NUTR*3210: Second Half

carbohydrates (CHO)

• Macronutrient

• Energy source

• Not “essential” per se

• Dietary goal: increase intake of non-digestible content (more fibre), decrease intake of simple sugars

• Health Canada recommended intake: 45-65%

lipids (fat)

• Macronutrient

• Rich source of energy (high energy on a “per gram” basis)

• Two essential: alpha-linoleic (ω-3) and linoleic (ω-6)

• Key roles as precursors for signalling molecules, structural role in membranes, etc.

• Dietary goal: decrease intake of total fat (esp. saturated and industrial trans fats), increase intake of MUFA and ω-3 fats, improve ω-6/ω-3 ratio

• Health Canada recommended intake: 25-35%

protein

• Macronutrient

• Provides amino acids (AAs) for protein synthesis

• Unlike CHO and lipids, are not stored in reserves

• Source of energy (if needed)

• Substrate for glucose synthesis

• In humans: 21 proteinogenic AAs, 9 essential AAs

• Health Canada recommended protein intake: 10-30%

proteinogenic

Able to be incorporated into a protein during translation.

proteinogenic amino acids

21 in humans (includes selenocysteine). All but selenocysteine are part of the standard genetic code.

non-proteinogenic amino acids

Amino acids that are not incorporated into a protein during translation; not used to make protein.

• e.g. some neurotransmitters like GABA

essential amino acids

9 in humans.

blood (RBC), connective tissue, eye lens

Tissues with high protein content.

protein in human diets

Percent content in animal-derived foods is generally higher than plants.

On average: more from animal-derived foods.

If consumed from plants, must think more thoroughly about how and where it is coming from.

amino acid structure

Amino terminal: amine functional group.

Carboxyl terminal: carboxylic acid functional group; contains a carbonyl carbon.

Side chain (R): has a variable composition, which may or may not contain functional groups; the only varying component.

amino acids

The basic building block of proteins. Can be considered a monomer.

Two types in the body: standard and non-standard.

standard amino acids

• All are used to make protein.

• AAs for which tRNAs exist.

• 20 AAs are encoded in the genetic code (except for selenocysteine).

non-standard amino acids

• Many exist in the body, but they are rarely used to make proteins.

• Usually formed by post-translational modification of other AAs or as intermediates in the metabolic pathways of standard AAs.

  • e.g. neurotransmitter GABA is a metabolite of the amino acid glutamate.

• Will not go into much detail about these!

amino acid enantiomers

• D vs L.

• How all standard AAs exist (except for glycine).

• L configuration of AAs is naturally occurring.

• D configuration of AAs is made through post-translational modifications.

Zwitterions

• At physiological pH, amino acids are ionized:

  • Protonated amine group

  • Deprotonated carboxyl group

• No overall charge (except R group).

• This increases polarity (makes AAs more water soluble).

peptide bonds (amide bonds)

A type of covalent bond that connect amino acids.

The carboxyl group of one AA reacts with the amino group of another AA, releasing H₂O (condensation reaction).

To break: add water (hydrolysis reaction).

dipeptide

Two amino acids.

tripeptide

Three amino acids.

oligopeptide

~50 amino acids.

polypeptide

>50 amino acids.

biologically active protein

1 or more polypeptide(s).

primary structure of proteins

Structure determined by the DNA sequence.

Refers to the polypeptide chain of amino acids.

AAs are held together by peptide bonds; translation is helped by chaperone proteins in the cell.

A polypeptide chain has a carboxyl and amino terminus. Counting of the AAs always starts from the amino end.

chaperone proteins

Proteins in the cell that assist in correct folding and translation of polypeptides.

secondary structure of proteins

Structure determined by the hydrogen bonds that create a more stable structure.

• Interestingly, certain AAs prefer one structure over the other.

Bonds don’t involve side chains, only backbone atoms.

Two “types” of stabilized structures exist:

1. α-helix

2. β-pleated sheet

α-helix

Secondary structure in which an amino group makes a hydrogen bond with a carboxyl group 4 AAs down the chain, creating a helical shape in the polypeptide.

