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Urea is the end product of nitrogen metabolism

Introduction

  • The continuous degradation and synthesis of cellular proteins occur in all forms of life.
  • Each day, humans turn over 1–2% of their total body protein, principally muscle protein.
  • Of the liberated amino acids, approximately 75% are reutilized.
  • Since excess amino acids are not stored, those not immediately incorporated into new protein are rapidly degraded to amphibolic intermediates.
  • The excess nitrogen forms urea (figure-1).

 Fate of amino acids

Figure-1- Fate of Amino acids, the ammonia released from the amino group of the amino acids is detoxified through urea formation and the carbon skeleton is used either for the synthesis of glucose, ketone bodies or is completely oxidized in TCA cycle to provide energy.

Urea formation (Urea cycle)

Characteristics of urea cycle

  • Urea is the major disposal form of amino groups
  • It accounts for 90% of the nitrogen containing components of urine
  • The urea cycle is the sole source of endogenous production of arginine
  • Urea formation takes place in liver,
  • Urea excretion occurs through kidney

Substances required for urea formation

1) Amino acids- 6 amino acids participate in urea formation, which are-

  • Ornithine
  • Citrulline
  • Aspartic acid
  • Argininosuccinic acid
  • Arginine and
  • N-Acetyl Glutamate

Of the six participating amino acids, N-acetyl glutamate functions solely as an enzyme activator. The others serve as carriers of the atoms that ultimately become urea.

2) Energy

  • Synthesis of 1 mol of urea requires 3 mol of ATP

3) Amino group

  • 1 mol each of ammonium ion and of the α-amino nitrogen of aspartate.

4) Enzymes

  • Five enzymes catalyze the reactions of urea cycle

5) Carbon dioxide

  • CO2 and ammonia both are waste products and are eliminated as urea from the body
  • CO2 is added in the form of bicarbonate ion

 Site of urea formation

  • Urea synthesis is a cyclic process.
  • The first two reactions of urea synthesis occur in the matrix of the mitochondrion, the remaining reactions occur in the cytosol

Steps of urea formation

Step-1- Formation of Carbamoyl-Phosphate (figure-2)

  • Condensation of CO2, ammonia, and ATP to form Carbamoyl phosphate is catalyzed by mitochondrial Carbamoyl phosphate synthase I (CPS-1)
  • Formation of Carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphoryl donor.
  • Carbamoyl phosphate synthase I, the rate-limiting enzyme of the urea cycle, is active only in the presence of its allosteric activator N-acetyl glutamate, which enhances the affinity of the synthase for ATP.

Step-2- Formation of Citrulline (figure-2)

  • The Carbamoyl group of Carbamoyl phosphate  is transferred to ornithine, forming Citrulline and Ortho Phosphate
  • The reaction is catalyzed by Ornithine trans Carbamoylase
  • Subsequent metabolism of Citrulline take place in the cytosol.
  • Entry of ornithine into mitochondria and exit of citrulline from mitochondria involves mitochondrial inner membrane transport systems

Clinical Significance

  • Ornithine Transcarbamoylase deficiency causes enhanced excretion of Uracil.
  • Excessive excretion of Uracil or its precursor Orotic acid, results from an accumulation of Carbamoyl phosphate in the mitochondria.
  • In the absence of Ornithine Transcarbamoylase, Carbamoyl phosphate accumulates and leaks in to the cytoplasm, where it can be used to make Carbamoyl Aspartate, the first intermediate in the pathway of pyrimidine nucleotide biosynthesis.

Step-3- Formation of Argininosuccinate (figure-2)

  • Argininosuccinate synthase (ASS) links L- Aspartate and Citrulline via the amino group of aspartate and provides the second nitrogen of urea.
  • The reaction requires ATP, production of argino-succinate is an energetically expensive process, since the ATP is split to AMP and pyrophosphate.
  • The pyrophosphate is then cleaved to inorganic phosphate using pyrophosphatase, so the overall reaction costs two equivalents of high energy phosphate per mole.

