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Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
These amino acids, although never found in
proteins, perform several biological important

These NPAAs and D-AA speculated to be
related to auto immune disease and to aging

understand the role of NPAAs and D-AA s in
auto immune disease and aging, the
determination of these NPAAs and D-AA s is
any nonprotein amino acid can be chemically
incorporated into peptides, provided that
appropriate methods are designed for
protecting the functional group.
Nonprotein amino acids with no cytotoxicity
have been known to be incorporated into
proteins. For examples,

tyrosine and tryptophan residues in some
proteins have been substituted with m-
fluorotyrosine and 4-fluorotryptophan
respectively, without any effects on the
protein functions, and the 19F nuclei have
been used as magnetic resonance

One NPA that has received some attention is canavanine, (L-
2-amino-4-(guanidinooxy)butyric acid), the guanidinooxy
structural analogue of arginine.
These non protien amino acids are classified as alpha and non
alpha amino acids
1) Alpha amino acids:
a) ornithine
b) citruline
c) arginosuccinic acid
d) thyroxine
e) triodothyroxine
f) S-Adenosylmethionine
g) Homocysteine
h) 3,4-Dihydroxy phenylalanine ( DOPA)
I ) creatinine
j) ovathiol
k) Azaserine

2) NON ALPHA amino acids:
a) beta alanine
b) beta aminoisobutyric acid
c) gama aminobutyric acid(GABA)
d) aminolevulinic acid (ALA)
e) taurine
1) ornithine :
ornithine is precursors of polyamine
Hydrolytic cleavage of the guanidino group of arginine,
catalyzed by liver arginase, releases urea .
The other product, ornithine, reenters liver mitochondria
and participates in additional rounds of urea synthesis.
Ornithine and lysine are potent inhibitors of arginase, and
compete with arginine.
Arginine also serves as the precursor of the potent muscle
relaxant nitric oxide (NO) in a Ca
-dependent reaction
catalyzed by NO synthase
2) Citrulline :
Citrulline is intermediates in the biosynthesis of urea

L-Ornithine transcarbamoylase catalyzes
transfer of the carbamoyl group of carbamoyl
phosphate to ornithine,forming citrulline &
orthophosphate While the reaction occurs in the
mitochondrial matrix, both the formation of
ornithine and the subsequent metabolism of
citrulline take place in the cytosol.
Entry of ornithine into mitochondria and exodus
of citrulline from mitochondria therefore involve
mitochondrial inner membrane transport systems

Arginosuccinic acid is intermediates in the
biosynthesis of urea
Argininosuccinate synthetase links
aspartate and citrulline via the amino group
of aspartate and provides the second
nitrogen of urea.
The reaction requires ATP and involves
intermediate formation of citrullyl-AMP.
Subsequent displacement of AMP by
aspartate then forms arginosuccinate.

Tyrosine forms norepinephrine and epinephrine,
and following iodination the thyroid hormones
triiodothyronine and thyroxine.
Use of measurement of blood thyroxine or
thyroid-stimulating hormone (TSH) in the
neonatal diagnosis of congenital
The amino acid tyrosine is the starting point in
the synthesis of the catecholamines and of the
thyroid hormones tetraiodothyronine
(thyroxine; T4 ) and triiodothyronine (T

thioredoxin reductase, glutathione peroxidase,
and the deiodinase that converts thyroxine to
The clinical history, physical examination, and
lab results were all consistent with primary
hypothyroidism. Accordingly, the patient was
started on a low dose of thyroxine (T4 ).
It is important to begin therapy with a small
dose of T
, as larger doses can precipitate serious
cardiac events, due to the changes in
metabolism caused by administration of the
Thyroxine (T
), free: 4.0 pmol/L (normal 10.3
21.9 pmol/L)

S-Adenosylmethionine, the principal source of methyl groups in
metabolism, contributes its carbon skeleton to the biosynthesis
of the polyamines spermine and spermidine.

Homocystinuria Cystathionine -synthase Lens dislocation,
thrombotic vascular disease, mental retardation, osteoporosis AR

5,10-Methylenetetrahydrofolate reductase
Mental retardation, gait and psychiatric abnormalities, recurrent
strokes ,Mental retardation, hypotonia, seizures, megaloblastic

Pathways, enzymes, and coenzymes involved in the
homocystinurias. Methionine transfers a methyl
group during its conversion to homocysteine.
Defects in methyl transfer or in the subsequent
metabolism of homocysteine by the pyridoxal
phosphate (vitamin B6)-dependent cystathionine b-
synthase increase plasma methionine levels.
Homocysteine is transformed into methionine via
remethylation. This occurs through methionine
synthase, a reaction requiring methylcobalamin and
folic acid.
Deficiencies in these enzymes or lack of cofactors is
associated with decreased or normal methionine
levels. In an alternative pathway, homocysteine can be
remethylated by betaine:homocysteine methyl

Life-threatening vascular complications
(affecting coronary, renal, and cerebral arteries)
can occur during the first decade of life and are
the major cause of morbidity and mortality.