β-pleated sheets

Secondary structure in which an amino group makes a hydrogen bond with a carboxyl group in the folded back peptide chain. Can be parallel or anti-parallel. More “2D” of a structure.

tertiary structure of proteins

Structure that corresponds to the arrangement of the secondary structure in 3D space. Many proteins become biologically active at this stage!

Consists of one polypeptide chain.

Involves interactions between AA side chains (near or far).

• For example, disulfide bonds can form between Cys AAs.

Hydrophobic AAs tend to be placed towards the centre of a protein to help ensure that the protein is water soluble - allows proteins to move around in aqueous cellular environment.

quaternary structure of proteins

Structure that corresponds to a combination of two or more tertiary structures that are required to make a functional protein.

When tertiary structures combine, the individual structures are referred to as “subunits”.

Forms a multi-subunit complex (i.e., multiple polypeptides): e.g. insulin, immunoglobulins.

Not all proteins have this level of structure (only those that have subunits that are required to make a functionally active protein),

subunit

The individual structures, when tertiary structures combine.

native protein

A protein in its normal 3D conformation.

denaturation of proteins

Can happen in a number of ways: for example, heat, salt treatment, detergent, pH (stomach acid). The protein will lose its bioactivity (3D structures are lost).

Imperative to allow for digestion of proteins and absorption of AAs in our digestive tracts.

Affects secondary, tertiary, and quaternary structures but NOT primary - doesn’t lead to breakdown/digestion of polypeptide chain.

different ways to classify amino acids

• Essential vs non-essential

• Basic, acidic, or neutral

• Polar vs non-polar

essential amino acid (indispensable)

Amino acids that are not made by the body or can’t be made quickly enough to meet demands.

9 AAs: Lys, Thr, Iso, Leu, Met, Phe, Trp, Val, His

conditionally essential amino acids

Amino acids that are not normally required in the diet in a healthy individual, but become essential under specific contexts.

For example:

• A genetic problem (phenylketonuria)

• Development of a disease (liver disease)

phenylketonuria

An inborn error of metabolism whereby a person is unable to break down Phe into Tyr.

• A buildup of Phe in the body causes intellectual disability.

• The solution is to limit Phe in the diet and supplement with Tyr.

liver disease (cirrhosis)

Disease impairs Phe and Met catabolism.

• Tyr and Cys are synthesized from Phe and Met, respectively.

• Tyr and Cys become indispensable in this context.

non-essential (or completely dispensable) amino acids

Amino acids that can be synthesized in the body and are not essential to obtain from the diet.

basic amino acids

Lysine, Arginine, Histidine.

• POLAR: because of charge present inside chains.

• Positive charge on NH₃ group on side chain, enables DNA binding.

• Important in histone proteins, which interact with DNA.

lysine

Essential, basic, polar AA.

• Limiting in grain products (if you consume only grain products, you would not get enough).

• Involved in the production of carnitine, which is important for fatty acid metabolism.

arginine

Conditionally essential, basic, polar AA.

• Preterm infants are unable to synthesize (first few days-months of life).

• Non-essential in healthy adults.

histidine

Essential, basic, polar AA.

• Ring structure.

• Used to produce histamine (inflammation).

acidic amino acids

• Aspartate, Glutamate

• Negative charge on side chain carboxyl group.

• Polar.

aspartate

Non-essential, acidic, polar AA.

• Important for amino acid catabolism.

• Transaminated to oxaloacetate (Krebs Cycle) - allows NRG to be generated.

• A “source” of nitrogen in the urea cycle.

glutamate

Non-essential, acidic, polar AA.

• Important for amino acid catabolism.

• Transaminated to alpha-ketoglutarate (Krebs Cycle) - allows NRG to be generated.

• Used to produce GABA (neurotransmitter).

neutral amino acids

Asparagine, glutamine, glycine, alanine, leucine, isoleucine, valine.

• No charge on side chain.

• Non-polar.

