NUTR - Topic 4

• EAR = 0.66g/kg/d
• RDA = 0.8g/kg/d
• Usual intake = 80-100g/d

• Indispensable (PVT TIM HLL)
• phenylalanine
• methionin
• leucine
• isoleucine
• valine
• lysine
• threonine
• tryptophan
• histididne

• Conditionally Indispensable
• tyrosine
• arginine
• glutamine
• glycine
• proline

• Dispensable
• alanine
• aspartate
• asparagine
• glutamate
• serine

By following "tracers"

• Bath represents all the free amino acids in the body
• Tap is open = e.g., we are eating

• Assumptions
• 1. System at steady state
• Water level in the bath tub remains constant – homeostasis
• Rate of appearance (Ra) = Rate of disappearance (Rd)
• Rate of inflow (influx) = rate of outflow (outflux)
• We are either at fed, nibbling, or fasting state

• 2.  Homogeneity of pool
• Complete mixing of the pool
• Tracer goes all over the body

• 3.  Massless tracer = tracee
• No influence in metabolism and in behaviour
• Labeled ones act just like un-labeled ones

• 4.  No tracer recycling
• Drained water stays out
• Fast flow of water --> light colour
• Slow flow of water --> dark colour

• Carbon = ¹²C, ¹³C, ¹⁴C
• Hydrogen = ¹H, ²H, ³H
• Nitrogen = ¹⁴N, ¹⁵N
• Oxygen = ¹⁶O, ¹⁸O
• Sulfur = ³²S, ³⁴S, ³⁵S

• 1st column =  Most common forms
• 2nd column =  Heavier than common forms – due to an extra neutron /  Ditirium (2H) is a part of heavy water
• 3rd column =  Radioactive – not stable & might cause a health issue

Cannot be synthesized by the animal organism out of materials ordinarily available to the cells at a speed commensurate with the demands for normal growth

• Growth
• Nitrogen Balance
• Plasma AA response
• Direct AA oxidation: tracer technique
• Indicator AA oxidation: tracer technique
• 24h balance
• or measure of organ or system function

• All methods should give the same answer
• Subjects should be studied at >6 test AA intake levels above and below requirement: To account for individual variability
• Endpoint should show a clear response to change in test AA intake: g., deficient à adequate
• Question of adaptation to test AA intake
• - N: 7-10 days for urea pool – slow turnover
• - CO2: hours to a couple of days – fast turnover
• -  Go out in breathe
• -  Rapid equilibrium

• 0 nitrogen balance (healthy): intake = excretion
• Protein absorption rate = 95%
• Miscellaneous losses (e.g., sweat secretion, hair growth, finger nail growth)

Protein is 16% nitrogen 6.25g protein contains 1g nitrogen

Nitrogen Balance = N intake - Fecal N (5%) - Urinary N (95%)

• ex. 100g of protein
• = 16g of N - 1G - 15g
• = 0 = healthy adult

0 balance : intake = output (healthy adult)

• +ive = intake > output
• growing children, pregnancy
• depositing protein (recovering from an illness)

• -ive = intake < output
• low protein diet - low intake
• GI problem - low absorption
• injury - high catabolism
• losing lean muscle mass
• muscle wasting

• 300 g
• Broken down amino acids are recycled to be used again
• Any excess protein intake will be broken down
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• There is no effect
• There will be a normal protein absorption rate of 95%

                                                   

• Positive nitrogen balance only if in a building state
• Growing or gaining lean mass
• Simply eating more proteins or regular exercise won’t result in positive nitrogen balance

• Negative nitrogen balance
• E.g., malnourished or losing lean mass

• Minimizes catabolism
• Maximises protein synthesis
• Oxidation is minimal

                                                   

• Limits protein synthesis
• Going from sufficient ---> deficient
• Increase ↑ in catabolism of the excess AA (Extra protein excreted/broken down)
• Loss of lean body mass
• Loss of immune proteins
• Plant-based protein sources are limiting in different amino acids

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• 1. Clinical/metabolic
• COMPLETE intake and collections: Missing one urinary collection hugely affects the result
• Adaptation of urea pool

• 2. Analytical
• Routine analysis of total nitrogen

• 3. Modelling
• Miscellaneous losses would increase requirement: eg., sweat, hair growth
• Sensitivity: small number calculated from 2 large and similar numbers
• (A huge nitrogen intake (e.g., 200g) – a huge urinary nitrogen loss (e.g., 190g) = nitrogen balance (e.g., 10g))
• Curvilinear response as balance approaches zero
• Between-subject variance is high therefore repeated measurements on each subject needed but adaptation issue

                                                   