Classic homocystinuria can be diagnosed with
analysis of plasma amino acids, showing
elevated methionine and presence of free

Total plasma homocysteine is also extremely
elevated (usually >100 M). Treatment consists of a
special diet restricted in protein and methionine
and supplemented with cystine.
In approximately half of patients, oral pyridoxine
(25500 mg/d) produces a decrease in plasma
methionine and homocystine concentration in
body fluids.
Folate and vitamin B12 deficiency should be
prevented by adequate supplementation. Betaine
is also effective in reducing homocystine levels.

Neural cells convert tyrosine to epinephrine and
norepinephrine. While dopa is also an intermediate in the
formation of melanin, different enzymes hydroxylate
tyrosine in melanocytes.
Dopa decarboxylase, a pyridoxal phosphate-dependent
enzyme, forms dopamine. Subsequent hydroxylation by
dopamine -oxidase then forms norepinephrine.
In the adrenal medulla, phenylethanolamine-N-
methyltransferase utilizes S-adenosylmethionine to
methylate the primary amine of norepinephrine, forming
epinephrine .
Tyrosine is also a precursor of triiodothyronine and
DOPA ... related to dopaminerelationship to Parkinson's

Dopamine, Norepinephrine, and Epinephrine
These three neurotransmitters are synthesized in a
common pathway from the amino acid L-tyrosine.

The first and rate-limiting step in the synthesis of these
neurotransmitters from tyrosine is the hydroxylation of
the tyrosine ring by tyrosine hydroxylase, a
tetrahydrobiopterin(BH4)-requiring enzyme. The product
formed is dihydroxyphenylalanine or DOPA.
The phenyl ring with two adjacent OH groups is a catechol,
andhence dopamine, norepinephrine, and epinephrine are
called catecholamines.

The second step in catecholamine synthesis is the
decarboxylation of DOPA to form dopamine. This
reaction, like many decarboxylation reactions of
amino acids,equires pyridoxal phosphate.
Dopaminergic neurons (neurons using dopamine as a
neurotransmitter) stop the synthesis at this point,
because these neurons do not synthesize
the enzymes required for the subsequent steps.
Neurons that secrete norepinephrine synthesize it
from dopamine in a hydroxylation reaction catalyzed
by dopamine -hydroxylase (DBH). This enzyme is
present only within the storage vesicles of these cell
Although the adrenal medulla is the major site of
epinephrine synthesis, it is also synthesized in a few
neurons that use epinephrine as a neurotransmitter.

7)Creatinine :
Creatinine is formed in muscle from creatine
phosphate by irreversible, nonenzymatic
dehydration and loss of phosphate.
Since the 24-h urinary excretion of creatinine is
proportionate to muscle mass, it provides a
measure of whether a complete 24-h urine
specimen has been collected.
Glycine, arginine, and methionine all participate in
creatine biosynthesis. Synthesis of creatine is
completed by methylation of guanidoacetate by S-
Creatinine : 200 mol/L (4480 mol/L)

Female -- 4480 mol/L 0.50.9 ng/mL

male -- 53106 mol/L 0.61.2 ng/mL
Sulfur containing amino acid found in fertilized
Eggs, and acts as an antioxidant

9) Azaserine: (antibiotic)
Purine deficiency states, while rare in humans,
generally reflect a deficiency of folic acid.
Compounds that inhibit formation of tetrahydrofolates
and therefore block purine synthesis have been used
in cancer chemotherapy.
Inhibitory compounds and the reactions they inhibit
include azaserine, diazanorleucine, 6-
mercaptopurine , and mycophenolic acid .

