biochemistry week 11 , 12, and 13

what are amino acids?

* amino acids contain C, H, N, and O
* the carbons, hydrogens, and oxygens can be metabolized for energy when there is an excess protein or not enough carbohydrates

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ammonium

* toxic (especially to neural tissues) and must be removed from the body

transamination

* transfers nitrogen from one molecule to another
* this cann allow tissues to use the carbon skeleton of amino acid for something else (i.e energy metbolism)
* allows our body to produce some of the non-essential amino acids
* PLP, pyridocxal phosphate, is the coenzyme required for these reactions and is the active form form of vitamin B6

deamination

* removes a nitrogen from a molecule but generates nitrogenous waste
* can allows tissues to use the carbon skeleton of amino acid for something else (energy metabolism or stored as fat)
* NH4 is toxic to our bodies and must and must be removed
* this is reversible
* if there is too much NH4 around, it will go in reverse using up all the a-ketoglutarate in the mitochondria
* this prevents the TCA cycle from functioning and results in a lack of energy for the cell

what converts ammonia to urea?

the liver

nitrogen exceretion

* nitrogen must be converted into urea in the liver to be excreted in the urine by the kidneys
* because ammonium is toxic, it can’t freely travel in the blood
* to transport in the blood, nitrogen is packaged as the amino acid alanine, which is non-toxic and polar, meaning it can freely travel in the blood
* in the liver, alanine is broken down, releasing ammonium, which is packaged into urea for excretion

alanine to pyruvate

* in the liver
* alanine transaminates to glutamate
* goes to pyruvate for energy
* glutamate demaninates NH4→ urea

is the amount of amount of nitrogen in the protein taken is more, less, or equal to what is excreted as urea

equal

what happens to muscle protein during fasting?

* muscle protein is broken down to produce energy and maintain blood glucose levels, generating nitrogenous waste
* urea is high while fasting (short term)
* it starts to decrease with long-term fasting

what do high levels of ammonia indicate?

problem with liver

what do high levels of urea indicate?

problems with kidneys

urea cycle

* multistep cycle that takes place in the liver
* any issues with any of these steps cause cause detrimental consequences to homeostasis
* ammonia can build up in the blood

what happens if a healthy individual goes on a low carb, high protein diet?

the liver expresses more urea cycle enzymes

reactive oxygen species

* oxygen can be toxic
* oxygen has 2 single electrons in two different orbitals, both with the same spin, it is a biradical
* these 2 electrons can’t easily oxidize with the bonds in organic compounds, but because of spin restriction- an enzyme is usually required
* if oxygen gets an extra electron, it gets VERY reactive
* radicals are atoms the have one unpaired electron in their outer valence shell

free radicals

* free radicals are those that can exist independently of an enzyme
* radicals can be made in an enzymatic reaction trapped in the enzyme's active site and not able to react with anything else in its surrounding
* can take electrons from other compounds and initiate chain reactions that damage substances within the cell
* generated naturally by cellular processes

sources of ROS: ETC

* the mitochondrial ETC produces ATP to run cellular processes
* this works by transferring electrons between carriers and complexes until it eventually reaches oxygen
* occasionally an electron can escape forming superoxide

source of ROS: ionizing radiation

* xrays and radioactive chemicals
* UV radiation can also cause ROS to form in the skin

sources of ROS: drugs metabolism

* cytochrome P450 enzymes metabolize drugs (and alcohol) and chemical toxins
* the job of these enzymes is to oxidize the drugs and other substrates to make them more soluble for excretion
* while most enzymes trap radicals in their active site, these enzymes can be leaky
* radical intermediates of these enzyme reactions can escape and become free radicalss, causing cell damage

sources of ROS: inflammation

* free radicals are used to destroy invading pathogens or clean up dead cells in damaged tissues
* in activated neutrophils, the respiratory burst consumes oxygen to create the reactive substances that will kill phagocytosed bacteria
* the release of these free radicals in areas of inflammation can lead to damage in surrounding tissues

reactive nitrogen-oxygen species (RNOS)

* these are free radicals as well and can cause damage to cellular components: DNA, cell membranes, enzymes, the ETC, etc.
* produced in the cell or can be from the outside sources and they ae damaging to the cells

ROS damage

* they trigger a chain reaction that produce more radicals and cause more damage
* the free radicals pull an electron from a molecule turning that molecule into a radical, which in turn pulls another electron from another molecule, turning that into a radical…

DNA damage

* can be damaged by ROS due to strand breaks or 1 of 20 different alterations that can lead to mutations
* one such alteration is the oxidation of a guanine to 8-hydroxyguanine
* DNA repair systems can fix some of these alterations but if they are missed, mutations accumulate

antioxidant scavenging molecules and enzymes

* endogenous antioxidants such as superoxide dismutases and catalases have different isoforms that exist in different parts of the cells

why are minerals required in our diets?

