8+9. Acidic, Basic and Branche Chain Amino Acids+Aromatic Amino Acids

Question 1

Iso enzymes of NOS and Therapeutic Applications of NO

Answer 1

There are 3 isoforms of NOS, these are products of 3 different genes. All forms are seen in almost all tissues.
Neuronal NOS: NOS1 or nNOS or neuronal NOS is seen in central and peripheral neurons. Nitrogenic neurons are seen especially in cerebellum and gastrointestinal tract. It is mainly a cytoplasmic enzyme. It is activated by calcium. The gene for neuronal NOS is seen on chromosome 12. NOS1 is implicated in the long QT interval syndrome.
Macrophage NOS: NOS-2 or iNOS or inducible NOS or macrophage NOS is mainly seen in macrophages and neutrophils; but is also present in hepatocytes. It is induced by cytokines (interleukin-1 and tumor necrosis factor) and during inflammation. It is a cytoplasmic enzyme. Calcium does not activate this iso-enzyme. Gene for iNOS is on chromosome 17.
Endothelial NOS: NOS-3 or eNOS or endothelial NOS is seen in endothelial cells, platelets, endocardium and myocardium. In these sites, the NO is constantly produced and released, so that arterial relaxation occurs. It is localized in the plasma membrane. It is activated by calcium. The gene for endothelial NOS is on chromosome 7.

Nitric Oxide in Diseases and Treatment

1. Angina Pectoris: Nitroprusside can directly release NO’. Nitroglycerine (glyceryl trinitrite) requires glutathione to produce NO’. These will dilate coronary arteries; and are beneficial in treating angina pectoris.
2. Pulmonary Hypertension: Inhalation of NO’ is useful in the treatment of pulmonary hypertension and high altitude pulmonary edema. NO’ produces pulmonary vasodilatation, without lowering systemic blood pressure.
3. Impotence: NO relaxes smooth muscles in the corpus cavernosum and increases blood flow into the penis and makes it erect. Sildenafil citrate (Viagra) selectively inhibits the specific phosphodiesterase type 5 (PDE-5); thus inhibiting hydrolysis of cGMP; and increasing the concentration of cGMP in corpus cavernosum.
4. Shock: Induction of NOS from macrophages is increased in sepsis and is responsible for severe vasodilation and shock. This hypotension is refractory and not responding to vasoconstrictor drugs.
5. Enhancers of NO activity: S-nitroglutathione (GS-NO) is a NO donor which inhibits platelet agglutination. N-acetyl cysteine (NAC) is a glutathione precursor; it protects NO from being metabolized by free radical scavengers, and hence enhances NO activity.
6. Antagonists of Nitric Oxide: Development of specific inhibitors of different isoforms of NOS promises therapeutic uses. N-monomethyl arginine (NMMA) competitively inhibits NOS. ADMA (asymmetric dimethyl-arginine), an endogenous arginine analogue acts as an NOS inhibitor; ADMA is seen to be increased in hyper-homocysteinemia and pre-eclampsia. In eclampsia, the hypertension is due to the lowered production of NO.

Question 2

Polyamines – Eg and Functions

Answer 2

Polyamines are compounds having many amine groups. They are aliphatic amines. They are synthesised from ornithine and methionine(SAM).
Example: Putrescine, Spermidine, Spermine.

Functions:
• Polyamines play an important role in stabilization of structures like DNA, ribosomes, tRNA, certain subcellular organelles. Since polyamines are cations, they bind with anionic structures like DNA, ribosomes, tRNA etc.
• Polyamines are involved in synthesis of DNA, RNA and proteins.
• Polyamines required for cell division, proliferation and growth.

Clinical significance: Excretion of polyamines is found in all types caner. E.g. leukemias, cancer of lungs, kidney, gall bladder etc. Monitoring the levels of polyamines in serum and urine is useful in detection of severity of cancer.

Key enzyme of polyamine synthesis is ornithine decarboxylase (ODC). DFMO (difluromethyl ornithine) is a powerful inhibitor of polyamine synthesis. It is an example of suicide inhibition.
It is used as an anti cancer drug.

Question 3

Maple Syrup Urine Disease - explain?

