Module 8-Amino Acids PDF

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1 ANSC 315: Amino Acids Lecture 1 – Introduction Protein vs amino acids vs crude protein (nitrogen) -

Proteins are polymers made up of amino acids Amino acids are monomers that contain amino groups (nitrogen)

Crude protein -

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Makes the assumption that all proteins are 16% Nitrogen or Nitrogen x 6.25 = protein Not true all the time o Milk 6.38 o Rice protein 5.12 Says nothing about the amino acid content of a diet BUT - it’s not totally useless

Crude protein problems -

Animals don’t require nitrogen, they require amino acids During protein synthesis, all the amino acids in the protein must be present o Example: somatostatin We can synthesize some of the amino acids so they will always be present for protein synthesis

Essential vs Nonessential vs Conditionally essential Amino acids -

Essential amino acids: ones that animals can’t synthesize or cannot synthesize in sufficient amounts to maximize production Nonessential amino acids: ones that animals can synthesize in amounts that meet their requirements to maximize production Conditionally essential amino acids o Essential in some species but not all o Essential in some life stages o Can partially replace the requirement for an essential amino acid

Taurine -

Synthesized from Cysteine Beta-sulfonic amino acid Often considered with the amino acids It is essential in cats

Essential amino acids -

Mnemonic: PVT TIM HALL Phe, Val, Trp, Thr, Ile, Met, His, Arg, Leu, Lys

Different animals have different essential amino acid requirements -

Humans: don’t require histidine Cats: require glutamine and taurine (amino sulfonic acid synthesized from Cysteine)

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Trout/salmon: require taurine

Conditionally essential amino acids -

Dietary requirement at certain stages of life Gly: required by broiler chickens in first 2-4 weeks after hatch

Amino acid sparing -

Cys and Tyr are conditionally essential because they can meet about 50% of the requirement for Met and Phe, respectively Cys can replace ~50% of the Met requirement Tyr can replace ~ 50% of the requirement for Phe This means you the requirement for Met is given as Met and Met + Cys

Why not just feed Essential amino acids? -

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To synthesize a non-essential amino acid, you need 2 things o Carbon skeleton o Amino group NEAA are the major source of amino groups for synthesis of NEAA What happens if you don’t have enough NEAA in the diet?

Lecture 2 – Amino Acid Functions Key Concepts -

The metabolic functions of the essential amino acids Balancing the amino acid content of a diet Mixing ingredients with different amino acids Using synthetic amino acids

Lysine -

More research has been done on lysine than all the other amino acids combined Google search: o 12.8 million hits for lysine o 2.2 million hits for methionine

Lysine functions -

Its only function is as a component of protein Other amino acids have important metabolic functions as well It is used as the standard against which we calculate the requirement of the other essential amino acids

Lysine requirements -

Why is the fact that it is only used in protein synthesis significant? o Only calculated in terms of growth

Lysine in animal nutrition -

First limiting amino acid in pigs Second limiting amino acid in poultry Low in grains

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Animals raised on wheat/corn/barley diets must have supplements High in pulses: soybean/pea High in canola meal

Methionine -

Methionine  contains sulfur, First limiting amino acid in poultry Second limiting amino acid in pigs (mammals) Can be spared by cysteine so usually give requirement as Met + Cys Low in legume-based diets; soybean meal Important in DNA metabolism Precursor of taurine

Taurine -

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Required by cats and salmonids ** Not used in protein synthesis Important in o bile acid conjugation, o membrane stabilization, o modulation of intracellular Ca2+ levels Not present in plants

Taurine deficiency -

Retinal degeneration causing blindness Impaired reproduction and fetal development Hearing loss, impaired nervous function Feline dilated cardiomyopathy (usually fatal)

Threonine -

Third limiting amino acid Can be deficient in animal diets Pulses are high in threonine; grains are low High levels in mucous lining of small intestine Disproportionately high in endogenous losses During mucosal diseases more mucous is produced; more Threonine is required

Tryptophan -

4th limiting amino acid in most diets Particularly low in corn Aromatic amino acid, similar to phenylalanine The B-vitamin niacin can be synthesized from Tryptophan Precursor for serotonin-important in brain, gut function and sleep

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Precursor for melatonin-controls circadian rhythms

Tryptophan Metabolism -

L-tryptophan  melatonin  niacin

Arginine -

Needed in urea cycle to break down amino nitrogen Carnivores have a high requirement for Arg (Cats and salmon for example) o They can’t produce enough arginine for the urea cycle to upregulate after a meal.