• Aliphatic (C and H atoms joined in straight or branched chains).

asparagine

Non-essential, non-polar, neutral AA.

Has an amide group in its side chain.

Aspartate + basic amino group.

glutamine

Non-essential, non-polar, neutral AA.

• Important in AA catabolism because it is an inter-organ carrier of nitrogen (to the liver and kidney).

• Glutamate + polar amino group.

glycine

Non-essential, neutral, non-polar AA.

• No enantiomers.

• Used primarily to produce porphorin (a component of heme, which is found in hemoglobin).

alanine

Non-essential, neutral, non-polar AA.

• Important in AA catabolism because it is an inter-organ carrier of nitrogen (to liver and kidney).

• Important role in the glucose-alanine cycle.

branched chain amino acids

• Leucine, isoleucine, valine.

• Neutral, non-polar.

• ALL ARE ESSENTIAL.

• Not catabolized in the liver, so high levels found in circulation.

  • Liver doesn’t express enzymes for BCAA catabolism, so they bypass the liver and go directly to muscle to promote protein synthesis.

• Promote protein synthesis.

• Levels are high in protein supplements.

leucine

Essential, branched, neutral, non-polar AAs.

• Not catabolized in the liver, so high levels found in circulation.

  • Liver doesn’t express enzymes for BCAA catabolism, so they bypass the liver and go directly to muscle to promote protein synthesis.

• Promote protein synthesis.

• Levels are high in protein supplements.

hydroxylated amino acids

• Serine, Threonine (also Tyrosine).

• OH group on side chain: important for protein phosphorylation.

• Polar.

serine

Non-essential, polar, hydroxylated AA.

• -OH group on side chain is important for post-translational phosphorylation of proteins.

threonine

Essential, polar, hydroxylated AA.

• -OH group on side chain is important for post-translational phosphorylation of proteins.

sulfur-containing amino acids

• Cysteine, methionine.

• Contain a sulfur-group.

• Non-polar (S-H bond not strong enough).

cysteine

Non-essential, non-polar, sulfur-containing AA.

• Made from methionine.

• “Spares” methionine when this is consumed in the diet.

• Used to form disulfide bonds (when incorporated in peptide chains; tertiary structure).

• Used in glutathione synthesis (oxidant defence system).

methionine

Essential, non-polar, sulfur-containing AA.

• First step in the synthesis of all proteins (without this, we cannot synthesize proteins).

• Limiting in legumes (won’t get enough if all you eat is legumes).

aromatic amino acids

• Phynylalanine, tyrosine, tryptophan, proline.

• Contain aromatic rings.

• Non-polar (except for Tyrosine because of its -OH group in the side chain).

phenylalanine

Essential, non-polar, aromatic AA.

• Used to make Tyrosine.

tyrosine

Non-essential, polar, aromatic, (hydroxylated) AA.

• Made from Phenylalanine.

• When consumed, “spares” Phe - Phe can go off and do work around the body without having to synthesize this AA.

• Used to synthesize neurotransmitters.

tryptophan

Essential, non-polar, aromatic AA.

• Used to make serotonin (mood).

• Used for niacin (Vitamin B3) synthesis (only a little of this AA can be used).

proline

Non-essential, non-polar, aromatic AA.

• Important for collagen production (extracellular matrix).

• Aliphatic side chain.

post-translational modifications (PTM)

Most proteins require some type of modification before they are biologically functional.

These modifications take place in polypeptide chains, not free amino acids.

Happens through:

• Phosphorylation by kinase enzymes

• Hydroxylation (creation of a new hydroxyl group)

• Gamma-carboxylation

• Iodination

• ADP-ribosylation

phosphorylation

The addition of a phosphate group by kinase enzymes. A common PTM of polypeptide chains → phosphorus dependent.

• Serine-OH

• Threonine-OH

• Tyrosine-OH

Can be modified due to their OH groups!

hydroxylation

The creation of a new hydroxyl group. Common PTM that takes place in polypeptide chains.

• Lysine → hydroxylysine (very important in elastin subunits, copper dependent; associated with aortic rupture).

