W30 Drug metabolism and NMR Analysis

Question 1

Lactulose (1,4 β-galactoside-fructose)
Used to treat what?

Answer 1

• Used to treat Portal-Systemic Encephalopathy (PSE) / Hepatic Encephalopathy (HE) and constipation
• Synthetic disaccharide derivative of lactose
• Consists of a Galactose and a Fructose ring
• Can be broken down into organic acids such as: lactic acid, formic acid, acetic acid
• Can also be broken down to release methane and hydrogen

Question 2

Lactulose – Mechanism of Action?

Answer 2

• Site of action is the large intestine as the small intestine does not have the enzymes needed to break down
• Saccharolytic bacteria break down the lactulose into organic acids, hydrogen, and other metabolites (increase in intraluminal gas)
• Ammonia, NH3 (weak base) diffuses into the acidic colon and is ionised into ammonium ions (NH4+)
• Ammonium ions cannot cross the biological membranes back into the blood and so must be excreted out of the system

Question 3

Lactulose – Mechanism of Action:
The Result:

Answer 3

• Increased pressure from gasses
• Osmolality
• Reduction of ammonia (accumulation
  of nitrogenous waste products is a principle feature of PSE)
• Need for NH4+ to be excrete

This all results in a general laxative effect and reduction of harmful nitrogenous waste products

Question 4

Vitamin K

Answer 4

• A group of fat-soluble compounds (K1, K2, K3)
• Deficiency often results from a decrease in intestinal absorption of fats, due to a decrease in bile secretion (liver disease, obstruction of bile ducts)
• Involved in blood clotting with calcium, so deficiency results in bleeding, bruising,
  hematomas, cartilage calcification, deposition of insoluble calcium salts in arteries
• This process involves Vit K acting as a cofactor for the enzyme γ-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid (Glu)

Question 5

Vitamin K - Availability
What is the mechanism to recycle vit K called?

Answer 5

• Although Vitamin K is fat soluble, the
  body stores very small amounts
• These stores are rapidly depleted
  without regular dietary intake
• The body therefore has a mechanism
  to recycle Vitamin K – The Vitamin K
  – Epoxide Cycle
• This cycle allows for a small amount
  of Vit K to be reused many times for
  protein carboxylation

Question 6

The Vitamin K – Epoxide Cycle
What are the steps? (2)

Answer 6

• The reduced form of Vit K donates a pair of electrons to the γ-glutamylcarboxylase which carboxylates glutamic acid residues
• The oxidised form of the Vit K(epoxide) is then converted back to hydroquinone in a two-step reaction
• 1st Step:** Conversion of epoxide to
  Vitamin K, catalysed by Vitamin K-epoxide reductase (VKOR)**
• 2nd Step: Reduction of Vit K to Vit K
  hydroquinone

Question 7

Vitamin K – Which form is present?

Answer 7

• As mentioned, Vit K exists in three common forms
• NMR is one technique that can be used to determine which compound, K1, K2, or K3 that you have
• Let’s revisit some principles of NMR

Question 8

Why is there a difference between
where absorbance happens?

Answer 8

Shielding.
Less shielded protons absorb at lower field

Question 9

What is Shielding?

Answer 9

• Magnetic forcefield exerted by electrons: electrons are circulating and generating a small induced magnetic field that opposes the external field (B0)
• Decrease in the magnetic field felt by the nucleus: this results in a stronger field being needed for resonance to occur
• Electronegative atoms such as oxygen pull electrons away from neighbouring atoms, deshielding them
• Shielded atoms need higher field energy to cause resonance

Question 10

Chemical Shift

Answer 10

• The difference in magnetic field strength between the resonance field of the proton
  being observed and that of tetramethylsilane (TMS) measured in parts-per-million (ppm)
• Si is less electronegative than C, so CH3 groups are relatively electron rich and so H
  well shielded (absorb at higher field strength)
• Therefore, most NMR signals appear downfield (to the left, more deshielded) of
  TMS (note: all 12 H in TMS absorb exactly the same so give one strong signal)
• The distance a proton resonates at in comparison to TMS is known as the Chemical Shift

Question 11

Chemical Shift

Answer 11

• The Chemical Shift (in ppm) of a given proton is the same regardless of the operating field and frequency of the spectrometer 1450 Hz
  Chemical shift ppm = shift downfield from TMS (in Hz)/ Spectrometer frequency (in MHz)

Question 12

Chemical Shift - Variations

Answer 12

1. Electronegativity (deshielding effect)
   With increasing electronegativity, there is more deshielding of neighbouring
   atoms as the electrons are drawn towards the more electronegative atom.
2. Distance from Electron-Withdrawing Group
   The further away an atom is from the electronegative group, the less deshielding
   effect is felt by the protons of that atom.
3. Addition of More Electron-Withdrawing Groups
   If more than one EWG is present, the deshielding effects are nearly (but not quite) additive.

Question 13

Number of Signals

Answer 13

The number of NMR Signals present represent the number of proton environments
Protons in identical chemical environments have the same chemical shift – Chemically
Equivalent

Question 14

Integration: Areas of the Peaks

Answer 14

The area under the peak is proportional to the number of protons in that environment
The integral trace cannot specify the number of protons but instead shows the ratio