Answer 3

i. It is also called branched chain ketonuria. The incidence is 1 per 1 lakh births. The name originates from the characteristic smell of urine (similar to burnt sugar or maple sugar) due to excretion of branched chain keto acids.
ii. The basic biochemical defect is deficient decarboxylation of branched chain keto acids (BKA).
iii. Clinical findings: Disease starts in the first week of life. It is characterized by convulsions, severe mental retardation, vomiting, acidosis, coma and death within the first year of life.
iv. Laboratory findings: Urine contains branched chain keto acids, valine, leucine and isoleucine. Rothera’s test is positive, but unlike in cases of ketoacidosis, even boiled and cooled urine will give the test. Diagnosis depends on enzyme analysis in cells. Diagnosis should be done prior to 1 week after birth.
v. Treatment: Giving a diet low in branched chain amino acids. Mild variant is called intermittent branched chain ketonuria. This will respond to high doses of thiamine. This is because the decarboxylation of the BKA requires thiamine. Liver transplantation has been succ

Question 4

How tyrosine is both glucogenic and ketogenic?

Answer 4

In the final step of tyrosine catabolism(hydrolysis), fumaryl acetoacetate (formed in the previous isomerisation step) is hydrolysed to form fumarate(a glucogenic product) and acetoacetate (a ketone body). Hence tyrosine is both glucogenic and ketogenic.

Question 5

What are the special products obtained from tyrosine?

Answer 5

1. Melanin
2. Thyroxine
3. Catecholamines

Question 6

Sparing action of tyrosine on phenylalanine?

Answer 6

The requirement for phenylalanine becomes reduced, if increased amounts of tyrosine are present in the blood. This is called sparing action of tyrosine on phenylalanine.

Question 7

Synthesis of Melanin?

Answer 7

There is only one enzyme involved, which catalyzes the first two steps. The remaining reactions are non- enzymatic and occur spontaneously.

i. Formation of DOPA: The first step is the hydroxylation of tyrosine by tyrosinase. It is a mono-oxygenase containing copper (step 1, Fig.17.3). Molecular O2 is used for the reaction, of which one atom is incorporated in the product, to form dihydroxy phenyl alanine or DOPA (Box 17.1).
ii. Formation of DOPA quinone: Tyrosinase again acts on DOPA to form dopaquinone (step 2, Fig.17.3).
iii. Formation of indolequinone: It is converted to indolequinone through a series of reactions involving decarboxylation and oxidation of the side chain. The indolequinone is polymerized to form melanin. Melanin is a group of polymers of random structure formed from indole- quinone. Melanin when reduced changes from black to a tan color.

Question 8

Basis of albinism in Cu deficiency?

Answer 8

i. Copper deficiency: Since tyrosinase is a copper containing enzyme, there may be disturbances in pigmentation during copper deficiency. Hair syn- thesized at the time of deficiency may be depigmented.
In albinism, tyrosinase is absent in melanocytes all over the body.

Question 9

What is VMA? Name any 2 conditions in which it is increased.

Answer 9

Vanillyl mandelic acid (VMA) is the major end product of the degradation of adrenaline.

Normal level of excretion of VMA is 2–6 mg / 24 hr. It is increased in pheochromocytoma (epinephrine excess) and in neuroblastoma (nore- pinephrine excess).

Question 10

Phenylketonuria - explain?

Answer 10

Deficiency of phenyl alanine hydroxylase (Fig.17.1) is the cause for this disease. The genetic mutation may be such that either the enzyme is not synthesized, or a non-functional enzyme is synthesized. It is a recessive condition.

Biochemical Abnormalities
A. Phenylalanine could not be converted to tyrosine. So phenylalanine accumulates. Phenylalanine level in blood is elevated.
B. So alternate minor pathways are opened (Fig.17.5). Phenyl ketone (phenyl pyruvate), phenyl lactate and phenyl acetate are excreted in urine.

Clinical Manifestations
A. The classical PKU child is mentally retarded with an IQ of 50. About 20% inmates of lunatic asylum may have PKU.
B. Agitation, hyperactivity, tremors and convulsions are often manifested. This may be because phenylalanine interferes with neurotransmitter synthesis.
C. Thechildoftenhashypopigmentation,explainedbythe decreased level of tyrosine.
D. Phenyllacticacidinsweatmayleadtomousybodyodor.

