MSAP Renal Biochemistry Flashcards
How hydrogen bonds function in the structure of water
Permanent dipole where H forms a bond with O and forms polar covalent bond
When two H2O molecules interact with once another it sets up a weak attractive force called Hydrogen bonding (H-bonding is relatively strong among weak attractive forces )
If each H20 molecule forms 4 unchanging hydrogen bonds we have ice
- In liquid water there are about 3.5 hydrogen bonds between each H2O (constantly changing, water molecules tumbling against each other)
When two H2O molecules bind together forces being pulled towards to Oxygen (net dipole)
Low frequency can form hydronium ion (H3O+)+Hydroxide ion (OH-)
Water as a solvent
H-bonds allow H2O to dissolve polar substances (NaCl)
pH and pOH
pH= a way to describe the concentration of H+ in water solution
pH scale:
1M HCL=pH 0 (acidic) (more H+, less OH-)
1M NaOH= pH 14 (alkaline) (less H+, more OH-)
pH of 7 is neutral
Auto-ionization of water
pkw=pH +pOH
w(constant) describes the equilibrium of water dissociation
In auto-ionization the concentration of H3O+ is 10-7; under conditions the concentration of H3O+ has to be the same for hydroxide (OH-)
Multiply them together and you get 10^-14
Concentration of hydronium (H+) is 10^-7 in pure water; creates pH of 7
Strong Acids
HCL- Hydrogen chloride
HBR- hydrogen bromide
HI- hydrogen iodide
HNO3- nitric acid
HCLO4 – Perchronic acid
H2SO4 (only first dissociation considered strong)
Strong Bases
LiOH- lithium hydrozide
NaOH- sodium hydroxide
KOH- potassium hydroxide
Ba(OH)2- Barium hydroxide
Mg(OH)2- magnesium hydroxide
Calculate the pH of a strong acid (strong base) solutions if given the concentration (integer values)
[H+] is approximately equal to the molarity of a strong acid
strong acids dissociate completely in water
HA–> [H+] + [A-]
Questions: What is the pH of a 0.001M HCL?
ph=-log[H3O+]
ph= -log [H+]
-log (10^-3) = 3
Titration curve of a strong base (and vice versa)
DRAW OUT TIRATION CURVE ON WHITEBOARD
Weak acids and bases
Weak acids do not completely dissociate (H2O)
- Most acids and bases in the body are weak
The higher the Ka…..
The stronger the acid
High Ka= more dissociation occured
The lower the pKa……
The lower the pKa the stronger the acid
smaller pKa=stronger acid
Titration of a weak acid by a strong base
DRAW OUT THE TITRATION CURVE
What is the equivalence point?
Equivalance poit occurs when an amount of acid is mixed with an equal amount of base (salt water at pH=7)
pKa and how it relates to the ka
On a titration curve the pKa is the 1/2 equivalence point
when the pH=pKa of the weak acid there is equivalent amounts of the weak acid and its conjugate base (50% dissociated)
Acid strength refers to the extent of the proton dissociated and is measured by the value pKa (or Ka)
pKa=-logKa (lower pKa stronger acid)
If Ka is very large= strong acid= HA is almost completely dissociated in water (sHA)
pKa of a wHA (weak acid) will be LARGER than the pKa of a strong acid
Biological danger of a weak acid
A wHA may be in food while dilute (think vinegar in salad dressing), but the same wHA might be toxic if concentration
-Example: Glacial acetic acid
Danger is not always related to acidity strength
Example: HF is a weak acid
- HF is toxic (and a weak acid)
- HF is toxic (and dangerous) at any concentration
Buffer
When a weak acid and a similar amount of its conjugate base are mixed, a buffer is form; resist a change in pH
Buffer region in weak acid (titration curve)
Buffering region on the curve is where pH changes slowly as H+ or OH- is added
POINT THIS OUT ON DRAW OUT TITRATION CURVE
Henderson Hasselbach equation
pH= pKa + log {[A-]/ [HA]}
A-= base form
HA= acid form
What happens to Henderson Hasselbach equation when the concentration of weak acid is equal to the concentration of its conjugate base?
