Amino acids metabolic disorders and methods of evaluation the levels of those amino acids

1. Inborn Error Of Amino Acids Metabolism
Prepared By
Revised By
Dr/ Hala Abdelrahman
Teaching Fellow of Biochemistry
at NHTMRI
Dr/ Mona Youssef
Teaching Fellow of Analytical Chemistry
At National Nutrition Institute
Dr/ Lamiaa Mohamed Kamal
MD Consultant of Clinical & Chemical Pathology
CDC Laboratory Manager

2. Contents
 Introduction
 Amino Acids Metabolic Disorders
 Amino Acids Quantifications Methods
 References

3.  Small organic molecule have –COOH & NH2 groups
 About 500 amino acids in nature only 20 amino acids
form our body proteins according to gene
instructions
Introduction
 The metabolism of those amino acids under tight control, the loss
of this control can lead to severe cellular dysfunction and disorder
1. Definition
2. Sources & Functions

4. 1. Phenylketonuria (PKU)
2. Tyrosinemia (TYRI, TYRII, TYRIII)
3. Alkaptonuria (AKU)
4. Maple syrup urine disease (MSUD)
5. Homocystinuria (HCU)
6. Non-ketotic Hyperglycinemia (NKH)
7. Urea cycle disorders
These diseases mostly inherited as
autosomal recessive.
Amino Acids Metabolic Disorders

6. Cause: inborn error in Phenylalaninemetabolism
due to mutation in phenylalaninehydroxylase
gene
Biochemical features: Build up Phenylalanine &
Phenylketons
Clinical features: Normal at birth, severe mental
disability approximately by end the 1st year (so
The necessity of early neonatal Screening) .
Treatment: Most common treatment is by
dietary restriction & special milk formulas.
1. Phenylketonuria (PKU)

7. 2. Tyrosinemia (TYR)
Cause: inborn error in tyrosine metabolism due to
mutation in fumarylaceoacetate hydeolyase gene .
Biochemical features: Build up tyrosine &
Succinylacetone.
Clinical features:
 liver & kidney Failure due to SA accumulation
Treatment:
 diet restriction & special milk formulas
 Inhibition of Succinylacetoneproduction via drugs
Note: About 10 % of newborns have temporarily
elevated levels of tyrosine (transient tyrosinemia).
In these cases, the cause is not genetic. The most
likely immature liver enzymes due to premature
birth resolves in about 2 months

8. 4. Maple syrup urine disease (MSUD)
Cause: inborn error in branched-chain amino
acids (leucine, isoleucine & Valine) metabolism
due to mutation in branched chain ketoacid
dehydrogenase complex genes
Biochemical features: Accumulation of
leucine, Allo-isoleucine & valine & their
ketones
Clinical features: Brain, lung , liver failure
(death in few months)
Treatment:
 Diet restriction & special milk formulas

9. Urea cycle disorders
Cause: Inborn error in Ammonia disposal
due to mutations in different genes of urea
cycle enzymes
Most common types usually included in
newborn screening programs:
1. Ornithine transcarbamylase (OTC) deficiency
2. Argininosuccinate synthetase (ASS) deficiency
(Citrulinemia )
3. Arginase deficiency (Argininemia)
Biochemical features: high level of ammonia
Clinical: loss of appetite, vomiting, behavioral
abnormalities, hallucinations, and liver
failure.
Treatment hemodialysis or hemofiltration
are the most efficient treatments for reducing
the plasma ammonia concentration.
1
X
2 X
3
X

10. Determination amino acids levels in neonatal Blood
1. Tandem Mass spectrometry 2. Cation exchange chromatography
Using Plasma Amino acid analysis

11. Mass spectrometry Principle
 Micro analytical instrumental technique for
separation of electrically charged species in
the gas phase according to mass/charge.
 Ionization: Electrically neutral atoms or molecules
are converted to electrically charged atoms or
molecules (ions) by gaining or losing an electron.
The product of ionization can be positive or
negative.

12. Triple Quadrupole mechanism of action
Q1 Q2 (Collison cell) Q3
Fast bombarding Nitrogen
atoms
Detector

13. Quantification
 Using an isotopic standard IS (same atomic
number,
 different mass number).
 MS can separate different isotopes.
(analyte cps) / (IS cps) X conc µM/L X (RRF) = [Analyte].

14. Cation exchange chromatography principle of the amino acid analyzer
 The principle of separation is based on
the reversible exchange of ions
between the ions present in the
sample and those available in the ion
exchange resin. Mainly, ion exchange
separations take place in a column
covered with an ion exchanger.

15. Isoelectric point of amino acids
pH where the amino acid turns neutral.
pI = 1/2 (pKa1 + pKa2)

16.  In cation exchange chromatography, a negatively charged ion exchange resin is utilized that has an
affinity towards molecules with overall positive surface charges.
Cation exchange chromatography

17. Quantification
 Ninhydrin Degraded by oxygen, light and heat.
 Reacts with primary amino acid @ 130 C forming
Rheumann’s compound.
 Rheumann’s compound (purple) absorbance @ 570 nm.
 Ninhydrin reacts with secondary amino acids (Proline)
giving a yellow color (@440 nm)
 The intensity of the absorbance is directly proportional to
the amount of the eluted amino acid.

18. High resolution detected amino acids chromatogram

19. References
 Sahai, I. and Erbe, R.W., 2022. Neonatal Screening. In Emery and
Rimoin's Principles and Practice of Medical Genetics and
Genomics (pp. 57-86). Academic Press.
 Matsumoto, S., Häberle, J., Kido, J., Mitsubuchi, H., Endo, F. and
Nakamura, K., 2019. Urea cycle disorders—update. Journalof
human genetics, 64 (9), pp.833-847.
 Domon, B. and Aebersold, R., 2006. Mass spectrometry and
protein analysis. science, 312(5771), pp.212-217.
 Le Boucher, J., Charret, C., Coudray-Lucas, C., Giboudeau, J. and
Cynober, L., 1997. Amino acid determination in biological fluids by
automated ion-exchangechromatography: performance of Hitachi
L-8500A. Clinical chemistry, 43(8), pp.1421-1428.