Dr. SaidunnisaProfessor of Biochemistry

Protein turn over, Nitrogen balance

Learning objectives At the end of session the student shall be

able to:1. Explain protein turnover and nitrogen

balance.2. Define nitrogen balance and factors effecting 3. Explain inter organ relationship of amino

acids in fasting and fed state.4. Define transamination and deamination,

mechanism of action with enzymes and co enzymes involved with suitable examples and its clinical application and its limitations.

5. Clinical importance of Glucose –alanine cycle.

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

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

AMINO ACID: SequenceAmino acids link

in specific sequences to form strands of protein

One amino acids is joined to the next by a PEPTIDE bond

AMINO ACID: Sequence

Dipeptide – 2 amino acidsTripeptide – 3 amino acidsOligopeptides – 4-10 amino acidsPolypeptide – more than 10 amino

acidsProteins in the body and diet are

long polypeptides (100s of amino acids)

DENATURING of PROTEINSAcid, 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)

PROTEINS: FunctionStructural

Functions:Collagen – is the

most abundant protein in mammals, and gives bone and skin their strength

Keratin – provides structure to hair and nails

ENZYMESEnzymes 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

PROTEIN: Functions

HORMONESHormones are

chemical messengers that are made on one part of the body, but act on cells in other parts of the body

Insulin, GlucagonAntidiuretic Hormone

(ADH)

IMMUNE FUNCTIONThe 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

PROTEIN: Functions

FLUID BALANCEBlood 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

TRANSPORTLipoproteins

(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)

PROTEIN: Functions

ENERGY SOURCEIf 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

Digestion of ProteinsTakes place in: StomachPancreasIntestinal cellsEnzymes: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:

DIGESTIONNo 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

DIGESTIONPancreas 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)

Specificity of Ser-Protease Family

COO-

CAsp

COO-

CAsp

Active Site

Trypsin Chymotrypsin

Elastasecut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

Non-polarpocket

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Shallow andnon-polar

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O O–C–N–C–C–N– C C C C NH3+

O O–C–N–C–C–N– C

O O–C–N–C–C–N–

CH3

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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

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.

1. Neutral amino acids2. Basic amino acids3. Acidic amino acids4. Imino acids5. Beta amino acids

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.

Clinical applicationFood 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

NITROGEN EXCRETIONAmino acid breakdown yields an amino

group (containing nitrogen)This molecule is unstable and is

converted to ammoniaAmmonia 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.

PROTEIN: Health Effects

INSUFFICIENT DIETARY PROTEINProtein-Energy Malnutrition (PEM) can

occur anywhere in the world, but is most common in developing countries

KwashiorkorMarasmusIn industrialized nations, PEM may exist

in the elderly population, in the poor, and those with anorexia, cancer, AIDS, or malabsorption syndromes

PROTEIN: Health EffectsEXCESS DIETARY PROTEINMay strain the kidneysMay 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

ATKIN’S DIETHow does it work?What are some of the possible

negative consequences?

Amino acid poolSources: 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.

About 300-400g 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.

Nitrogen Balance

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

Nitrogen BalanceDietary 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

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.

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.

Other factors influencing nitrogen balance:

Hormones: Positive nitrogen balance: growth

hormone and insulinNegative nitrogen balance:

corticosteroids, cancer and uncontrolled diabetes

Inter organ transport of amino acidsIn 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.

In Fasting StateThe 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.

Fasting The brain predominantly takes up branched chain amino acids.

In Fed StateAmino 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.