Step-4- Cleavage of Argininosuccinate (figure-2)

  • Cleavage of argininosuccinate catalyzed by argininosuccinate lyase (ASL), proceeds with retention of nitrogen in arginine and release of the aspartate skeleton as fumarate.
  • Addition of water to fumarate forms L-malate, and subsequent NAD+-dependent oxidation of malate forms oxaloacetate (figure-3).
  • Transamination of oxaloacetate by glutamate aminotransferase then re-forms aspartate. carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea

 

 Urea cycle

Figure-2-Steps of urea formation, the first two reactions are mitochondrial and the remaining reactions take place in the cytoplasm.

Relationship of Urea cycle with TCA cycle

 Fate of fumarate

Figure-3- Urea cycle and TCA cycle are linked together through fumarate.

Step-5- Cleavage of Arginine(figure-2)

  • Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver Arginase (ARG1) releases urea, the other product, Ornithine, reenters liver mitochondria for additional rounds of urea synthesis.
  • Arginine also serves as the precursor of the potent muscle relaxant nitric oxide (NO) in a Ca2+-dependent reaction catalyzed by NO synthase

Regulation of Urea formation

  • The activity of Carbamoyl phosphate synthase I is determined by N-acetyl glutamate, whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate.
  • These reactions are catalyzed by N-acetyl glutamate synthase and N-acetyl glutamate hydrolase, respectively (figure-4).

Formation and degradation of N-Acetyl glutamate 

Figure-4-Formation and hydrolysis of N-Acetyl glutamate

  • Major changes in diet can increase the concentrations of individual urea cycle enzymes 10- to 20-fold.
  • Starvation, for example, elevates enzyme levels, presumably to cope with the increased production of ammonia that accompanies enhanced protein degradation
  • Regulation is also achieved by linkage of mitochondrial glutamate dehydrogenase with CPS-1

Fate of Urea (figure-5)

  • Urea formed in the liver is transported through circulation to kidneys for excretion through urine.
  • It is also transported to intestine where it is decomposed by Urease produced by microbial action.
  • Ammonia liberated by this activity is transported by portal circulation to liver where it is detoxified back to urea.
  • A fraction of ammonia goes to systemic circulation.

 Fate of urea

Figure-5- Urea formed in the liver is mainly excreted through kidney in urine. A fraction of urea is transported to intestine where it is acted upon by bacterial urease. The ammonia thus released either goes through portal circulation to liver for reconversion to urea or enters systemic circulation.

 

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Serine

Serine is a hydroxyl group containing, nutritionally non-essential, one carbon donor and glucogenic amino acid.

Structure– Chemically it is α- Amino –β- Hydroxy Propionic acid (figure-1)

 serine

Figure-1- Chemical structure of serine

Synthesis

1)  From Glycine

Glycine and serine can be inter- converted by the action of serine hydroxy methyl transferase (figure-2).

serine to glycine conversion 

Figure-2– Serine to glycine interconversion

2) From 3- Phospho Glycerate (figure-3)

This is the major source of serine in the body. The reactions involved are as follows

Step-1- Dehydrogenation

The reaction is catalyzed by dehydrogenase enzyme

3- Phosphoglycerate——–> 3-Phosphohyroxy pyruvate

Step-2- Transamination

The reaction is catalyzed by phospho serine transaminase enzyme; the alpha amino group is donated by glutamate

3-Phosphohyroxy pyruvate <———–> Phosphoserine

Step-3 Dephosphorylation

The reaction is catalyzed by phosphatase enzyme

Phosphoserine——–> Serine + H3PO4

Synthesis of serine from 3-phosphoglycerate

Figure-3- Synthesis of serine from 3-Phosphoglycerate

3) From Hydroxy pyruvate

Hydroxy pyruvate can be transaminated to form serine

Alanine + Hydroxy pyruvate——–> Pyruvate +Serine

Catabolism of Serine

1)  Non oxidative deamination- Serine can be non oxidatively deaminated to form pyruvate, hence it is glucogenic amino acid (figure-4)

 Non oxidative deamination of serine

Figure-4- The first step of the reaction sequence is catalyzed by dehydratase enzyme that requires B6-P as a coenzyme. The second step is same as oxidative deamination i.e. hydration followed by deamination.

2)  Conversion to Glycine- It can also be converted to glycine depending upon the cellular requirement.

