Carbohydrate, Protein and Lipid Metabolism Notes

Part 1 – Metabolism Concepts and Measurement

Carbohydrates, protein and fat are macronutrients.

In the human body metabolism is the oxidization of carbohydrates, protein and fat to give CO2, H2O and energy.

What is Metabolic Rate?

Metabolic Rate is the amount of energy liberated per unit time.

The Basal Metabolic Rate is the rate of energy expenditure at rest in a neutrally temperate environment, in the post-absorptive state (meaning that the digestive system is inactive, which requires about twelve hours of fasting in humans).

  • The Basal Metabolic Rate is the largest component of total caloric expenditure in humans: 70%
  • Physical activity contributes: 20%
  • Thermogenesis & digestion contributes: 10%

Units used for Metabolic Energy

  • calorie (cal – note lowercase) is the standard unit of metabolic heat energy, being the amount of energy needed to raise 1g of water by 1 degree, from 15o to 16o C.
  • Calorie (kilocalorie, kcal, big calorie, large calorie, kilogram calorie) is more commonly used, representing 1000 calorie.
  • Joule is the SI unit for energy, such that 1 calorie = 4.2 joule.
  • To convert from Calories (kilocalories) to kilojoules, multiply by 4.2.

How do we measure Metabolic Energy and Metabolic Rate?

Direct calorimetry

A Bomb Calorimeter, or constant-volume calorimeter, is used to measure the energy released by food during complete oxidization.

The food is placed in a sealed metal container surrounded by water in an insulated container. The food is ignited by an electrical spark and the temperature change of a known volume of water is used to calculate the energy released by the food.

Standard caloric values for macronutrients are:

  • Carbohydrates: 4.1 kcal/g
  • Protein: 5.3 kcal/g
    (but in the body is only 4.1 kcal/g due to incomplete oxidation)
  • Fat: 9.3 kcal/g
  • Ethanol: 7 kcal/g

Indirect calorimetry

Calculates energy release by measuring the products of metabolism such as CO2, H2O and urea/nitrogen compounds or O2 consumption. O2is general used as it is neither stored or produced, so O2 consumption is directly proportional to the energy liberated.

Approximately 4.82 kcal is liberated for each 1L of O2 consumed, although this varies depending on the macronutrient composition of food consumed.

Respiratory Quotient (RQ) is the ratio of the volume of CO2 produced to the volume of O2 consumed per unit time, in the steady state. This is different from the ‘Respiratory Exchange Ratio’ (R or RER), which quantifies the same ratio at any point in time, not only at equilibrium. 

RQ = V­producedCO2 / VconsumedO2

RQ of carbohydrate is 1.0 and for fat 0.7. Why the difference? Because carbohydrates have the same 2:1 ratio of hydrogen to oxygen as does water, whereas fats require extra oxygen for the formation of H2O. Protein has an approximate ratio of 0.82, but is complex to calculate as protein is incompletely oxidized in vivo.

  • With a modern diet RER is approximately 0.8 at rest. With exercise this will approach 1.0 as muscle glycogen (and thus glucose) is metabolized. With intense exercise RER can exceed 1.0 because muscle CO2 production and O2 consumption increases. RER is an inaccurate way to estimate RQ during exercise because of the bicarbonate buffering of hydrogen ions, depressing actual CO2 expiration.
  • The proportional metabolism of each of the macronutrients in the body can be estimated at any time by measuring both the RQ and urinary nitrogen secretion. We can take this further, measuring regional and organ-specific RQ and so infer specific metabolic preferences of different organs. This is how we know that the brain is a preferential glucose metabolizer by calculating the brain RQ as 0.97-0.99.

Which organ has a negative RER?

The stomach, because it takes up more CO2 from arterial blood than it releases into venous blood, during the secretion of acid.

We can also use similar techniques to determine total regional energy expenditure:

Liver 27%
Brain 19%
Heart 7%
Kidneys 10%
Skeletal muscle 18%
Other organs 19%

To measure the systemic metabolic rate of an individual historically we would use a Modified Benedict apparatus. This is essentially an oxygen-filled spirometer in series with a CO2 absorber, used to measure oxygen consumption.