Laboratory Diagnosis
A. Blood phenylalanine: Normal level is 1 mg/dl. In PKU, the level is >20 mg/dl. This may be demonstrated by chromatography. Tandem mass spectroscopy is the most reliable test; but is costly.
B. Guthrie test is a rapid screening test. See Box 17.4.
C. Ferricchloridetest:Urineofthepatientcontains phenyl ketones about 500–3000 mg/day. This could be detected by adding a drop of ferric chloride to the urine. A transient blue-green color is a positive test. But this is a less reliable test.
D. DNA probes are now available to diagnose the defects in phenylalanine hydroxylase and dihydrobiopterin deficiency.
7. Treatment
A. Early detection is very important. About 5 units of IQ are lost for each 10-week delay in starting the treatment.
B. The treatment is to provide a diet containing low phenyl alanine (10–20 mg/kg body weight per day). Food based on tapioca (cassava) will have low phenyl alanine content.
C. This special diet is to be continued during the first decade of life; after which the child can have a normal diet. Life-long compliance of special diet is advised, though not mandatory.
D. Female child, on growing to adulthood may become pregnant (maternal hyper phenyl alaninemia). Then again special diet is to be given, because the increased phenylalanine level will affect the brain development of the fetus.

Question 11

Explain alkaptonuria.

Answer 11

Biochemical Defect

1. Alkaptonuria is an autosomal recessive condition with an incidence of 1 in 250,000 births.
2. The metabolic defect is the deficiency of homogentisate oxidase (step 4, Fig. 17.2 and item 2, Fig.17.7). This results in excretion of homogentisic acid in urine.
3. It is compatible with fairly normal life. The only abnormality is the blackening of urine on standing. The homogentisic acid is oxidized by polyphenol oxidase to benzoquinone acetate (Fig.17.6). It is then polymerized to black colored alkaptone bodies.
4. By the 3rd or 4th decade of life, patient may develop ochronosis (deposition of alkaptone bodies in intervertebral discs, cartilages of nose, pinna of ear). Black pigments are deposited over the connective tissues including joint cavities to produce arthritis.
5. No specific treatment is required. But minimal protein intake with phenylalanine less than 500 mg/day is recommended.

Diagnosis of Alkaptonuria

1. Urine becomes black on standing when it becomes alkaline. Blackening is accelerated on exposure to sunlight and oxygen. The urine when kept in a test tube will start to blacken from the top layer.
2. Ferric chloride test will be positive for urine.
3. Benedict’s test is strongly positive. Therefore, alkaptonuria comes under the differential diagnosis of reducing substances in urine

Question 12

Explain albinism?

Answer 12

Albinism is an autosomal recessive disease with an incidence of 1 in 20,000 population (Fig.17.7).

2. Tyrosinase is completely absent, leading to defective synthesis of melanin.
3. Theocularfundusishypopigmentedandirismay be grey or red. There will be associated photophobia, nystagmus and decreased visual acuity.
4. The skin has low pigmentation, and so skin is sensitive to UV rays. The skin may show presence of naevi and melanomas. Hair is also white.
5. Manifestations are less severe in tyrosinase positive type, where the abnormality is in the uptake of tyrosine by melanocytes.

Question 13

5-HIAA. When is it increased?

Answer 13

HIAA (5-Hydroxy indole acetic acid) in Urine:
Normal value is 2-7 mg/day. It is increased in carcinoid tumors in the gut or bronchus, tropical sprue, Whipple’s disease, oat cell carcinoma of the bronchus. If urine 5 HIAA exceeds 25 mg/day, diagnosis of carcinoid syndrome can be made. Metastatic carcinoid tumor (functioning) shows higher values (>350 mg/day). Since 5 HIAA secretion may be intermittent, repeated testing is required. It is decreased in depression, small intes

Question 14

Special Products from Tryptophan?

Answer 14

Substances produced from tryptophan are:

1. Alanine (glucogenic)
2. Acetoacetyl CoA (ketogenic)
3. Formyl group (One-carbon unit)
4. Niacin and NAD+
5. Serotonin
6. Melatonin
7. Hydroxy indole acetic acid (excretory product) 8. Indican (excretory product)

(MISFHAAN)

Question 15

Explain Hartnup’s Disease

Answer 15

It is an inherited autosomal recessive disease.

ii. Absorption of aromatic amino acids(most importantly, tryptophan) from intestine as well as reabsorption from renal tubules are defective. So amino acids are excreted in urine.
iii. The pellagra like symptoms are due to the deficiency of niacin derived from tryptophan. The common manifestations are dermatitis and ataxia.
iv. A diagnosis is based on amino aciduria and increased excretion of indole compounds detected by the Obermeyer test.
v. Patients improve when put on a high protein diet with supplementation of niacin and minimum exposure to sunlight. The neuropsychiatric manifestations of Hartnup’s disease are said to be responsible for the sadistic and bizarre behavior of emperors like Nero and Caligula.