• Proline → hydroxyproline (very important in collagen subunits, Vitamin C dependent, associated with scurvy).

gamma-carboxylation

PTM required for calcium homeostasis and blood clotting. Certain proteins are modified to become Ca²⁺ binding proteins. Another carboxyl group is added to glutamate residue within a protein.

Vitamin K dependent.

iodination

PTM critical in the formation of thyroid hormones and crucial for regulation of the metabolic rate. About 2 billion humans are iodine deficient.

Iodine dependent.

ADP-ribosylation

PTM that adds ADP-ribose to an acceptor protein. Critical for DNA repair and regulation of protein function.

Vitamin B3 (niacin) dependent.

Niacin used to form NAD⁺. When NAD⁺ is broken down in the cell, ADP-ribose and nicotinamide are the products.

protein digestion: mouth

• No enzymatic digestion (unlike CHO).

• Mechanical breakdown.

protein digestion: stomach

• Stomach produces “gastric juice”.

• HCl in gastric juice:

  • Denatures proteins: disrupts hydrogen bonds, electrostatic bonds.

  • Activates pepsin (initially produced as pepsinogen).

• Pepsin (endopeptidase): starts to break down dietary protein.

protein digestion: pancreas

• Pancreatic juice containing zymogens (inactive digestive proenzymes).

zymogensq

Inactive digestive proenzymes found in pancreatic juice. Activated in the small intestine.

protein digestion: small intestine

• Zymogens are activated.

• Enzymes break down peptides.

• Absorption of AAs.

  • Mostly fragments of AA chains that must now be further broken down, usually only absorbs singular AAs but can absorb di- and tripeptides → if big peptides are absorbed → allergy.

HCl

Secreted from parietal cells; its release is triggered by gastrin, acetylcholine, and histamine.

Two functions:

1. Denatures proteins - disrupts hydrogen bonds and electrostatic bonds (doesn’t affect AA chain, but downgrades or eliminates 3D configuration).

2. Activates pepsin.

pepsin

Secreted as pepsinogen, which is an inactive zymogen.

• Active in an acidic pH, inactive at a neutral pH.

• HCl causes a conformational change in pepsinogen, allowing it to then autoactivate itself.

• The activated form is an endopeptidase (i.e., in other words, it cleaves peptide bonds within a polypeptide chain).

• Generated mostly oligopeptides (smaller fragments) and some free amino acids (which go to the small intestine).

exopepsidase

Targets AAs at the end(s) of chains for cleavage.

trypsin

Secreted by the pancreas as trypsinogen (inactive). Once it reaches the small intestine, is activated by enteropeptidase (located in brush border).

Targets basic AAs in peptide chains.

Once activated, activates other zymogens in the small intestine:

• Chymotrypsinogen → chymotrypsin

• Proelastase → elastase

• Procarboxypeptidases → carboxypeptidase

• EXCEPTION: aminopeptidase, which is synthesized and released from the small intestine itself.

pepsinogen

Zymogen; inactive form of pepsin. Activated at acidic pH (activated by HCl in the stomach).

trypsinogen

Zymogen, inactive form of trypsin. Activation is triggered by enteropeptidase in the small intestine brush border.

pancreas

Where the zymogens and proenzymes are made.

proteases

Trypsin, elastase, pepsin, parapepsins, chymotrypsin.

trypsin

Protease that attacks basic AAs.

elastase

Protease that attacks neutral aliphatic AAs.

pepsin, parapepsins, chymotrypsin

Proteases that attack large neutral AAs.

carboxy-peptidases

Enzyme that attacks the carboxyl terminal of a peptide chain.

amino-peptidases

Enzymes that attack the amino terminals on a peptide chain.

amino acid absorption

Most amino acids are absorbed in the upper small intestine.

Two ways:

• Facilitated diffusion (along favourable concentration gradient).

• Active transport (>60% of AAs are absorbed this way); via peptide transporter 1 (PEPT1).

Evidence suggests that:

• Essential AAs may be absorbed faster than non-essential AAs.

