can still provide energy from these molecules
if needed
Goal: mobilize aa’s as fuel molecules to tissues for energy:
glucogenic or ketogenic
Signaled by glucagon
Liver: make glucose
Waste products (free nitrogen) eliminated by urea cycle, and as free
NH4+ in urine and feces (gut and kidney).
Most will be in skeletal muscle
Branched chain AA amide absorber  Glutamine (cant enter
gluconeogenesis)  get rid of nitrogen waste Alanine Glucose
Valine and isolucine are ketogenic precursors for brain (energy)
Glutamine is nitrogen acceptor  lets tissue get rid of nitrogen
waste
Inter-organ flux of amino acids in the posadsorptive state: during an overnight fast, protein synthesis continues at a
diminished rate. Skeletal muscle has a net degradation of labile protein.
Release of amino acids from skeletal muscle during fasting
muscle is the largest amount of tissue in the body
provides large amount of aa into blood pool
muscle processes BCAA for energy and to make glutamine.
Ala and Gln account for 50% total nitrogen released by muscle, but muscle produces most of this by metabolism of aa
rather than by direct loss of Ala and Gln from protein
fall of insulin during fasting causes net proteolysis and release of aa to blood. Ubiqutin synthesis is also induced.
insulin causes net aa uptake and protein synthesis
Skeletal Muscle
o uses branched-chain amino acids (BCAA) for energy, nitrogen sent to the kidney for elimination
o aa, α-ketoacids and ala sent to liver
for glucose production
 Amino acid metabolism in liver during fasting
o Muscle donates Ala, and Gln but
o glucagon and glucocorticoids stimulates
uses BCAA (holding on to BCAA
o uptake of aa from blood
tighter, increases, not released)
o stimulates gluconeogenesis, aa degradation and
ureagenesis.
 liver is major site of alanine uptake: amino nitrogen is
Liver
discarded as urea.
o Glucagon greatly stimulates alanine uptake by liver
and upregultaes the urea cycle
 liver takes up other aa, α-keto acids and glutamine from blood
o Glucogenic and ketogenic roles
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Other Tissues
o Glucose produced by liver is oxidized in muscle to pyruvate, which can be used to make alanine. By
accepting nitrogen
o Glutamine generated in skeletal muscle and brain is taken up by tissues with high turnover rates, like
immune system. (lots of growth)
o Used as energy source, nitrogen donor for purine synthesis and as a substrate for ammoniagenesis.
o unused nitrogen from glutamine is transferred to pyruvate to form alanine, which carries the
nitrogen back to the liver
o Brain uses glucose primarily, but can use BCAA for fuel, and the nitrogen is used in neurotransmitter
synthesis during fasting.
o Amino acids released by muscle during fasting can be used as precursors for neurotransmitters.
Principles Governing AA Flux between tissues
 NH4 is toxic.
o Transported between tissues as alanine or glutamine.
o Alanine primary carrier to the liver, where urea is produced and excreted
o Glutamine in the blood serves essential metabolic functions. The utilization of blood glutamine pool is
prioritized based on need.
 BCAA can be converted to TCA cycle intermediates and used as fuel by most tissues.
o Apart from BCAA and Ala, Asp and Glu, all aa’s are metabolized in liver.
 Amino acids are major gluconeogenic substrates leading to glucose.
o Some aas used to produce acetyl CoA or ketone bodies.
o Hormones control glucose homeostasis and utilization of aa’s for glucose synthesis in liver
 Relative rate of protein synthesis versus degradation (protein turnover) determines the size of the aa pool in blood.
Utilization of AAs by tissues
 Each organ / tissue will utilize specific amino acids based on metabolic state and function of organ / tissue.
 All tissues have the same requirement for essential amino acids for protein synthesis during protein turnover.
Kidney
 Kidneys accept glutamine  NH4 via glutaminase  Glutamate  a-KG via glutamate dehydrogenase  glucose 
comes back and feeds this kidney
 Takes up glutamine:
o Gln is deaminated by glutaminase, forming NH3 and glutamate.
o NH3 is released into urine, where it forms NH4+ using protons from amino acid degradation. Picks up a lot of
free H+.
o Produces free amonia amonium  buffers urine
 Therefore, NH4+ buffers the urine: NH4+  H+ + NH3
 Alanine and serine are produced from glutamine
 α-ketoglutarate is formed from glutamate which is deaminated or transaminated.
o α-ketoglutarate enters TCA cycle  malate…malate  PEP
o PEP enters gluconeogenesis and can form glucose or alanine or serine, which are released into the blood.
o Glucose used by the kidney for energy
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pH regulation
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Bicarbonate for the blood
 Helps maintain blood pH
NH3 / NH4+ for the tubule cell
 Maintain tubule pH and control HCO3- reabsorption level.
NH3 / NH4+ and phosphate for the urine
 Excess taken away in urine.