Branched chain amino acids - Ile, Leu, Val -

Major components of muscle Enhance intestinal development, intestinal amino acid transportation and mucin production. Up-regulating innate and adaptive immune responses Risk of having a dietary deficiency in practical diets is low

Histidine -

Not required in humans but required in all monogastric agricultural species Rarely deficient in diets Histamine is formed by the decarboxylation of histidine o Involved in local immune responses (allergies), chemotaxis of white blood cells, vasodilation, regulation of gut function

Phenylalanine -

Used for production of melanin and melatonin Tyr can be converted to Phe Tyr can meet ~100% of the Phe requirement Inability to do this conversion is a common genetic defect in humans called Phenylketonuria

Aspartame -

Discovered by accident in 1965 180 x as sweet as sucrose Metabolized to Asp, Phe and MeOH Is it safe?

Cats glutamine

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Glutamine is required by cats ** Involved in the amino acid deamination when amino acids are used for energy Cats consume high protein diets and need more Gln than other species They can synthesize it but not at an adequate rate

Broilers and glycine -

Nitrogen excretion differs in different classes of vertebrates Birds (and reptiles) lay eggs and need to conserve water more than mammals Excrete N as uric acid as a concentrated paste which requires less water to produce than urea Requires glycine to synthesize

D and L Amino Acids -

All amino acids except Gly have D and L isomers L-amino acids are the natural isomers Some D-amino acids can be converted to L-amino acids (Met) Synthetic Lys, Thr and Trp are all L-isomers o Produced by fermentation (biological process) Synthetic met is a mixture of D and L isomers o Produced by an industrial process

Hydroxy Analogues of Amino Acids -

You can buy two kinds of synthetic Met: Methionine and the hydroxy analogue of methionine (MHA) Methionine hydroxy analogue is a commonly used Met supplement Can be converted to Methionine but… Does MHA = Methionine nutritionally?

Lecture 3 – Amino Acids Digestion Protein Digestion -

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Two objectives o Digest dietary protein o Reclaim digestive enzymes-they are protein too Endogenous protein is 30-50% of digesta protein If endogenous protein is lost in excess-> Negative N balance even though dietary protein is adequate

Stomach or Proventriculus -

Acidic environment (HCl-pH 1-3) Solubilizes and denatures protein Disrupts H-bonding (2o protein structure) Protein denaturation assisted by pepsin Peristalsis mixes the gastric juices to aid breakdown of proteins

Pepsin -

Proteolytic enzyme with wide specificity Most efficient breaking bonds between aromatic aa--Phe-Trp, Phe-Phe or Phe-Tyr Attacks protein regions resistant to low pH denaturation

6 Duodenum and jejunum -

Acidic gastric digesta is buffered by HCO3- ion to a slightly basic pH Pancreatic proteases are released These enzymes are either: Endolytic Exolytic

Endolytic enzymes -

Trypsin Chymotrypsin Elastase Hydrolyze peptide bonds in the middle of the protein

Exolytic enzymes -

Carboxypeptidase A and B Amino peptidase Hydrolyze peptide bonds at the carboxy or amino ends of the protein

Enzyme synergy -

Endolytic enzymes create more carboxy and amino ends Exolytic enzymes have more ends to work on

Steps in protein digestion -

1: Brush border peptidases 2: Brush border amino acid transporters 3: Brush border di and tri-peptide transporters 4: Intercellular peptidases 5: Basolateral membrane amino acid transporters 6: Basolateral membrane di and tri-peptide transporters

Absorption of Digestion Products -

In jejunum, 2/3 AA are present as di and tripeptides; 1/3 as free AA Peptides and AA are absorbed by two processes: Diffusion and active transport Diffusion is important when the concentration of AA is high Active transport is efficient even at low conc. of AA

Active Transport of Amino Acids -

Requires energy There are many different AA transporters Several amino acids may use the same transporter (ex. Lys and Arg use System y+) Excess of Arg may restrict absorption of Lys by competition for AA transporter Amino acid balance is important