Ex. If [A-]=0.1M and [HA]=0.1M
pH=pKa + log{[A]/[HA]}
pH=pKa + log (1)
pH=pKa +0
pH=pKa
Major biochemical buffers in mammals
CO2, Proteins, phosphate
By regulating the amount of CO2 dissolved, we can regulate our physiological pH
- Phosphate as H3PO4- can act as a physiological buffer
side chain of histidine has a physiologicaly useful pKa
Acid base chemistry of ammonia
Ammonia has pKa about 9.3
NH3–> NH4+ +OH-
At Physiological pH there is a 100 fold more NH4+ than NH3
Too much ammonia is toxic to humans; changed to urea (not charged, no acid base activity, carries x2 nitrogen atom)
3 reactions that may fix free ammonia in mammals
- Glutamate dehydrogenase
Glutamate dehydrogenase freely reversible reaction that liberates or incorporates free NH3
If glutamate is formed it is called reductive amination
- Glutamine synthetase
Amino acid glutamate uses free ammonia and ATP (donates phosphate to create a high energy intermediate, coupled reaction allow endergonic RXN to progress) to form glutamine (donates amino groups for many biosynthetic reactions)
Scavenger of ammonia in peripheral tissue (everything except for the liver)
- Carbamoyl phosphate synthetase 1 (CPS-1)
First reaction of the Urea cycle
Able to take free ammonia and CO2 to make organic molecule called Carbomyl-P (Phosphate)
Main reactions that release ammonia in mammals
Glutamate dehydrogenase (fix or release free ammonia):
In the liver ammonia formation (and alpha KG) is the predominate direction of this reversible reaction b/x the ammonia may be delivered to the urea cycle for non-toxic disposal
Glutaminase
Releases free ammonia (NH3) from glutamine (carrier of two nitrogens)
What happens to ammonia in the liver?
IF in the liver NH3 eneters the urea cycle (non-toxic disposal)
What happens to ammonia in the kidney?
If in the kidney some NH3 may be excreted in urine (neutralize metabolic acids, buffer urinary pH)
What happens to free ammonia after it is releaesd?
As free ammonia acids are released they must be detoxified: NH3 –>Converted to urea in the liver–>urea released to blood–>carried to the kidney and filtered–> excreted in the urine
Function Glutimase Enzyme
Low blood pH then kidney can activate Glutaminase which releases free ammoniaà ammonia converted to ammonium which is excreted to urine (carries H+ ion)
Response to metabolic acidosis
Acid base affect
Transamination
End fate of NH4+–>the urea cycle
Most amino acids in the amino group are transferred to glutamate (in the liver)
Idea: Collection of the N group into glutamate
Reaction: transamination with alpha-KG
Results in: Formation of glutamate with alpha keto acid
After transamination what happens to keto acids?
Keto acids are shunted off; always being converted into glucose or ketone bodies for energy purposes
After transamination what happens to glutamate?
Glutamate is being always being deaminated by glutamate dehydrogenase to release ammonia so it can be converted into urea
Aminotransferase
- _Aminotransferase i_s always going towards the formation of glutamate and responding keto acid
How many NH3 are released from a) glutamate and b) glutamine
Glutamate: 1
Glutamine: 2
Glutamine
NH3 collected in glutamine delivered to the liver
- First- Glutaminase (GLN–> GLN + NH3)(1st ammonia released)
- Then- glutamate dehydrogenase (GLT–>alpha KG+ NH3) (2nd ammonia released)
- Oxidative deamination in liver
- Regeneration of alpha KG
The urea cycle
NH3 is detoxified through the urea cycle
How is the urea cycle useful during the fasting state?
Very active during the fasting state
Body protein degraded
Amino acids flood into the blood
Amino acids go to the liver
NH3 is removed (forms keto acids; keto acid carbon skeletons are converted into glucose or keto bodies which help support the energy needs of the body)
Where do the first two steps of the urea cycle take place?
First two take place in mitochondrial matrix
Where do the last three steps of the urea cycle take place?