* they contain metals that enzymes use

free radical scavengers

* vitamins such as the fat-soluble vitamin E and the water soluble vitamin C are involved in neutralizing free radicals
* carotenoids, derived from the diet have antioxidant properties as well

what is an enzyme that could prevent some DNA damage?

* superoxide dismutase (SOD)

non-communicable diseases

mainly cancer, cardiovascular disease, chronic respiratory diseases, and diabetes

recommendations on alcohol consumption

* less is better
* risks outweigh any benefits
* male vs female have differences in metabolism
* standard drinks = 0.6 oz of ethanol

alcohol and the CNS

* alcohol is a psychoactive agent and increasing amounts start to impair judgment (frontal lobe)
* next, the speech and vision centres are affected
* hand eye coordination and voluntary muscle function are then impaired
* finally, the heart is affected as is respiration, the individual usually passes out at this point

alcohol metabolism: ADH and ALDH systems

* ethanol is metabolized mainly by the alcohol dehydrogenase (ADH) enzyme in the cytosol of the liver
* acetaldehyde is the product of alcohol metabolism and is toxic
* acetaldehyde dehydrogenase (ADH, in the mitochondria) converts this toxin to non-toxic acetate which can be used by muscle and other tissues as fuel (it is converted to acetyl CoA
* alcohol metabolism can produce up to 5 NADHs and 1 FADH2
* this can generate 17 ATPs in the ETC
* 2 ATPs are used up to make acetyl CoA, so there is a net gain of 15 ATP

Alcohol metabolism: MEOS system

* when the ADH and the ALDH system is overwhelmed the MEOS system turns on to compensate
* the microsomal ethanol oxidizing system is part of the cytochrome P450
* these enzymes are embedded in the membrane of the ER and are inducible
* this is the system that is used when there are higher levels of alcohol consumed
* free radicals are generated form MEOS causing oxidative stress and can interfere with drug metabolism

alcohol and Tylenol

* acetaminophen is normally metabolized in the liver and excreted in the kidney
* however, when someone consumes alcohol, the cytochrome P450 enzyme tat is part of MEOS gets expressed at a high level
* this enzyme can turn acetaminophen into a toxic metabolite called NAPQI
* many medications are metabolized by cytochrome P450 enzymes before being excreted
* alcohol can compete with the medication for cytochrome P450 enzymes, decreasing its metabolism and excretion- effectively increasing its dose (ie. overdose when mixed with alcohol)

what happens if someone has an alcohol addiction?

the CYP enzymes may always be expressed, changing the way they metabolize drugs

alcohol and energy metabolism

* the conversion of alcohol to acetate generates plenty NADH molecules, impact the ratio of NADH/NAD+
* the increased levels of NADH signal to the cell that there is a surplus of energy, which causes energy metabolism to shut down and switch to energy storage
* the lack of NAD+ causes a shift in metabolism, leading pyruvate to be converted to lactate to regenerate NAD+

binge drinking and fatty liver

* binge drinking is defined as 4 drinks in one sitting by a woman or 5 drinks in one sitting for a man
* fat deposits can be seen in the liver after one episode of binge drinking

which metabolic systems will be involved to break down alcohol ingested?

ADH and MEOS

how does ethanol metabolism affect drug metabolism?