More modern, advanced systems such as the “Quark RMR” from COSMED (below), measure VO2 and VCO2, and automatically calculate RQ, REE and substrate utilization. These can be used with a canopy hood in research subjects or connected directly to mechanically ventilated subjects in an ICU or critical care setting.

More commonly we simply estimate an individual’s Basal Metabolic Rate.

  • Estimate BMR (kilocalories) using the Harris-Benedict equation:
    • Men:  BMR = 66 + ( 13.7 x weight in kg ) + ( 5 x height in cm ) – ( 6.76 x age in years )
    • Women:  BMR = 655 + ( 9.6 x weight in kg ) + ( 1.8 x height in cm ) – ( 4.7 x age in years )
  • Lean body mass = 35 kcal/kg/d or 148 J/kg/d (~ 2500 kcal/d for 70 kg).

Then use the Harris-Benedict Principle to calculate daily needs

  • Little to no exercise, daily calories needed = BMR x 1.2.
  • Light exercise (1-3 days per week) = BMR x 1.375
  • Moderate exercise (3-5 days per week) = BMR x 1.55
  • Heavy exercise (6-7 days per week) = BMR x 1.725
  • Very heavy exercise (twice daily, heavy workouts) = BMR x 1.9

The Harris-Benedict equation was developed in 1919, but not further improved until 1990 by Mifflin. Since then further improvements have been made by other teams primarily to take into account the metabolic difference between lean body mass and body fat.

What factors influence metabolic rate?

  • Anaerobic exercise increases BMR by increasing lean body mass.
  • Aerobic exercise may increase BMR, although controversial.
  • Muscular exertion during or immediately before measurement.
  • Digestion of food
  • High or low environmental temperature
  • 14% increase in MR for each 1oC in environmental temperature.
  • Height, weight, surface area, sex, age
  • Growth
  • Reproduction
  • Lactation
  • Emotional state
  • Body temperature
  • Thyroid hormones
  • Adrenaline & Noradrenaline

Disease states 

Sepsis leads to a 30-60% increase in BMR. One study suggests the increase iz inversely proportional to sepsis severity, ie. BMR increase in uncomplicated sepsis was > sepsis syndrome > septic shock.
[ http://www.ncbi.nlm.nih.gov/pubmed/8319458 ]

Burns over a large surface area cause a dramatic increase in BMR.

  • Adults with a 25% (TBSA) burn increase metabolic rate to 120-210% of normal.
  • Those with over 40% TBSA always experience hypermetabolism.
  • At 50-60% TBSA there is further increase in hypermetabolism, after which there is minimal further increase.
  • Typically, post-burn respiratory quotients are 0.70-0.76.
  • Caloric intake in burns patients aims to be provided as 50% carbohydrate, 30% fat, 20% protein. Enteral feeding is preferred.

During sleep BMR falls 10% and during starvation 40%, known as the minimal metabolic rate.

The average adult requires approximately 2000 kcal/d for essential physiological functions, with a sedentary person requiring a further 500 kcal/d for low-level activity. ie. 35 kcal/kg/day total.

While 62% of differences between individual’s BMR is explained by lean body mass, 26% of the inter-individual variation in BMR is unexplained. Brown fat deposition and basal metabolic hormone profiles are likely implicated.

 

Part 2 – Cellular Metabolism

Cellular metabolism describes the process by which the products of digestion (intermediaries amino acids, fat derivatives and the hexoses/ monosaccharides fructose, galactose and glucose) are chemically modified to yield usable energy.

The final common pathway of metabolism is the citric acid cycle, which accepts fragments of the intermediaries, releasing CO2 and H+ ions. Most catabolic energy is not immediately used but rather temporarily stored in high-energy phosphate compounds as bonds between phosphoric acid residues. The most important of these is adenosine triphosphate (ATP). Hydrolysis of ATP to ADP yields energy directly for cellular and bodily function. Acetyl-CoA is another important energy source, and is molar equivalent to ATP in its ability to provide energy for cellular processes. ATP:

Citric Acid Cycle / Kreb’s Cycle / Tricarboxylic Acid Cycle

  • Occurs in the mitochondria.
  • Requires oxygen!
  • Common pathway of metabolism.
  • Yields CO2, H+ ions and energy.
  • Provides precursors for other compounds.
  • Begins with acetyl-CoA transferring it’s 2C acetyl group to the 4C acceptor oxaloacetate, yielding the 6C citrate – this is the rate-limiting step. Citrate goes through several transformative steps loosing 2C as CO2.
  • Energy released is transferred as energy-rich electrons to NAD+ to form NADH. Three NADH are produced for each AcetylCoA entering.
  • The cycle continues to end with the reformation of 4C oxaloacetate.
  • The Citric Acid Cycle produces one GTP (or ATP), three NADH, one QH2 and two CO2 for each Acetyl-CoA.
  • Two Acetyl-CoA are produced from one glucose molecule.
  • Regulation of TCA is almost entirely determined by substrate availability and product inhibition acting in a negative feedback loop (NADH, Acetyl-CoA, succinyl-CoA, ATP, Citrate). Calcium activates various enzymes in the TCA.
  • Hans Krebs received the Noble Prize in Physiology in 1953 for his discovery of the TCA in 1937.

A Mnemonic to remember the steps (if you must):

Oxaloacetate + acetylCoA -> citrate -> isocitrate -> alpha-ketoglutarate ->

succinyl-CoA -> succinate -> fumarate -> malate -> OA.

“Only Cool Icy Alcohol Seems to Satisfy Fellows Metabolically”

Glycolysis

  • 10 reactions that convert glucose C6H12O6 into pyruvate CH3COCOO + 2 NADH + 2 H+ + 2 ATP.
  • The most universal metabolic pathway, occurring in almost all organisms, both anaerobic and anaerobic.
  • The type of glycolysis that primarily concerns us medically is the Embden-Meyerhof pathway (pathway to pyruvate via trioses).
  • An alternative to glycolysis is the hexose monophosphate shunt (via 6-phosphogluconate and the pentoses), which although it involves oxidation of glucose it is primarily an anabolic pathway. It only occurs in the liver and adipose tissue, and makes up ~ 30% of glycolysis.
  • The reverse of the process is gluconeogenesis, which generates glucose from non-carbohydrate carbon substrates, such as lactate, glycerol and gluconeogenic amino acids. Many, but not all, of the steps of gluconeogenesis are the reverse steps of glycolysis.
  • In humans gluconeogensis only occurs significantly in the liver, and to a lesser degree in the renal cortex. It occurs in response to fasting, low-carbohydrate diets or intense exercise. Metformin used to treat type 2 DM works partly by inhibiting gluconeogenesis.
  • All Citric Acid Cycle intermediates can be used for gluconeogenesis. The carbon backbone of amino acids can also enter gluconeogenesis after their transamination or deamination. Odd-chain fatty acids can also be used for gluconeogenesis.

Cori cycle

  • Describes the cycle of lactate production in muscle occurring due to anaerobic glycolsysis, and the subsequent conversion of lactate back to glucose in the liver.
  • Muscles use glycogen as a store of glucose in the form of glucose-6-phosphatase. G-6-P is a ready substrate for glycolysis providing ATP. During anaerobic activity pyruvate is instead converted to lactate, yielding only 2 ATP. Conversion of lactate back to glucose through gluconeogenesis will consume 6 ATP nett. This net cost of 4 ATP per cycle cannot continue indefinitely and must at some stage be replenished. Oxygen debt must be repaid after muscle activity ceases.
  • The Cori Cycle effectively shifts the metabolic burden from the muscles to the liver. Oxygen debt must be repaid after muscle activity ceases.
  • Both the Citric Acid Cycle and Oxidative Phosphorylation require oxygen and do not function under anaerobic conditions.

Oxidative phosphorylation and the Electron transport chain

  • Oxidative phosphorylation uses energy released by other intermediary metabolic pathways (ie. Kreb’s Cycle, beta-oxidation of fats) to form ATP. It is a very efficient process for releasing energy.
  • Oxidative phosphrylation takes protons (H+) supplied by NADH and using a flavoprotein-cytochrome system translocates the protons across the mitochondrial inner membrane from the mitochondrial matrix into the intermembrane space. This creates a concentration gradient of H+ ions, down-which they travel creating ATP via ATP synthase.
  • ATP synthase activity is determined by the amount of ADP present, the availability of fats, lactate and glucose derivatives, and most importantly the availability of oxygen.
  • 90% of oxygen consumption in the basal state is mitochondrial, and 80% of this is coupled to ATP production. 27% of ATP is used for protein synthesis, 24% by Na-K-ATPase, 9% by gluconeogenesis, 6% by Ca-ATPase, 5% by myosin ATPase and 3% by ureagenesis.
  • At rest 70% of produced ATP is derived from fats and 30% from carbohydrates. As activity increases in intensity there is a shift from fat to carbohydrate.