Remaining three reactions occur in the cytosol
First Step of the Urea Cycle
- Formation of carbamoyl phosphate in the mitochondria by CPS1 (Ammonia and Aqueous carbon dioxide, and 2ATPà Carbamoyl phosphate)
- CPS1 is a mitochondrial reaction
- Rate limiting (and regulated) step of urea cycle
- One of three mammalian enzymes that can “fix “ ammonia into organic compound
2nd step of the urea cycle
Omithinin transcarbamoylase (OTC)
- Formation of citrulline from ORN and Carb-P
- Mitochrondrial reaction
- Citrulline is transported out of the mitochondria
- ORN and CIT are both amino acids
3rd step of the urea cycle
Angiosuccinate synthtase
- Aspartate reacts with Citrulline
- Cytoplasmic reaction
- Consumes x2 phosphoanhydride bonds
- AMP is release
- 2nd nitrogen released that will eventually go towards the formation of urea in the cycle
4th step of the urea cycle
Argiosuccinate lyase (cytoplasmic)
- Cleaves argiosuccinate into fumarate and arginine
- Arginine is the final compound in the urea cycle
- Represents the biosynthetic pathway for arginine
- Fumarate can be reused by urea cycle and can be converted to oxaloacetate (OAA) (carbon skeleton of aspartate) ; occurs in the TCA cycle
- OAA may be transaminate to aspartate (glutamate donor)
5th step of the urea cycle
Arginase
- Cleaves arginine to release urea and reform ornithine; transferred back to the urea cycle (mitochondria)
- Creates urea
- Urea diffuses to the blood and is excreted in the urine
Why is aspartate aminotransferase an exception to the rule?
Aspartate aminotransferase in the exception to the rule that the purpose of aminotransferase reactions is the net formation of GLU (glutamate transfers amino group to OAA which forms aspartate and reform alpha KG) (aspartate is eventual 2nd nitrogen into the urea cycle)
Sources of Nitrogen for Urea Cycle
1st nitrogen enters as free ammonia; formation of Carbamoyl phosphate by CPS1 is the regulated step of the urea cycle (mitochondrial reaction)
-2nd Nitrogen enters as aspartate (reaction in cytoplasm)
Whay does ATP hydrolysis ensure?
ATP hydrolysis ensures that the cycle only turns in one direction
How is the first step of the urea cycle regulated
Feed forward mechanism to regulate the urea cycle; dependent on glutamate and arginine concentration
- Allosteric regulation of CPS1 by N-Acetylglutamate
- Formation of N-Acetylglutamate is dependent on: 1) Glutamate and 2) Arginine
3 mechanisms of the positive regulation of the urea cycle
- Regulation of CPS1 by N-Acetylglutamate
- Synthesis of N-acetylglutamate dependent on [Glu]
- Synthesis of N-acetylglutamate by ARG
- Note that ARG in produced by the urea cycle
- Regulation by substrate []
* Rate of NH4+ production - Induction (synthesis) of the cycle enzymes with increase in protein metabolism (genetic regulation)
Liver has a huge capacity to dispose of NH4+
Net reaction of the urea cycle
1st N of urea from – NH4+
2nd N of urea from aspartate
- Glutamate- important to mediate the supply of both
- Consumption of ATP ensures that cycle does not turn backwards
Why is ammonia toxic to human?
Alters balance of NT in the brain
Osmotic effect
Deleterious effect on the mitochondria (reduces TCA cycle efficiency)
Alpha ketoglutarate is converted to glutamate:
- Results in decreased TCA cycle activity
- Reduced ATP synthesis (Brain needs ATP)
Glutamate converted to glutamine:
- Results in high circulating glutamine levels
Reduced GABA and glutamate levels, and increased levels of glutamine in the brain
Humans cannot release all nitrogen as ammonia
Acid base effect
Ammonium ion takes many water molecules to stay hydrated in solution (would incur a large obligate water loss)
Rmoval of amino group from amino acid
Idea: Collection of the N group into glutamate
Reaction: transamination with alpha-KG
Results in: Formation of glutamate with alpha keto acid
Genetic Disorders of the Urea Cycle and Liver Disease may lead to hyperammonemia
Inborn metabolic deficiencies of any of the 5 pathway enzymes and its regulatory (NAG synthase)
- Heredity
- All result in hyperammonemia
Secondary hyperammonemia (acquired)
- Liver disease e.g. hepatitis, hepatotoxins, cirrhosis
- Leads to an increase in blood [NH3]
Ammonia intoxication symptoms: vomiting, irritability, mental retardation, lethargy, blurred vision