* drug metabolized by CYP enzymes are interfered with

progression of liver damage

* alcohol hepatitis and fatty liver (called steatosis) are the first steps in liver damage and are reversible
* progression occurs to fibrosis and finally cirrhosis with continued drinking, which is not reversible
* the liver is responsible for the production of many blood proteins, including albumin, mineral transporting proteins (transferring) and the proteins responsible for blood coagulation, liver failure is the usual cause of death

catabolism (breakdown)

* releases energy and CO2, which must be removed
* breakdown of proteins produces nitrogen which must be disposed of

anabolism (build up)

eg. protein translation from individual amino acids

metabolism

* many processes occur between digestion and either the storage or use of dietary molecules
* tissues may be specialized for either
* storage (adipose tissue)
* anabolism (liver)
* waster removal (kidney)
* nutrients and wastes must circulate in the blood to reach these tissues

cellular respiration

* in the presence of oxygen, nutrients are broken down to form carbon dioxide, water, and ATP

Circulation and waste removal

* products of digestion enter the blood stream
* water-soluble molecules go to the liver directly via the portal vein before entering general circulation
* fat-soluble go to the blood directly via the lymphatic system
* unabsorbed molecules are excreted from the GI tract
* nutrients, hormones and waste products are transported through the body by the circulatory system
* the respiratory system disposes of CO2
* the urinary system eliminated nitrogenous wastes

urea production

* nitrogenous wastes arise from the catabolism of amino acids
* the carbon skeleton of amino acids can be used as energy immediately or stored
* the nitrogen must be removed because it is toxic
* the liver packages the nitrogen as urea which can be safely transported in the blood

kidney function

* water, minerals and metabolic wastes (urea) are excreted by the kidney, through the bladder
* the kidney controls excretion to maintain homeostasis
* the RAAS is a critical regulator of blood volume and systemic vascular resistance
* the kidney also participates in the maintenance of pH homeostasis
* the kidney can remove bicarb and hydrogen ions from the blood, directly impacting blood pH

maintaining the energy supply

* the cells of the body need a constant supply of energy
* this source of this energy can be from the diet or body storage
* metabolism shifts depending on how long it has been since a meal and what fuel sources are available
* the two different states are called “fed” or “fasted” state

what is the most critical fuel source in the body?

glucose

* primary energy source for the brain and RBC
* glucose levels in blood must remain almost constant to keep body processes functioning by supplying neergy
* even during a prolonged fasted state, blood glucose levels do not fall below 3.3mmol/L

how does a human body maintain the energy supply and where does all this glucose come from?

insulin and glucagon

Fed state

* after a meal
* lasts until absorption of food is complete
* lasts about 1-2h
* as glucose enters the blood from the GI tract the concentration of glucose in the blood increases
* the glucose can enter many cells including the pancreas

steps in fed state

1. blood glucose levels rise signaling the pancreas to increase= insulin and decrease- glucagon
2. chylomicrons are the source of lipoproteins from diet, formed in the intestines
3. amino acids are used by the liver to make blood and liver proteins
4. blood glucose levels rise signaling the pancreas to increase= insulin and decrease- glucagon
5. the liver is first to receive blood from the GI tract. in the liver glucose is: used for immediate energy needs, replenishes glycogen stores, excessing is converted to triglycerides
6. ^
7. the liver forms VLDL for transport through the blood. The triglycerides in VLDL are from excess carbohydrate and protein that ha been converted for storage
8. glucose in the blood is delivered to al other tissues. all cells can use it as fuel. Adipose and muscle will only use glucose when insulin is present
9. ^
10. ^


13. tissues can use these fatty acids for energy or adipose tissue can store the excess
14. free amino acids enter the amino acid pool and can be used by all tissues to make protein or converted into other nitrogen-containing molecules. excess protein can be converted into glucose for glycogen storage or converted to fatty acids and stored as triglycerides if glycogen stores are full

glycogen

* stored form of glucose in humans
* consists of chains of glucose molecules
* liver cells store glucose as glycogen to help regulate blood glucose levels
* muscle cells store glucose as glycogen for use as energy during activity and muscle contraction

what are the two main places for storage of glycogen?