Intestinal Energy Metabolism

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The gut is 2-6% of Body weight o It is responsible for 10-20% of whole-body CO2 production and 50-75% of maintenance energy requirements

The Fate of Absorbed Amino Acids -

Once absorbed into mucosal cells, most peptides are broken down by proteases to free AA Free AA Are then transported across the mucosal membrane into the portal vein to liver Used for: Anabolism: protein synthesis Catabolism: excess AA are oxidized for energy (controlled by insulin)

Amino Acids vs. Keto Acids -

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The amino group on glutamate gets replaced with a ketone group o Double bonded O Makes ammonia

Catabolism of Amino Acid Nitrogen in Mammals -

The amino groups produced by catabolism are extremely toxic They are converted into less toxic forms in mammals by the following steps Transamination Oxidative Deamination Ammonia transport Urea cycle

Transamination -

An amino acid has its amino group removed and transferred to alpha-ketogluarate You end up with 1) glutamate and 2 an alpha-ketoacid This step requires energy

Oxidative Deamination -

Amino groups of most aa. are transferred to α-ketoglutarate Release of this N as ammonia is catalyzed by L-glutamate dehydrogenase This regenerates α-ketoglutarate

Ammonia Transport -

The free amino group is added to a glutamate molecule giving glutamine This is transported to the liver

Why don’t we just excrete the ammonia? -

Ammonia is very toxic By keeping the amino group attached to amino acids, this toxicity is avoided Urea is relatively non-toxic and easy to excrete in a concentrated form

Ammonia Intoxication

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In ruminants, caused by the absorption of excess ammonia from the rumen Insufficient glucose to form α-ketoglutarate and therefore glutamine In cats, low arginine diets can cause ammonia intoxication Can be life threatening Symptoms are tremors, blurred vision, coma, death

Fate of Carbon Skeletons -

Source of energy and glucose Amino acids are either ketogenic or glucogenic or both Ketogenic amino acids form ketone bodies Acetone, acetoacetate or β-hydroxybutyrate Can be used to synthesize fatty acids Glucogenic amino acids form glucose-essential during starvation

Protein Deficiency in Humans -

Condition resulting from protein deficiency Serum protein is severely decreased causing edema (swollen abdomen) Often occurs in children after weaning in famine areas

Lecture 4 – Amino Acids Digestibility Protein is insufficient as an energy source -

Energy cost of o Protein metabolism o Nitrogen excretion o Heat increment o Conversion of protein to fat

Energetic efficiency of using protein for energy -

High protein diets are poor for finishing animals. Why? o Energy cost of converting different substrates to fat Fatty acids  fat 480 kJ/mol Carbohydrate  fat 6104 kJ/mol Protein  fat 12767 kJ/mol

Protein Quality -

Two things determine the nutritional quality of a protein o The amino acid content of the protein relative to the requirement of the animal o The digestibility of the amino acids in protein

Protein Digestibility -

Feed proteins are digested in stomach and small intestine Absorption occurs in Small intestine No absorption of AA in Large intestine o Undigested feed protein is digested by microbes in large intestine and turned into microbial protein o Microbial protein is mainly excreted in feces

9 Total AA vs. Digestible AA -

The percent AA that are absorbed in the gut differs between feed ingredients Therefore, diet formulations based on total levels of AA are inaccurate

Absorption vs. digestibility -

Absorption is the amount of a dietary component passing across the gut o Hard to measure Digestibility is the amount of a dietary component removed from the digesta o Easier to measure It is assumed that amino acids that are digested are also absorbed - not always true

Apparent Fecal Digestibility -

If amino acids found in feces are subtracted from AA in feed, the apparent digested amount can be calculated Example 10 g lysine ingested, 2 g lysine in feces 10 g – 2 g = 8 g absorbed Apparent Fecal Digestibility is 80 %

Without Microbial Metabolism of Amino Acids -

Microbes in ceca and large intestine may change amino acids by deamination and transamination Therefore, apparent fecal digestibility coefficients may be inaccurate Example with no bacteria: 10 g Lys ingested: 2 g Lys found in feces. Apparent digestibility = 10 g – 2 g = 8 g or 80%

Microbial Metabolism of AA -

Example with bacteria: 10 g Lys ingested: 1 g of Lys converted to other AA by microbes 2 g Lys found in feces. Apparent digestibility = still 80% True digestibility = 1- ((Lysine ingested – (Lysine in feces - Lysine metabolized))/Lysine ingested 1 – ((10 – (2 + 1))/100 = 70% **exam Note that Lysine metabolized is positive if destroyed and negative if created**