3) Transamination– Serine can be transaminated to form hydroxy pyruvate

Metabolic role of serine

1) One carbon donor- During the conversion of serine to glycine one carbon fragment is transferred to THF forming N5N10 Methylene THF (figure-2).

2) Synthesis of Cysteine- Serine contributes its carbon skeleton for the synthesis of cysteine, the- SH group is donated by Methionine (figure-5). It is a two-step process and the reactions are as follows-

Synthesis of cysteine form serine and methionine 

Figure-5- Synthesis of Cysteine from Serine and Methionine. Homocysteine is a metabolic product of Methionine

3) Synthesis of phosphoproteins- Serine acts as a carrier of phosphate group in phosphoproteins- like casein, vitellin etc.

4) Synthesis of phospholipids- Phosphatidyl serine is biologically an important phospholipid (figure-6).

 phosphatidylserine

Figure-6-Chemical structure of Phosphatidyl serine

5) Synthesis of Sphingosine- Sphingosine the alcohol present in sphingolipids is synthesized by the condensation of Palmitic acid and serine.

6) Synthesis of Ethanolamine- Serine is decarboxylated to form ethanolamine. Ethanolamine can be used either for the synthesis of choline by subsequent methylation reactions or it is as such used for the synthesis of Phosphatidyl ethanolamine, an important phospholipid and a lipotropic agent.

7) Regulation of enzyme activity- The hydroxyl group of serine can be reversibly phosphorylated or dephosphorylated to regulate the enzyme activity (Figure-7).This is covalent modification and is an important mechanism to regulate the activity of many enzymes. For example- Glycogen synthase a key regulatory enzyme for glycogen synthesis gets activated upon dephosphorylation, whereas phosphorylase, an enzyme of glycogen degradation becomes active upon phosphorylation. All the enzymes under the influence of Insulin are active in the dephosphorylated form while the enzymes under the influence of glucagon are active in the phosphorylated form.

 covalent modification

Figure-7- Reversible phosphorylation and dephosphorylation of serine residues for regulation of enzyme activity (covalent modification)

Apart from that serine is also found at the active site of many enzymes- Serine proteases- Coagulation factors and trypsin.

8) Formation of O- glycosidic linkages- In glycoproteins the carbohydrate groups are generally linked either by O-Glycosidic linkages or by N- Glycosidic linkages. The O-Glycosidic linkages are provided by –OH group of either serine or threonine, whereas the N-glycosidic linkages are provided by NH2 group of Asparagine (figure-8).

 Glycosidic linkages

Figure-8- O and N-Glycosidic linkages

9) Incorporation in to tissue proteins- Like other amino acids serine is also incorporated in to  tissue proteins.

10) Glucogenic- Serine undergoes non oxidative deamination to form Pyruvic acid that can be channeled towards pathway of gluconeogenesis.

Serine analogues- Azaserine and Cycloserine are serine analogues. They are used as drugs to inhibit nucleotide biosynthesis. Azaserine is an anticancer drug, whereas Cycloserine is used as an antitubercular drug.

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Metabolic role of glycine

Although nutritionally glycine is a non-essential amino acid but functionally it is very essential. The important functions of Glycine can be summarized as follows

i) As a constituent of protein

Glycine being a simple amino acid is found where the polypeptide bends as in beta bends or loops.

A striking characteristic of collagen is the occurrence of glycine residues at every third position of the triple helical portion of the alpha chain. This is necessary because glycine is the only amino acid small enough to be accommodated in the limited space available down the central core of the triple helix. This repeating structure, represented as (Gly-X-Y)n (figure-1), is an absolute requirement for the formation of the triple helix. While X and Y can be any other amino acids, about 100 of the X positions are proline and about 100 of the Y positions are hydroxy proline. Proline and hydroxy proline confer rigidity on the collagen molecule.

Collagen structure

Figure-1- Glycine is the most abundantly found amino acid in the structure of collagen. Every third amino acid in the structure of alpha helix is glycine.

ii) One carbon donor

The major pathway of glycine catabolism involves the cleavage of glycine to form CO2, NH4+ and N5N10 Methylene tetra hydro folate (Figure-2). Hence it acts as a donor of one carbon fragment.