liver and muscle cells

glycogen synthesis

uses excess glucose-6-P to form glycogen chains

glycogenolysis

breaks off these glucose molecules, regenerating glucose-6_p which can reenter glycolysis

fasted state

* begins about 2 hours after a meal
* blood glucose levels have returned to fasting levels
* the fasted state continues until food is consumed again and a new fed state begins
* the return of blood glucose to baseline signals the pancreas to decrease insulin secretion and glucagon secretion increases

fasted state steps

1. this signals the pancreas to decrease insulin secretion and glucagon secretion increases
2. the liver responds to these hormonal signals to control blood glucose levels. Glucagon signals the need for glycogenolysis so blood levels do not fall too low
3. for brain and neural tissue, glucose is the preferred fuel substrate
4. the RBC can only use glucose for fuel since they lack mitochondria. The livers role is crucial in maintaining blood glucose levels during fasting- it is the fuel control centre. The liver can produce glucose from glycogenolysis or as glycogen stores are depleted, new glucose can be made from alternative sources (gluconeogenesis)
5. during fasting the major source of fuel for many tissues is fatty acids. Adipose triglycerides are broken down (lipolysis), yielding glycerol for gluconeogenesis and free fatty acids- lipolysis is occurring at the same time as glycogenolysis
6. ^
7. muscle and many other tissues can use either fatty acids or ketone bodies for energy

9-12. the liver will use lactate, amino acids and glycerol to produce glucose, helping maintain blood sugar levels. This will also result in the production of urea (filtered by the kidney)

gluconeogenesis

* liver stores of glycogen are limited- this supply is exhausted first
* the use of non-carbohydrate sources to produce glucose
* reverse of glycolysis
* glycerol, lactate, and amino acids can be used as precursors for glucose
* this provides a steady source of glucose to maintain blood glucose levels
* however, amino acid breakdown will generate ammonia/urea
* kidneys help with this process

ketone bodies (keto acids)

* released from the liver for the break down of fatty acids
* they can be used as an alternative fuel source for some tissues during fasting
* they are converted to acetyl-CoA and used in the TCA cycle

starved state

* during a continued fast, the body must change its fuel metabolism
* blood glucose is critical
* gluconeogenesis relies on protein- this is being taken from muscle leading to muscle wasting
* muscle increases its use of fatty acids as fuel and decreases its use of ketone bodies (so they build up in the blood)
* the brain uses a greater percentage of ketone bodies for its energy needs
* this decreases the demand for glucose and gluconeogenesis slows (muscle is spared)
* urea builds up in the kidneys because they are not being broken down

starvation

* when about 40% of body weight is lost, death due to starvation occurs
* blood and urine levels of the different substances are used to determine the metabolic state of the individuals
* reflecting in the amount of fuel being used

change in fuel usage during a prolonged fast: muscle

change: decrease

fuel: use of ketone bodies

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change: decrease

fuel: protein degradation

change in fuel usage during a prolonged fast: brain

change: increase

fuel: use of ketone bodies

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change: decrease

fuel: use of glucose

change in fuel usage during a prolonged fast: liver

change: decrease

fuel: gluconeogenesis

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change: decrease

fuel: production of urea

urea excreation

* urea production is a good indication of what fuels are available for a person

investigation of a patient

* patient history
* physical examination
* laboratory tests
* imaging techniques

core lab facility

* found at virtually all hospitals
* operates 24/7
* provide the most essential and most requesting
* highly automated environment
* instruments with multi-analyte capabilities

special chemistry

* less frequently ordered tests
* labour intensive and often manual methods
* generally non-stat tests (result not required immediately)

point of care testing (POCT)

* instruments located outside of chemistry laboratory such as CCU, ER, ICU, or satellite centre (clinic)

electrolytes

sodium (Na), potassium (K), chloride (Cl)

blood gases

pO2, pCO2, pH, HCO3, oxygen saturation

endocrine

* thyroid hormones, prolactin, testosterone

lipids

total cholesterol. LDL, HDL, triglycerides

proteins

* total protein
* specific proteins such as albumin, immunoglobulins

tumour markers

* prostate specific antigen (PSA)

toxicology

* ethanol, methanol
* drug of abuse generally conducted as a screen

what is involved in a point of care testing ?

* tests are of urgent importance and results will affect the immediate management of the patient
* instruments are available that can perform certain tests at remote locations, such as at the bedside on in a clinical care unit
* blood glucose
* urinalysis
* blood gases
* electrolytes
* cardiac markers
* drug screens
* POC are early always more expensive than the same tests performed in the central laboratory
* many are immunoassy based
* can be qualitative or quantitative

why are laboratory tests ordered?