ATP – Adenosine Triphosphate

There is only about 0.1 mole of ATP in the body, but daily energy needs require 100 to 150 moles, or about 50 to 75 kg. The average human adult will use their body weight in ATP each day – so ATP must be recycled as it is used.


ATP formation (per glucose molecule)

  • Glycolysis: 2 ATP (2 used, 4 produced)
  • Kreb’s: 2 ATP
  • 24 H+ ions produced overall
  • 20 of these produce 30 ATPs.
  • remaining 4 H+ -> 4 ATP
  • TOTAL: 38 ATP per aerobic glucose (so the textbooks say!)
  • Efficiency of 66% – compared with internal combustion engine efficiency of 20% at best and rocket engines of 70%.

Or is it 31 ATP produced?

Recent research suggests the yield is only 31 ATP per glucose molecule, as the ATP pay-out ratios for NADH & FADH2 are 2.5 and 1.5 respectively, rather than 3.0 and 2.0 as has been assumed for the past 50 years! 

Carbohydrate metabolism

  • Most dietary carbohydrates are polymers of hexoses, primarily glucose, galactose and fructose.
  • Glucose is stored in its phosphorylated form glucose-6-phosphate, the formation of which in muscles is catalyzed by hexokinase, and in the liver by glucokinase.
  • Glucokinase is important because its activity is stimulated by insulin and it’s activity reduced in starvation, and glucokinase has a stronger affinity for glucose than does hexokinase.
  • Only hepatic (and insignificant amounts of renal) glycogen is accessible to the body as a glucose source. Glucose-6-phophatase is only found in the liver, converting Glc-6-P to glucose.
  • Both adrenaline and glucagon stimulate hepatic phosphatase and so increase blood glucose levels by stimulating release of hepatic glucose.
  • In this way the liver functions as the hepatic glucostat, releasing glucose when blood glucose levels are low and taking up glucose (stimulated by insulin) when blood glucose is elevated.

While carbohydrates can be converted to fats via glucose and acetylCoA, the textbooks tell us that the conversion of pyruvate to acetylCoA is an irreversible reaction, so supposedly fats cannot be converted to significant amounts of glucose. (though insignificant amounts of glycerol can be converted to glucose.)

However in 2011 a German research group re-examined whether fatty acids can be converted to carbohydrate. They conducted a computational analysis of human biochemistry and identified 22 pathways by which acetone could be converted to pyruvate. Although these pathways are less efficient than producing glucose from amino acids or glycerol, they probably serve as supplementary methods of glucose production.

[ http://blog.cholesterol-and-health.com/2012/01/we-really-can-make-glucose-from-fatty.html#more ]

  • 5% of dietary glucose intake is converted to glycogen, 30-40% to fat and the remainder metabolized directly in muscle and other tissues.
  • A typical 70 kg male has total carbohydrate stores of about 2500 kcal (2% of total energy stores), equivalent to the energy required for a day of sedentary living. This exists as 400 g of muscle glycogen, 100g of hepatic glycogen and 20 g of ECF glucose.
  • In contrast ,112,000 kcal (80%) of energy is stored in body fat and 25,500 kcal (18%) as protein.
  • Resting and post-exercise muscle utilizes fatty acids for metabolism. At rest the brain uses 70-80% of the total glucose consumption with RBCs accounting for most of the remainder.

Three main hexoses:

  • Glucose – enters glycolysis
  • Galactose – from the disaccharide lactose (comprises glucose and galactose)
  • Fructose – metabolism is unaffected by insulin, so had been advocated as a suitable carbohydrate source for diabetics. However most fructose is simply metabolized by the liver and intestines. Readily converted into palmitic acid.