Glycine cleavage system

Figure-2- The reaction is catalyzed by glycine cleavage system, N5,N10 Methylene tetra hydro folate acts as a carrier of one carbon fragment

iii) Synthesis of Glutathione

Glutathione is a tripeptide containing three amino acids- Glutamic acid, cysteine and glycine (gamma glutamyl cysteinyl glycine) – Figure-3. It is an important reducing agent, helps in maintaining the integrity of the red blood cells; also acts as a coenzyme in many reduction reactions.

Structure of glutathione

Figure-3- Structure of glutathione, GSH represents the reduced form of glutathione, the –SH group is contributed by cysteine

iv) Synthesis of creatine

Creatine (methyl guanido acetic acid ) is synthesized from three amino acids-Methionine, arginine and glycine. Methyl group is donated by Methionine; guanido group is contributed by Arginine and Acetic acid group comes from glycine. Creatine is stored in the muscle in the phosphorylated from- Creatine-P, a high energy compound. Creatinine is the anhydrous form of creatine (figure-4)

Synthesis of creatine

Figure-4- Role of glycine in the synthesis of creatine. Creatine –P also called phosphocreatine,  is a high energy compound that can be non enzymatically converted to creatinine, the excretable form of creatine.

v) Synthesis of purine nucleotide

Glycine contributes its entire structure for the formation of C4, C5 and N7 of purine nucleus (figure-5)

Components of purine ring

Figure-5- C4,C5 and N7 are derived form Glycine. Three amino acids – Glycine, aspartic acid and glutamine, contribute towards formation of  purine ring.

vi)  Synthesis of bile salts

Cholyl co A derived from cholesterol conjugates with glycine to form Glycocholic acid, a bile acid which is secreted in the bile in the form of sodium salt- Sodium glycocholate(figure-6).

Bile salts are required for the digestion and absorption of fats.
Role of glycine in bile salt formation

Figure- 6-Taurene is derived from cystiene. Glycocholic acid, tauro and glycochenodeoxy cholic acid are primary bile acids. The primary bile acids are converted to secondary bile acids by 7-Alpha dehydroxylation and deconjugation. Deoxy cholic acid and lithocholic acid are secondary bile acids.

vii) Detoxification

Aromatic compounds like benzoic acid obtained from diet are detoxified by conjugation with glycine to form hippuric acid (figure-7) which is excreted in urine.  This reaction takes place exclusively in liver. Hippuric acid excretion test is carried out to determine the functional status of liver.

Many drugs, drug metabolites, and other compounds with carboxyl groups are excreted in the urine as glycine conjugates.

Hippuric acid synthesis

Figure-7- Sodium benzoate is given as a loading dose and the amount of hippuric acid excreted in urine is estimated to determine the functional status of liver.

viii) Synthesis of heme

The two starting materials for heme synthesis are succinyl-CoA, derived from the citric acid cycle in mitochondria, and the amino acid glycine. By a series of reactions heme is synthesized (figure-8) that can be used for the synthesis of hemoglobin and other hemo proteins.

Role of glycine in heme synthesis

 

Figure-8- By a series of steps porphoblinogen is converted to heme.

ix) Synthesis of Glucose

Glycine is glucogenic in nature. During the course of its metabolism it is converted to serine (figure-9) which is non oxidatively deaminated to from pyruvate. Pyruvate is further channeled towards pathway of gluconeogenesis.

Serine and glycine interconversion

Figure-9- Glycine and serine are inter convertible. Serine forms pyruvate upon non oxidative deamination  which is a substrate for gluconeogenesis.

x) Glycine as a neurotransmitter

Glycine itself acts as neurotransmitter to regulate brain activities.

 

Clinical significance

Non ketotic hyperglycinemia- It is due to defect in the glycine cleavage system. Glycine level is found to be higher in blood, C.S.F and urine. Severe mental retardation and convulsions are observed. There is no permanent cure for this disorder only symptomatic treatment can be given.

Glycinuria

The disease is characterized by excessive excretion of glycine in urine. Urinary excretion of glycine ranges from 600-1000 mg/dl. Plasma level of glycine remains normal.

Biochemically there is no enzyme deficiency. The defect is attributed to renal tubular reabsorption of glycine. The tendency to oxalate stone formation is increased.

