1. Professor of Biochemistry Protein turn over, Nitrogen balance
Dr. Saidunnisa
Professor of Biochemistry
Protein turn over, Nitrogen balance

2. Learning objectives
At the end of session the student shall be able to:
Explain protein turnover and nitrogen balance.
Define nitrogen balance and factors effecting
Explain inter organ relationship of amino acids in fasting and fed state.
Define transamination and deamination, mechanism of action with enzymes and co enzymes involved with suitable examples and its clinical application and its limitations.
Clinical importance of Glucose –alanine cycle.

3. WHAT IS PROTEIN? Proteins are a sequence of amino acids
Of the 20 amino acids that exist, 9 are essential amino acids, and 11 are non-essential
In recent years Selenocysteine (21) and Pyrrolysine (22) are two more amino acids added to the list.
Amino acids, hence proteins, contain C, H, O, N,& S

4. AMINO ACIDS: Structure
Consist of a central carbon atom bonded to: a hydrogen, a carboxylic acid, an amino group, and an additional side group that is unique to each amino acid

5. AMINO ACID: Sequence
Amino acids link in specific sequences to form strands of protein
One amino acids is joined to the next by a PEPTIDE bond

6. AMINO ACID: Sequence Dipeptide – 2 amino acids
Tripeptide – 3 amino acids
Oligopeptides – 4-10 amino acids
Polypeptide – more than 10 amino acids
Proteins in the body and diet are long polypeptides (100s of amino acids)

7. DENATURING of PROTEINS
Acid, alkaline, heat, alcohol, and agitation can disrupt the chemical forces that stabilize proteins and can cause them to lose their shape (denature)
Denaturing of proteins happens during food preparation (cooking, whipping, adding acids) or digestion (in the stomach with hydrochloric acid)

8. PROTEINS: Function Structural Functions:
ENZYMES
Enzymes are proteins that catalyze chemical reactions without being used up or destroyed in the process
Used in – digestion, releasing of energy from nutrients for fuel, triggering reactions that build muscle and tissue
Structural Functions:
Collagen – is the most abundant protein in mammals, and gives bone and skin their strength
Keratin – provides structure to hair and nails

9. PROTEIN: Functions IMMUNE FUNCTION
The Immune Response is a series of steps your body takes to mount an attack against invaders
Antibodies are blood proteins that attack and inactivate bacteria and viruses
HORMONES
Hormones are chemical messengers that are made on one part of the body, but act on cells in other parts of the body
Insulin, Glucagon
Antidiuretic Hormone (ADH)

10. PROTEIN: Functions TRANSPORT Lipoproteins (chylomicrons, LDL, HDL)
Albumin transports a variety of nutrients such as calcium, zinc, and Vitamin B6
Transferrin transports iron (hemoglobin – a protein, contains iron, but it transports oxygen)
FLUID BALANCE
Blood proteins like albumin and globulin help to regulate this balance by remaining in the capillaries and attracting fluid
Edema is the result of fluid imbalance

11. PROTEIN: Functions ENERGY SOURCE
If the diet does not provide enough energy, the body must begin to break down its own protein
The proteins are broken down into individual amino acids, then deaminated, and the remaining carbon, hydrogen, and oxygen compounds are used to make energy or glucose
If the diet contains too much protein, the excess will be converted to glucose, or stored as fat

12. Digestion of Proteins Takes place in:
Stomach
Pancreas
Intestinal cells
Enzymes:
Endopeptidases: act on peptide bonds inside the protein molecule making the molecule smaller. These are pepsin, Trypsin, chymotrypsin and Elastase.
Exopeptidases: act an peptide bonds only at the end region. These include:
Carboxypeptidases:
Aminopeptidases:

13. DIGESTION
No digestion of protein takes place in the mouth, it begins in the stomach
Hydrochloric acid denatures protein and also converts pepsinogen to pepsin
Pepsin breaks the protein by hydrolysis of the bonds formed by carboxyl groups of aromatic and methionine.
Proteins are broken into proteases and peptones.
Pepsin completes ~ 10-20% of digestion

14. DIGESTION
Pancreas makes Trypsin, chymotrypsin, Elastase and Carboxypeptidases on stimulation by peptide hormones cholecystokinin and pancreozymin in the small intestine.
Proteases break down polypeptides into smaller peptides (very few peptides have been broken down to amino acids at this stage)

15. Specificity of Ser-Protease Family
Trypsin
Chymotrypsin
Elastase
cut at Lys, Arg
cut at Trp, Phe, Tyr
cut at Ala, Gly
O O
–C–N–C–C–N– C
NH3 +
O O
–C–N–C–C–N– C
O O
–C–N–C–C–N–
CH3
Shallow and
non-polar
pocket
Deep and negatively
charged pocket
COO- C
Asp
Non-polar
pocket
Juang RH (2004) BCbasics
Active Site
E5-26