Lipid Metabolism

  • Triglycerides are the largest dietary fat source, comprising three fatty acids joined to a glycerol backbone.
  • Hydrolysis of triglycerides in the small intestine results in Free Fatty Acids & glycerol. Absorbed into enterocytes and packaged with cholesterol into chylomicrons, excreted into the lymph system, and subsequently reach the circulation via the thoracic duct and the left subclavian vein.
  • Glycerol is converted to glycerol-3P which enters the glycolytic pathway and can then be converted to glucose.
  • Beta-oxidation of fatty acids produces Acetyl-CoA.

Beta oxidation involves:

  1. Activation of the fatty acids in the cytosol through reactions with ATP and Coenzyme A.
  2. Carriage of FFA into mitochondria by carrier-protein ‘carnitine’.
  3. Beta oxidation in the mitochondrial matrix
  • AcetylCoA enters Krebs cycle, combing with OA to form citrate and subsequently release CO2 and H+.
  • Produces lots of energy, with 1 stearic acid molecule (-> 9 acetyl CoA) producing 146 ATPs.
  • In the liver only some of the acetylCoA is used in Krebs, with a large proportion:
    • 2 AcetylCoA molecules condense to form acetoacetate
    • Some acetoacetate -> ß-hydroxybutyrate
    • and then a very small amount ß- hydroxybutyrate -> acetone.
  • Acetoacetate, beta-hydroxybutyrate and acetone are ketone bodies.
  • These ketone bodies are transported in the blood to tissues,where the reverse reaction occurs, releasing AcetylCoA.
  • Oxaloacetate is required to bind AcetylCoA for its use in the Krebs Cycle, which becomes deficient if carbohydrate metabolism ceases (as occurs in a big switch to FFA metabolism), hence causing ketosis due to excess acetyl-CoA condenses to acetoacetyl-CoA.

Ketosis occurs in three conditions:

  1. Fasting or Starvation
  2. Diabetes mellitus
  3. High-fat/protein low-carbohydrate diets

Fatty acids can be readily used as an energy source by most body tissues, except: the brain, erythrocytes and the adrenal medulla.

Protein Metabolism

  • Proteins are chains of amino acids linked by peptide bonds between amino and carboxyl groups. There is much variation and complexity in different protein structures.
  • Most proteins are digested in the gastrointestinal tract and absorbed as amino acids. These amino acids, combined with those resulting from turnover of endogenous body protein, are termed the amino acid pool.
  • Interconversion between amino acids and products of fat and carbohydrate metabolism at the level of the common metabolic pool revolve around amino group transfer (transamination), removal (deamination) or formation.

 

Part 3 – Fasting, Starvation and Re-Feeding

Effects of insulin on cellular metabolism
  • Increases glucose uptake in muscle and adipose tissue (about 67% of body cells).
  • Increased glycogen synthesis.
  • Increased fatty acid synthesis, stimulating fat cells uptake of lipids.
  • Increased esterification of fatty acids, stimulating adipose tissue to make fats.
  • Increases amino acid uptake, DNA replication and protein synthesis.
  • Decreased proteolysis, hence “protein sparing” effect of glucose.
  • Decreased lipolysis by inhibiting hormone sensitive lipase.
  • Decreased gluconeogenesis.
  • Decreased autophagy (degradation of damaged organelles).
  • Increased potassium uptake.
  • Decreased arterial muscle tone, increasing blood flow, especially in micro-arterioles.
  • Increased acid secretion by gastric Parietal cells.
  • Increased cholesterol production by stimulating HMG-CoA reductase.

Vitamins and trace elements

  • Trace elements are elements found in tissues in minute amounts but necessary for normal body function.
  • Vitamins are any organic dietary component necessary for health and growth that does not function to supply energy.
  • Fat soluble vitamins: A, D, E, K

Total Parental Nutrition

TPN is a way of providing metabolic intermediaries intravenously by bypassing the GIT. It is generally only beneficial when there are long-term digestive and/or absorptive problems with the gut. TPN contains macronutrients, water, electrolytes, minerals, vitamins and insulin.