• Competition for absorption exists between AAs.

• Free AAs have no absorptive advantage (i.e., protein supplements) over AAs in foods (always better to take full AA source to avoid AA imbalance).

amino acids used in the small intestine

AAs are either transported out of the intestinal cell or used directly within the enterocyte for:

1. Synthesis of new protein

2. Energy

Estimates indicate 30-40% of essential AAs are used in the small intestine. Enterocytes reside in a very intense environment and are constantly dying and being replaced by stem cells → high AA/protein demand!

glutamine used in the small intestine

AA highly used in enterocytes to:

1. Generate energy for the cell

2. Stimulate cell proliferation (to replace shed enterocytes)

3. Increase synthesis of heat shock proteins (chaperones)

4. Drive mucus (essentially proteins) production, which helps to prevent bacterial translocation (happens if mucus layer is not sufficient)

amino acid metabolism

The liver is very effective at taking up amino acids from circulation. It uses 20% of the AAs to:

• Make new proteins/enzymes (also important for lipoproteins).

  • Albumin and other transport proteins

• Made peptide hormones.

Liver catabolizes the remaining 80% of AAs, where:

• NH_3, sent to the urea cycle.

• Carbon skeleton sent to Kreb’s cycle (for NRG) or used for gluconeogenesis or lipogenesis.

BCAAs are NOT taken up by the liver (liver doesn’t express proper enzymes) and are instead are anabolic signals for tissues like muscle.

protein quality

Highly linked with AA essentiality. Four aspects to consider:

1. Amino acid composition,

2. Digestibility.

3. Presence of toxic factors.

4. Species consuming the protein.

protein quality: amino acid composition

Any protein that provides all essential AA is considered “high quality”. Animal protein > plant protein. For example, grains are limiting in lysine, legumes are limiting in sulfur-containing AA (methionine).

protein quality: digestibility

Some proteins are more digestible than others. More digestible means higher quality. Animal protein > plant protein. Some materials, like hair, have a great amino acid balance but are indigestible.

protein quality: presence of toxic factors

Less toxic factors means higher quality. Animal protein > plant protein. Plants contain thousands of phytochemicals. For example, soybeans contain inhibitors that interfere with trypsin, thus preventing protein digestion.

protein quality: species consuming the protein

Humans, pigs, and chickens have similar protein needs. Ruminants have bacteria in the rumen that can make all AAs, so none are considered essential (remember that ruminants can use low quality protein sources).

assessing protein quality

• Protein Efficiency Ratio (PER)

• Chemical Score (CS)

• Nitrogen Balance

Protein Efficiency Ratio (PER)

• Official method in Canada for the evaluation of protein quality.

• With this method, young rats are fed a diet for 4 weeks. The diet has all nutrients present at adequate levels except for protein, which is included at 10% kcal of the diet.

• 10% protein is lower limit for health. If there is anything wrong with the protein source, the growth of rats will be impaired.

• Rats are weighed at the beginning and end of the 4 weeks. Food consumption is carefully monitored.

• At 10% “perfect protein” you get 2g of rat growth per gram intake (HIGHEST) → WHOLE EGG.

• Gelatin = 0 (no tryptophan); Wheat = 0.6 (little lysine); Raw soy = 0.5 (trypsin inhibitors)

• Cooked soy: inhibitors denature → 1.7

PER Pros and Cons

PROS: simple, cheap, very sensitive to AA balance, digestibility, toxic factors.

CONS: rats are not humans, done in growth stage and not maintenance stage, you don’t know WHY a protein is poor quality.

Chemical Score (CS)

• The test protein is chemically digested into free amino acids.

• These are then quantified by chromatography, and mathematically compared to the composition of whole egg protein (reference).

• LOWEST SCORE DETERMINES SCORE OF PROTEIN SOURCE.

CS Pros and Cons

PROS: simple and cheap, identifies the limiting AA in the food, used to optimize feeds by mixing different sources of protein.

CONS: doesn’t account for digestibility (e.g. hair) or toxins, assumes whole egg is an ideal protein.