Bicarb is reabsorbed via the kidney how pH is maintained by the kidney
If you got rid of bicarb it would be too acidic
pH of urine has to be tightly regulated bc its directly related to bicarb absorbed
Nitrogenous waste controls urine pH levels which helps maintain bicarb levels
If you don’t maintain level of nitrogen, you will constantly lose bicarb, individual will be metabolically
acidotic
Skeletal Muscle
 Muscle releases a lot of Glu and Ala, but not all comes from protein degradation-- some from metabolic intermediates
 BCAA can be used as energy (ketogenic).
 Muscle can convert glucose to pyruvate, which can be converted to alanine and sent to the liver. Liver will use Ala for
glucose production, send glucose back to muscle.
 Many of these AA can be broken down as ketones and enter metabolism (AA  keto acids)
o Isoleucine, leucine, and valine
 Only the glutamine and glutamate and alanine are secreted (off shoots of metabolism) 
o TRANSAMINATED: (adding free nitrogen that we want to get rid of)
 Pyruvate Alanine
 aKG glutamate  glutamine
 Can get rid of nitrogen without affecting metabolism
BCAA Metabolism
 BCAA to Glu, then to Gln or through purine nucleotide cycle to Gln.
 Both routes end at Gln
 BCAA nitrogen enters alanine-glucose cycle  urea and released from body
 The purine nuclotide cycle is where nitrogen is removed from purines during degradation back to IMP.
 BCAA breakdown products  Glutamate  Glutamine
The Gut
 Amino acid utilization is the same whether in the fed (post-prandial) state (aa from ingested protein) or fasted (post
absorptive) state (aa from blood pools).
 Note glutagenic amino acid utilization.
 Absorption of glutamine in intestinal epithelium free amonium comes off  glutamate citruline, ornithine
 Gut plays a role in amonium production by absorbing the glutamine from diet and blood and like any other tissue it
has to go through metabolism/tca/glycolysis
 Being the tissue adjacent next to dietary AA they are absorbed quickly versus pulling all their AA from blood
Brain: AA to Neurotransmitters
 Rapid turnover of many neurotransmitters requires a continuous supply of aa precursors from pool.
 Brain is net glutamine producer
 BCAA (esp. valine) are used to make Glu and Asp and TCA intermediates
 Glutamate is converted to glutamine which is transferred to neuronal cells or released into blood
 Glutamine is used to make glutamate and GABA (neurotransmitter) in neuronal cells
 GABA breakdown leads to succinate, which is a TCA cycle intermediate.
 BCAA sent to brain to make glutamate, aspartate, and TCA intermediates  glutamine nuerotransmitters
 Brain & Nerve Tissue
o BCAA cross blood-brain barrier
o Glutamate to glutamine. Gln can leave (as waste) or move to neuronal cells.
o In neuronal cells, Gln  Glutamate or GABA (neurotransmitters)
AA Flux after a High Protein Meal
 after high protein meal, liver and gut utilize most of the aa’s
o Gut uses most of Glu and asp, so little enter the portal vein.
o Gut uses some BCAA
o Liver takes up 60-70% of aas in portal vein
o Liver converts most of aa’s to glucose
o AA entering the peripheral circulation are mostly BCAA – liver doesn’t have transaminases to modify these
AAs so they don’t enter gluconeogenesis
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muscle does have these transaminases
 After a pure protein meal, higher levels of glucagon stimulate aa utilization:
stimulates aa uptake by liver
stimulate gluconeogeneis (no glucose in meal)
insulin is still released, but not much. BCAA uptake and protein synthesis continues but gluconeogenesis is
not inhibited.
Hypercatabolic States
 Defined as surgery, trauma, burns and septic stress
o Sistemic response– AA are key player in making energy available and proteins needed to respond
 Characterized by increased fuel utilization
 Negative nitrogen balance: due to increased protein turnover and increased degradation to meet aa needs for
energy
 Fuel stores mobilized to meet tissue needs when dietary intake is decreased
 Immune response and wound healing require energy, AAs
Sepsis: a catabolic state
 high energy and precursor needs for immune system, host defense mechanisms and wound healing.
 aas provide substrates for new protein synthesis, glucose production provides energy
 Protein synthesis decreases and protein degradation increases
o BCAA oxidation increases
o aa uptake is diminished (aa abundant from degradation)
 Amino acid utilization is prioritized
o immune and anti-inflamation systems receive top priority
o liver uptake of aa increased for the production of key proteins
o other protein synthesis is down regulated
 gluconeogenesis is increased to provide energy for cells of immune system
 Increased glutamine efflux from skeletal muscle provides energy and nitrogen.
 Elevated glucocorticoids and epinephrine and glucagon stimulates utility of FA from adipose to spare glucose
 This is a hormonal response tells muscle to get rid of AA (quickly mobilized in this response)  makes things
available to fight SISTEMIC infection
 The more sepsis increases the amount of white cells that interact
 When someone goes into sepsis there are a high level of bacterial products in blood body shuts down to deal with
infection
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