16. DIGESTION and ABSORPTION
The intestinal wall produces peptidases (Dipeptidases, and tripeptidases) which continue to split the remaining polypeptides into tripeptides, dipeptides, and some amino acids
These smaller units are transported into the enterocytes

17. ABSORPTION
In the enterocyte, other peptidases immediately digest everything into single amino acids which are absorbed into the bloodstream by 5 different carriers for amino acids.
Neutral amino acids
Basic amino acids
Acidic amino acids
Imino acids
Beta amino acids

18. Meister cycle (Gamma glutamyl cycle)
In intestines, kidney tubules, and brain the absorption of neutral amino acids is by gamma glutamyl cycle mail role is played by tripeptide glutathione.

19. Clinical application
Food Allergy: Is due to absorption of partially digested proteins.
Partial gastrectomy, pancreatitis, carcinoma pancreas and cystic fibrosis affect the absorption of proteins.
Hartnups disease: Absorption of aromatic amino acids from intestines as well as reabsorption from renal tubules are defective so amino acids are excreted in the urine

21. NITROGEN EXCRETION
Amino acid breakdown yields an amino group (containing nitrogen)
This molecule is unstable and is converted to ammonia
Ammonia is toxic, so it is excreted from the cells and sent to the liver, where it is converted to urea and water
The urea is transported to the kidney, where it is filtered from the blood and finally sent to the bladder for excretion in the urine
Nitrogen is also lost through hair, skin, GI cells mucus, nails, and body fluids like sweat.

22. PROTEIN: Health Effects
INSUFFICIENT DIETARY PROTEIN
Protein-Energy Malnutrition (PEM) can occur anywhere in the world, but is most common in developing countries
Kwashiorkor
Marasmus
In industrialized nations, PEM may exist in the elderly population, in the poor, and those with anorexia, cancer, AIDS, or malabsorption syndromes

23. PROTEIN: Health Effects
EXCESS DIETARY PROTEIN
May strain the kidneys
May cause mineral losses (especially calcium)*
May increase risk of obesity*
May increase risk of heart disease*
May increase risk of cancer*
*only with animal protein

24. ATKIN’S DIET How does it work?
What are some of the possible negative consequences?

25. Amino acid pool
Sources: adult has about 100g of Free Amino Acids which represent the amino acid pool of the body.
The body amino acid pool is always in a dynamic steady state.

26. About g of protein per day is constantly degraded and synthesized which represents the body protein turnover.
Control of protein turn over: a small protein called ubiquitin (mol wt 8,500) tags to proteins and facilitates degradation.
Certain proteins with amino acid sequences PEST (proline, glutamine, serine, threonine) are rapidly degraded.

27. Nitrogen Balance
In an adult the rate of synthesis of protein balances with rate of degradation so that nitrogen balance is maintained

28. Nitrogen Balance
Dietary protein is almost an exclusive source of nitrogen to the body.
The term nitrogen balance represents the protein utilization and its loss from the body.
Nitrogen balance is determined by comparing the intake of nitrogen (chiefly by proteins) and excretion in the urine, feces and sweat.
I = U+F+S

29. Individual said to be in a nitrogen balance if the intake and output of nitrogen are the same.
There are two other situations:
1. Positive nitrogen balance: nitrogen intake is higher than output. Observed in growing children, pregnant women or during recovery after serious illness.

30. 2. Negative nitrogen balance: nitrogen output is higher than the input
2. Negative nitrogen balance: nitrogen output is higher than the input. Seen in children suffering from kwashiorkor or marasmus , inadequate intake of protein and destruction of tissues.

31. Other factors influencing nitrogen balance:
Hormones:
Positive nitrogen balance: growth hormone and insulin
Negative nitrogen balance: corticosteroids, cancer and uncontrolled diabetes

32. Inter organ transport of amino acids
In plasma all amino acids are with range of 1mg/dl, except glutamic acid which is about 10mg/dl.
Breakdown of muscle protein is the source of amino acids for tissues.
Liver is the site of disposal.

33. In Fasting State
The muscle releases mainly Alanine and glutamine of which alanine is taken up by the liver and glutamine by kidneys.
Liver removes the amino group and converts it to urea and the carbon skeleton is used for Gluconeogenesis.
Alanine is a major gluconeogenic amino acid.

34. Fasting
The brain predominantly takes up branched chain amino acids.

35. In Fed State
Amino acids released by the digestion of dietary proteins travel through the hepatic portal vein to the liver where they are used for the synthesis of blood proteins like serum albumin.
Excess AA are converted into glucose and stored as glycogen or released into blood when glucose levels are low, or TAG which are packaged into VLDL.
AA that pass through the liver are converted to proteins in cells of other tissues.