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what are the protein requirements?
- EAR = 0.66g/kg/d
- RDA = 0.8g/kg/d
- Usual intake = 80-100g/d
how are Amino Acids classified?
- Indispensable (PVT TIM HLL)
- Conditionally Indispensable
how is metabolic flux of amino acids determined?
By following "tracers"
what are the tracer dilution principles (bathtub)?
- Bath represents all the free amino acids in the body
- Tap is open = e.g., we are eating
- 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
what are some stable isotope tracers (non-radioactive)?
- 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
how does a gas chromatograph mass spectrometer (GCMS) work?
What is a nutritionally indispensable AA?
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
Why are amino acids needed?
- Nitrogen Balance
- Plasma AA response
- Direct AA oxidation: tracer technique
- Indicator AA oxidation: tracer technique
- 24h balance
- or measure of organ or system function
What are the general aspects of experimental approaches for nitrogen analysis?
- 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
how does nitrogen balance work?
- 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%)
0 balance : intake = output (healthy adult)
- ex. 100g of protein
- = 16g of N - 1G - 15g
- = 0 = 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
how many grams of protein synthesis per day occurs in a healthy adult?
- 300 g
- Broken down amino acids are recycled to be used again
- Any excess protein intake will be broken down
what happens when there is a high protein consumption?
- 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
what is an optimal AA profile?
- Minimizes catabolism
- Maximises protein synthesis
- Oxidation is minimal
what is a limiting AA profile?
- 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
- 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
what is the metabolic response to intake of an essential AA: Nitrogen Retention (nitrogen balance)?
- 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
what is direct AA oxidation?
- 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
why is it not ideal to use the direct AA oxidation method?
- A lot of subject cooperation is requiredSpecialized 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
what is the metabolic response to intake of an essential AA: Direct oxidation?
- 1. Before requirement:
- Efficiently used for protein synthesis
- Minimally catabolized
- 2. At requirementOptimal level of protein synthesis
- Minimally catabolized
- 3. After requirement
- Optimal level of protein synthesis
- Excess AA are catabolized
Direct AA oxidation (ADD)
- 1. Clinical/metabolicRestricted 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. ModellingSteady state "nibbling" = 24h/meal feeding
- Breakpoint increase in oxidation with increasing intake
what is the Indicator AA oxidation (IAAO)?
- 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
what is the metabolic response to intake of an essential AA: indicator oxidation?
- 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
Indicator AA oxidation (IAAO)
- 1. Clinical/metabolicFree 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 ↑
what is the minimally invasive IAAO model?
- 1. Tracer administrationRepeated 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
Integrate concepts of determining AA requirement?
- 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 onInfants 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
Metabolic response to intake of an essential AA: Indicator oxidation
what is the EAR vs what is the RDA?
Estimates of LYS requirement (mg/kg/day)
Indispensable proteins in AA
- Potentially limiting AAs
Why is limiting AA important?
- 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
what happens when Histidine is removed from the diet for 48days?
- 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
- 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)
how are intravenous regimens designed?
- 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
how do we design the optimal AA profile (aromatic AAs)?
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
Nitrogen Retention in Baby Pigs (Study)
- 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
Tissue protein synthesis
- 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
what are the protein requirements based on N balance?
- EAR = 0.66 g/kg/d
- RDA = 0.80 g/kg/d
why is nitrogen balance (+) on the graph?
- 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
how does a low protein intake, specifically 0.6g/kg/d for 7 days, affect function?
- ↓ Protein turnover (↓ Protein metabolism)
- ↓ Albumin synthesis (negative acute phase protein)
- ↑ Fibrinogen synthesis : involved in tissue repair and the last process of blood coagulation
- Suggests that our current EAR is too low
how does a moderate protein intake, specifically 0.75g/kg/d for 7 days (a little over the EAR), affect function?
- ↓ Protein turnover
- ↓ Albumin synthesis
- ↓ Glutathione synthesis (tripeptide antioxidant)
- - antioxidant capacity, increased ↑ susceptibility to oxidative stress
- Studies suggest that our EAR/RDA is too low
what are the different organs involved in the metabolism of AAs?
Pepsinogen --> (acid) --> Pepsin
- Acid unfolds proteins (not functional) then pepsin breaks down the proteins
- 2. Proteases from pancreas in small intestine – luminal digestionBicarbonate 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
In general, they parallel glucose transporters
- Responsive to insulin
what happens in the fed state (post prandial)
: AA go to the liver by the portal vein, excess in the portal vein are catabolized
: ↑ protein synthesis, ↑ AA oxidation, promotes insulin secretion
: secretes insulin
Secretion of insulin
: ↑ protein synthesis and ↓ protein breakdown
what happens in the fasted state? (fixxxxxxxx)
= 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
what happens in a long term fast (ADD)?
- 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 vs Muscle
- 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
How does the liver play into amino acid metabolism?
- Anabolic: constitutive protein synthesis (proteins that stay in the liver, proteins that get exported), plasma protein synthesis, gluconeogenesis, lipogenesis
- Catabolic: AA catabolism, urea cycle
what are the differences in muscle mass?
- 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
insulin clamp studies (ASK)
What are the enzymes for AA synthesis?
- 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?