II) Non--Amino Acids:
Non--amino acids present in tissues in a free form include -
alanine, -aminoisobutyrate, and -aminobutyrate (GABA). -
Alanine is also present in combined form in coenzyme A and
in the -alanyl dipeptides carnosine, anserine and
homocarnosine .
1) Beta-Alanine & -Aminoisobutyrate :
Alanine and -aminoisobutyrate are formed during
catabolism of the pyrimidines uracil and thymine,
respectively . Traces of -alanine also result from the
hydrolysis of -alanyl dipeptides by the enzyme carnosinase. -
Aminoisobutyrate also arises by transamination of
methylmalonate semialdehyde, a catabolite of L-valine .
The initial reaction of -alanine catabolism is transamination
to malonate semialdehyde. Subsequent transfer of
coenzyme A from succinyl-CoA forms malonyl-CoA
semialdehyde, which is then oxidized to malonyl-CoA and
decarboxylated to the amphibolic intermediate acetyl-CoA.
Analogous reactions characterize the catabolism of -
aminoisobutyrate. Transamination forms
methylmalonate semialdehyde, which is converted to
the amphibolic intermediate succinyl-CoA by
reactions 8V and 9V of.

Disorders of -alanine and -aminoisobutyrate
metabolism arise from defects in enzymes of the
pyrimidine catabolic pathway.

Principal among these are disorders that result from a
total or partial deficiency of dihydropyrimidine

2) beta-Alanyl Dipeptides :
The -alanyl dipeptides carnosine and anserine (N -
methylcarnosine) activate myosin ATPase, chelate
copper, and enhance copper uptake. -Alanyl-
imidazole buffers the pH of anaerobically
contracting skeletal muscle.

Biosynthesis of carnosine is catalyzed by carnosine
synthetase in a two-stage reaction that involves
initial formation of an enzyme-bound acyl-adenylate
of -alanine and subsequent transfer of the -alanyl
moiety to L-histidine.

Hydrolysis of carnosine to -alanine and L -histidine
is catalyzed by carnosinase. The heritable disorder
carnosinase deficiency is characterized by

Homocarnosine, present in human brain at higher
levels than carnosine, is synthesized in brain tissue
by carnosine synthetase. Serum carnosinase does
not hydrolyze homocarnosine. Homocarnosinosis,
a rare genetic disorder, is associated with
progressive spastic paraplegia and mental

3) gama-Aminobutyrate

gama-Aminobutyrate (GABA) functions in brain
tissue as an inhibitory neurotransmitter by altering
transmembrane potential differences.

GABA is formed by decarboxylation of glutamate
by L -glutamate decarboxylase. Transamination of
-aminobutyrate forms succinate semialdehyde,
which can be reduced to -hydroxybutyrate by L -
lactate dehydrogenase, or be oxidized to succinate
and thence via the citric acid cycle to CO
and H

A rare genetic disorder of GABA metabolism
involves a defective GABA aminotransferase, an
enzyme that participates in the catabolism of
GABA subsequent to its postsynaptic release in
brain tissue.

Defects in succinic semialdehyde dehydrogenase
are responsible for another rare metabolic disorder
of -aminobutyrate catabolism characterized by 4-
hydroxybutyric aciduria.

4) amino levulinic acid (ALA) :
ALA is intermediate in the synthesis of
porphyrin (finally heme)
Taurine (2-aminoethylsulphonic acid) is a non-protein
aminoacid present in almost all animal tissues and
the most abundant free intracellular aminoacid in
human cells.
In humans, it is considered to be a semi-essential
aminoacid since it can be synthesized from other
sulfonic aminoacids such as methionine and cysteine,
the presence of vitamin B6,2,3 but endogenous
production is insufficient, so that it needs to be
provided through diet.
Taurine is found in association with bile acids.

Biological effects of taurine in the context of
Biological EffectMechanismof Taurine
Antioxidant action By inhibiting ROS generation at
mitochondria Osmoregulation By counteracting
osmotic inbalance through cellular membrane due
to hyperglycaemia
Antiinflammatory effects By interfering the
formation of inflammatory mediators Glucose
Homeostasis By interfering the insulin signalling
pathway acting upon UCP2 protein

Estimation of NPAAs :
1) The aim of our study was to analyze NPAAs and
D-AA s in biosamples by means of capillary
electrochromatogrphy (CEC) using a chiral
practicle- loded monolithiac column with
flurrosense detection for high sensitivity.
2) capillary electrophoresis
3) High perfromance liquid chromatography(HPLC)
4) laser-induced flurosecne (LIF)
5) scanning electro microscopy (SEM)

Scanning electro micrograph of CEC capillary
Location of the regions of ordered secondary structures for b-residues in
fqy space. The a-helix and b-sheet are the classical structures for poly a-amino
acids. b-residues occurring in the appropriate shaded region can be accommodated
without disruption of secondary structures. The 12-helix and 14-helix are well
characterised secondary structures for poly b-peptides.