• Nitrogen balance is progressively more positive as the requirement is met
• Actual requirement ≠ theoretical requirement
• The graph plateaus due to adaptation and individual variation

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• Growth increases until the requirement is met
• Excess amino acid intake will not make you grow more
• Nitrogen retention (nitrogen balance) has the same curve as growth curve

• Labeled amino acids (Phe is labelled with ¹³C) will either be synthesized into proteins or catabolized
• When the labeled amino acids are catabolized, they come out of breath
• Carboxyl groups from amino acids are chopped off to give CO₂ --> exhaled --> heavier ¹³C are measured

•  A lot of subject cooperation is required
• Specialized formula tastes bad
• Other diet sources need to be protein free
• People on this diet crave textures
• No fiber in diet causes constipation

• Researches are usually done on men
• Due to varying menstrual cycle and etc. 

• Fast procedure (-4 hours)
• Can be done on infants, children and animals

• 1.  Before requirement:
• Efficiently used for protein synthesis
• Minimally catabolized

• 2. At requirement
• Optimal level of protein synthesis
• Minimally catabolized

• 3. After requirement
• Optimal level of protein synthesis
• Excess AA are catabolized

• 1. Clinical/metabolic
• Restricted choice of test AAs (branched chain AA, LYS, PHE)
• Free pool changes with changing test AA intake
• Non-negligible tracer – can’t study very low intakes

• 2. Analytical
• Breath collection
• Blood samples

• 3. Modelling
• Steady state "nibbling" = 24h/meal feeding
• Breakpoint increase in oxidation with increasing intake

• Concept: when an indispensable AA is limiting, then all other indispensable AA will be oxidized (remember that AAs cannot be stored)
• Increasing ↑ intake of limiting amino acid will decrease ↓ IAAO

• Eg., test amino acid (lysine); indicator (phenylalanine)
• When lysine intake is zero, phenylalanine is in a huge excess and will be oxidized
• As lysine intake increases, oxidation of phenylalanine decreases until lysine intake meets its requirement

• 1.  Clinical/metabolic
• Free choice of test AA, can study 0 intake
• Tracer AA pool not perturbed with changing test AA intake
• PHE, LYS, LEU as indicator AAs

• 2. Analytical
• Breath collection
• Blood samples; urine samples (non-invasive)

• 3. Modelling
• Steady state “nibbling” = 24-h/meal feeding
• Breakpoint decrease ↓ in oxidation with increase intake ↑

• 1. Tracer administration
• Repeated oral “nibbling” of tracer solution after 4-h feeding equilibration

• 2. Sampling
• Breath collection for CO₂ enrichment
• Urine in place of blood for plasma AA enrichment :Taking out blood samples from infants is very stressful for the infants

• Common key principles
• Amino acids are either synthesized into proteins or catabolized in the body (NOT STORED!!)

• Ethics regarding sampling and specific diets that people are on
• Infants can undergo oxidation method (a few hours) but not nitrogen balance method (10 days)

• What is the key endpoint for each method? 
• 1. Nitrogen Balance: Follows urea production and excretion
• 2. Direct oxidation: Follows carbon skeleton catabolism
• 3. Indirect oxidation: Follows carbon skeleton catabolism

• Potentially limiting AAs
• Lysine
• Threonine
• Tryptophan
• Methionine
• Cysteine

• Omnivores with average protein intake – not an issue
• Vegans with lower protein intake and lower protein quality – limiting AA is significant issue especially for children (because they have a higher protein requirement)
• Complementary proteins: Beans and rice and Legumes and grains (e.g., peanut butter sandwich)
• International : food security and diet diversity – very important especially for children

• No effect on nitrogen balance
• Decreased protein turnover, PHE oxidation

• Decrease in HIS level in blood over time
• Hematocrit (Hct) – the percent occupied by cells in blood = decreased
• Hemoglobin (Hb) decreased = Causing iron-deficiency anemia
• Ferritin – binds iron when being stored = increased
• Ferritin level goes down in case of anemia
• Iron stores are getting higher
• Transferrin – transports iron in blood = decreased
• Transferrin level goes up when iron is deficient
• Albumin – the most abundant protein in plasma = decreased

• Conclusion
• Lack of histidine limits red blood cell production
• Histidine is very important for hemoglobin to bind oxygens
• Lower capacity to transport oxygen
• Decrease in these nutrient transport proteins that are high in histidine
• Accommodation process – more histidine can be available for other things (Functional adaptation)

• 1. Metabolism
• Bypass splanchnic control (gut & liver): All the digestions have to be taken place before infusion