Start by calculating daily caloric needs, and work from there:

  • Males 25-30 kc/kg
  • Females 20-25 kc/kg
  • + 10-30% for major trauma & sepsis
  • + 50-100% for major burns

Add in daily H2O requirements: 30 mL/kg/day

Macronutrient contributions are typically:

  • Carbohydrates 7 g/kg/d
  • Protein 2.5 g/kg/d
  • Fat 2 g/kg/d

Electrolyte requirements need also be considered:

  • Na+ 1-2 mmol/kg/d
  • K+ 0.7-1 mmol/kg/d
  • Ca2+ 0.1 mmol/kg/d
  • Mg2+ 0.1 mmol/kg/d
  • PO4 0.4 mmol/kg/d

Additional trace elements and vitamins:

  • Iron 1 mg
  • Vitamin k 1 mg
  • Trace elements 5 mL
  • Cernevit 5 mL
  • Insulin (adjust to maintain BSL)

TPN is an imperfect science, so close monitoring of BSL and electrolytes is mandatory!

Fasting

  • “The metabolic state achieved after complete digestion and absorption of a meal.”
  • The body readily copes with short periods of fasting, such as for pre-surgery fasting. In fact, with only 6 hours of pre-op fasting many patients have probably not entered a metabolic fast.
  • Initial consequences of short fast:
    • Water depletion – roughly 125 mL/hour for 70kg adult.
    • Energy depletion – body glycogen depleted within 24-48 h.
    • Glucagon-induced naturesis contributes to initial weight loss.

Consequences of starvation

  • A diet that is both protein and calorie inadequate will lead to fat and protein catabolism.
  • Brain moves to fat-derived keto acids as an alternative energy source, this has a sparing effect on the Branch Chain Amino Acids (BCAA: leucine, isoleucine and valine) which are important constituents of muscle protein.
  • Most of the protein catabolized during starvation comes from the liver, spleen and muscle, with little from the brain or heart.
  • BSL drops after depletion of glycogen stores, but hypoglycemia is avoided due to hepatic gluconeogenesis.
  • When fat stores (neutral fat, not structural lipid) are consumed, protein catabolism increases and death follows. In young, fit and healthy hunger strikers death has been observed 40-70 days after starting fast (data from Irish Republican hunger strikers in Maze Prison, Belfast 1981), assuming access to water is maintained.
  • Orthostatic hypotension common by day 20. Electrolyte disturbances are also common, particularly hyponatremia and hypokalemia.
  • It is unusual for hunger strikers to continue past 30-40 days because of the severity of symptoms.
  • Death is usually from myocardial infarct or multi-organ failure. The critical point appears to be reaching a BMI of 12.5 kg/m2.
  • Surprisingly, experimental evidence suggests lean subjects loose body weight faster than the obese.
    http://www.ncbi.nlm.nih.gov/pubmed/9353494
  • A lean 70 kg male has 100g of glycogen in his liver and 400g in his muscles, compared with 12 kg of fat.

Three Stages of Starvation 

1. Glycogen depletion – 24-48 hours

2. Protein catabolism – 10-14 days

  • Protein catabolized from skeletal muscle, liver & spleen over 10-14 days depending on how fat adapted the individual is. Brain & heart relatively protected.

3. Fat metabolism – 14 days onward

  • Fat metabolism spares further protein loss / only about 10% of energy source at this time. Once fat stores depleted death quickly follows.

Re-feeding Syndrome 

  • May be at risk after as little as 5 days of fasting;
 significant risk after 3 weeks.
  • Features:
    • Fluid and electrolyte disorders.
    • Cardiac – tachyarrhythmias (most common cause of death); cardiac failure.
    • Neurological – Wernicke’s encephalopathy; confusion; seizures.
  • May be precipitated by a fall in glucagon and rise in insulin in response to food intake, in particular carbohydrate intake.
  • This leads to fluid and electrolyte shifts, increased BMR and increased oxygen consumption. Changes not well tolerated by a cardiorespiratory system already stressed by starvation.
  • To avoid, re-feed with small amounts of low protein, low refined-carbohydrate foods, eg. boiled vegetables. Milk also a food of choice in early re-feeding.
  • Vitamin supplementation: thiamine, vitamin B, multivitamin.
  • Electrolyte replacement as needed: potassium, phosphate and magnesium.

 


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