* diagnosis
* monitor progression of disease
* monitor effectiveness of treatment
* screening population for diseases
* to identify complication of treatment
* for predicating survivability, employability
* to check the accuracy of an unexpected data
* to conduct research
* to prevent malpractice
* for educating residents
* to assess nutritional status and health of a healthy individual
* responding to total uncertainty

the test

* measuring an analyte as a marker to distinguish health and disease
* ideal marker

what is the ideal marker?

* absolutely specific for a specific disease
* easily measurable
* quantity reflective of severity of disease
* early detection following onset of disease not affected by other biological disturbances

highly specific marker: troponin T

* marker of myocardial infraction (heart attack)
* found predominately in cardiac tissue
* released into the blood stream following cell death

non-specific marker: low blood pH (acidosis)

* very important to know but can be caused by a hosts of events
* drugs
* respiratory problems
* renal problems

whole blood

the red fluid that is drawn from our veins into a tube containing an anti-coagulant (plasm) or no (serum)

plasma

the protein-containing fluid portion of the blood, after centrifugation to remove red and white blood ells and platelets

serum

* the clear fluid left after blood is allowed to clot and then centrifuged- it is plasma minus the blood clotting proteins
* comprises 55% of the total volume of whole blood
* contains proteins, sugars, vitamins, minerals, lipids, and lipoproteins
* no clotting factors
* 95% of serum is water

what are the three layers of whole blood?

1. plasma (PRP)
* plasma is fluid components of blood
* comprises \~55% of total volume of whole blood
* contains proteins, sugars, vitamins, minerals, lipids, lipoproteins and clotting factors
* 95% of plasma is water
2. buffy coat
* WBC and platelets
3. hemtocrit
* RBC

what happens if blood is allowed to stand without anti-coagulants?

it will form an insoluble fibrin clot

clotted blood

* RBC
* clotting factors (fibrin, platelets, etc)

blood analysis

* source
* veins
* arteries
* skin puncture- capillary blood
* collection method
* syringe
* evacuated tube
* additives
* separator gel
* intravenous lines

collection tubes

* negative pressure facilitates collection
* easy to use
* sterile
* universally used colour-coded rubber stoppers to denote tube type
* tubes can contain various anticoagulants for the collection of whole blood or plasma
* tubes can have additives for specific tests (glucose, metals)

red top tubes

* contain no anticoagulants or preservatives
* used for collecting serum
* 10-15 min is required to allow blood to clot before centrifuging
* used for blood bank specimens and chemistry

gold and tiger top tubes

* contain a gel that forms a physical barrier between the serum and cells after centrifugation
* no other additives are present
* gel barrier may affect some lab tests

gray top tubes

* used for glucose measurement
* after blood collection, glucose concentration decreases significantly because of cellular metabolism
* contain either:
* sodium fluoride and potassium oxalate
* sodium iodoacetate
* both preservatives stabilize glucose in plasma by inhibiting enzymes of the glycolytic pathway (glycolysis)
* NaF/oxalate inhibits enolase
* iodoacetate inhibits glucose-3-phosphate dehydrogenase

green top tubes

* contain either the Na, K, or lithium salt of heparin
* most widely used anticoagulant for chemistry
* can affect the size of integrity of cellular blood components and is not recommended for hematology studies
* heparin accelerates the action of antithrombin III, which inhibits thrombin so blood does not clot (plasma)

what is the advantage of plasma in green top-tubes?

* there is no time wasted waiting for the specimen to clot

lavender-top tubes

* contain the k-salt of ethylenediaminetetraacetic acid (EDTA), which chelates calcium (essential for clot formation) and inhibits coagulation
* used for hematology and some chemistries
* cannot be used for K or Ca tests

blue top tubes

* contain sodium citrate, which chelates calcium and inhibits coagulation
* used for coagulation studies because it is easily reversible

brown and royal blue top tubes

* specially cleaned for trace metal studies
* brown top tubes are used for lead analysis
* royal blue top tubes are used for the other trace elements studied (acid washed)

clinical variations

* variations within an individual and between individuals
* how we see “normal”

analytical variations

* no test is perfect\]all tests have some degree of variations for repeated measurements of the same sample

factors affecting the final test result

* pre-analytically
* at the time of the test
* after the test is complete