Bypassing the microbes -

Because of the microbial manipulation, fecal digestibility coefficients are inaccurate To avoid the influence of the microbes, digestive fluids are captured at the distal ileum (less bacteria in small intestine) Digestibility coefficients are calculated based on the disappearance of amino acids prior to the distal ileum

Apparent Ileal Digestibility -

Example: 10 g lys ingested, 1.5 g lys in ileal fluids 10 - 1.5 = 8.5 g lys absorbed Digestibility coefficient is 0.85 Effect of microflora on amino acids is smaller

10 Endogenous Contributions -

Amino acids are constantly lost from digestive enzymes, enterocytes, bile, mucus, and other sources These amino acids are secreted into the lumen of SI Some of them are not reabsorbed and end up in digesta captured at the distal ileum Taking them into account gives us True ileal digestibility

True Ileal Digestibility Coefficient -

If AID is corrected for the endogenous losses, then a true ileal digestibility coefficient (TID) can be calculated

Example -

Pig consumes 50 g of lysine Ileal lysine = 10 Endogenous losses = 5 What is True ileal digestibility? o Digestibility of the feed (%) = o Nutrients ingested – (Total Nutrients excreted - Nutrients from endogenous losses) o Nutrients ingested o = 50 – (10 – 5) o 50 o = 50 – 5 = 90% o 50

Measuring Endogenous Losses -

Endogenous losses in a pig are measured by capturing the digestive fluids at the distal ileum after a nitrogen free diet has been fed If you just feed starch you would still find protein This protein is the endogenous losses Feed something without protein, measure the protein at the end Animals don’t like eating protein free diets tho (less palatable)

Digestibility Measures and Feed Formulation **exam -

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Total amino acids in feed o Only if you don’t have any other information o Try to do a digestibility trial before you formulate diets Apparent fecal digestibility (fish) o Not corrected for bacterial metabolism or endogenous losses Apparent ileal digestibility (poultry and pigs) o Corrects for microbial metabolism (mostly) True ileal digestibility o Corrects for microbial metabolism and endogenous losses

Lecture 5 – Amino Acids Digestion Amino Acids Requirement of Monogastrics -

Animals require amino acids-not protein

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Amino acids are building blocks for protein Lean meat mainly consist of H2O and protein We want as much lean meat deposited as possible Protein synthesis is an “all or nothing” event All AA need to be available at the same time

Requirements for Amino Acids -

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AA requirements depend on o Species o Age o Production level Genetics determine the animals’ capacity for lean meat deposition Nutrition determines if the genetic capacity is utilized Different genetics have different requirements for amino acids Old methods o Crude protein, biological value, protein efficiency ratio New methods o Amino acid requirements, ideal protein balance

Crude Protein (N x 6.25) -

Nutrient requirements of pigs and poultry were given as % crude protein up to 1971 (poultry) and 1972 (swine) E.g. 10-20 Kg pigs require 18% crude protein (NRC, 1972) Assumption based on typical corn-soy diets

Protein Efficiency Ratio -

PER = amount of weight gained/amount of protein consumed Widely used in human and aquaculture nutrition Primitive method of assessing protein quality Not widely used in other agricultural species

Biological Value -

BV = nitrogen retained/nitrogen absorbed*100 Takes digestibility and amino acid balance into account Measures the utility of a protein to an animal without measuring the amino acids A BV of 100 indicates complete utilization of a protein (100% absorbed and retained)

First Limiting Amino Acid -

The First Limiting Amino Acid is the amino acid present at the lowest amount relative to the requirement The amount of this amino acid will determine the level of production and maximum growth rate of the animal Measure the requirement for every essential amino acid Formulate diets to meet these requirements

Measuring Amino Acid Requirements -

Animal growth studies generally used

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Requirement can be based on: Maximum gain Maximum feed efficiency Maximum yield of product eg. breast meat

Determining the Requirement for Lysine -

You must provide a basal diet that is adequate for all nutrients other than lysine The basal diet should be deficient in lysine You can add Lys-HCl to the basal diet to give different levels of lysine

Measuring the Requirements of ...