• 2. Composition (Solubility and Stability)
• Free AAs, glucose, vitamins and minerals in an infusion bag
• Tyrosine is not soluble
• Glutamine is not stable – deconstructed after being in the solution for a while
• Lipids in a separate infusion bag: TGs stabilized with lecithin (from soybean oils)
• Omega-6 fatty acids are pro-inflammatory – NOT GOOD

Issues to address: tyrosine insolubility, transient hyperphenylalaninemia and hypertyrosinemia

• 1.  Infuse extra phenylalanine
• – PHE will be hydroxylated to become tyrosine
• - Used for adults

• 2.  Infuse more soluble precursor of tyrosine (N-acetyltyrosine)
• - Used for babies

• 3.  Dipeptides (e.g., glycine + tyrosine = glycyl-tyrosine)
• - Cheapest

• 1. Vamin
• - Modeled after egg white
• - Extra PHE

• 2. Vaminolact (VL)
• - Modeled after breastmilk
• - Ignored the need of tyrosine: least nitrogen retention and growth

• 3.  VL + PHE
• - Increased nitrogen retention and growth

• 4. VL + NAT (N-acetyltyrosine)
• - No benefit 

• 5. VL + GT (dipeptide)
• - Increased nitrogen retention and growth

• FSR (fraction synthesis rate)
• -  Newly made per day
• -  Measured by using isotope tracers

• Lower protein synthesis in protein deficient (almost by half)
• -  Lower protein synthesis in muscles leads to decrease in growth
• -  Lower protein synthesis in visceral tissues leads to decrease in synthesis of immune-related proteins

• EAR = 0.66 g/kg/d
• RDA = 0.80 g/kg/d

• Nitrogen balance is positive due to uncounted miscellaneous losses
• Individual variability in protein metabolism is depicted as solid dots
• Break point = 0.9g/kg/day
• Break point + 2 standard deviation = 0.99g/kg/day
• Oxidation technique with different protein intakes is used

• ↓ Protein turnover (↓ Protein metabolism)
• ↓ Albumin synthesis (negative acute phase protein)
• ↑ Fibrinogen synthesis : involved in tissue repair and the last process of blood coagulation

• Conclusion
• Suggests that our current EAR is too low

• ↓ Protein turnover
• ↓ Albumin synthesis
• ↓ Glutathione synthesis (tripeptide antioxidant)
•   - antioxidant capacity, increased ↑ susceptibility to oxidative stress

• Conclusion
• Studies suggest that our EAR/RDA is too low

Digestion

• 1.  Pepsin in stomach
• Pepsinogen --> (acid) --> Pepsin
• Acid unfolds proteins (not functional) then pepsin breaks down the proteins

• 2.  Proteases from pancreas in small intestine – luminal digestion
• Bicarbonate increases ↑ pH – inactivating/denaturing pepsin
• There are many proteases exist – due to their specificity of cleavage sites

3.  Peptidases on brush border of intestinal epithelial cells

• 4.  Active transporters
• In general, they parallel glucose transporters
• Responsive to insulin

Anabolic state = insulin

Gut: AA go to the liver by the portal vein, excess in the portal vein are catabolized

Liver: ↑ protein synthesis, ↑ AA oxidation, promotes insulin secretion

Pancreas: secretes insulin

Secretion of insulin: ↑ protein synthesis and ↓ protein breakdown

• Catabolic state = glucagon secretion
• Glucagon = stimulates the Cori and Cahill Cycle to bring lactate and alanine to the liver
• ↓ PS, ↓ oxidation of AA, ↑ PB
• We take out blood glucose out of the store for the brain

Muscles: breakdown of AA

• Gut metabolism slows down
• β - oxidation of FA generating Acetyl-CoA
• ↑ Acetyl- Coa generates  in the absence of glucose producing ketones that can be used by the brain

• Liver is much more metabolically active
• Muscle is very slow in terms of protein and energy metabolism
• Exercising = increasing very slow process
• As we age, our muscle metabolism gets slower = less response to insulin

• Anabolic: constitutive protein synthesis (proteins that stay in the liver, proteins that get exported), plasma protein synthesis, gluconeogenesis, lipogenesis
• Catabolic: AA catabolism, urea cycle

• Hypertrophy (muscles getting bigger): Growth, anabolic, activation of PI3K-Akt pathway and MTOR
• Atrophy (protein intake ↓ and muscle loss): Wasting, catabolic, Activation of ubiquitin-proteosome pathway

• Essential AAs
• 59 enzymes total required
• We lost the capacity to make these enzymes
• Expensive to make 
• Survival advantage to be able to rely on diet

• Non essential AAs (up to 11)
• 17 enzymes required
• Easy to make : directly or indirectly from glucose (except for tyrosine)
• Survival advantage to maintain capacity to synthesize
• Are they more important?