Hyperoxaluria

The disorder is characterized by continuous high excretion of oxalates. Biochemically it is a protein targeting effect. There is excessive oxalate formation from Glycine. The patients present with progressive bilateral calcium oxalate urolithiasis, recurrent urinary infections and renal damage.

Death occurs in childhood or early adult life from renal failure or hypertension.

 

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Metabolism of Glycine

Characteristics

  • Simple amino acid
  • Optically inactive
  • Nutritionally non-essential
  • Glucogenic in nature

Chemistry

Chemically glycine is amino acetic acid.

Glycine structure

Figure-1- Structure of glycine, since it lacks an asymmetric carbon atom, hence it is optically inactive.

Synthesis of Glycine

Glycine can be synthesized in three different ways-

1) From Serine

The alpha carbon of serine becomes Alpha carbon of glycine, whereas the beta carbon is channeled to one carbon pool. The reaction can be represented as –

Serine to glycine conversion

Figure-2- The reaction is catalyzed by Serine hydroxy methyl transferase, tetra hydrofolate is converted to N5, N10 Methylene tetra hydro folate

2) From Threonine

Glycine can also be synthesized from threonine by the action of threonine aldolase

Threonine to glycine conversion

3) De novo synthesis

Glycine can also be synthesized from its precursor molecules i.e. from CO2, NH4+ and one carbon unit. The reaction is catalyzed by Glycine synthase system. (Figure-3).

Glycine synthase system

Figure-3- The reaction is reversible; the same enzyme catalyzes the degradation of glycine also.

Catabolism of Glycine

1) Oxidative deamination

Glycine undergoes oxidative deamination. The reaction is catalyzed by Glycine oxidase, an enzyme that requires FAD as a coenzyme (figure-4). The reduced form of FAD (FADH2) is not oxidized through electron transport chain, it is oxidized at the expense of molecular oxygen forming H2O2.. The decomposition of H2O2.takes place by Catalase forming water and molecular oxygen that can be reutilized. That is the reason that amino acid oxidases and catalases are found together so as to decompose H2O2 quickly as soon as it is generated.

Oxidative deamination of glycine

Figure-4- The reaction proceeds through two steps, initially an imino acid is formed that undergoes hydration and deamination to produce glyoxalate.

2) Transamination- Like other amino acids, Glycine can undergo transamination to form Alpha keto acid (Glyoxalate)- Figure-5. The reaction is catalyzed by Alanine glyoxalate transaminase that requires B6-P as a coenzyme.

Fate of glyoxalate- Glyoxalate can undergo decarboxylation to produce formate that enters one carbon pool, hence this way glycine is a one carbon donor .

Alternatively glyoxalate can also be converted to oxalate by oxidation

Clinical Significance

Genetic defects in alanine-glyoxalate transaminase (either low activity or, rarely, a mutation that leads to the enzyme being in mitochondria rather than peroxisomes) results in hyperoxaluria. The glyoxalate formed by glycine oxidase cannot be recycled to glycine by transamination, but accumulates, and is a substrate for oxidation catalyzed by lactate dehydrogenase, forming oxalate. Oxalate crystallizes in the liver and kidneys, leading, to stone formation in urinary tract or  in severe cases, to early death.

Transamination of glycine

Figure-5- Glyoxalate is the end product of both transamination as well as oxidative deamination reactions

3) Formation of serine- Glycine can be converted to serine, which by non oxidative deamination can produce pyruvate, thus glycine can be considered glucogenic (figure-6).

Non oxidative deamination of serine

Figure- 6- The first step of the reaction sequence is catalyzed by dehydratase enzyme that requires B6-P as a coenzyme. The second step is same as oxidative deamination i.e. hydration followed by deamination.

4) Glycine cleavage- Major pathway involves the cleavage of glycine to form CO2, NH4+and N5N10 Methylene tetra hydro folate. The reaction is reversible and the glycine cleavage system is a multienzyme complex comprising of-

i) Glycine decarboxylase- P protein

ii) Amino methyl transferase- T protein

iii) Hydrogen carrier protein- H protein

iv) Dihydrolipoyl dehydrogenase- L -Protein

The net reaction can be represented as follows-

Glycine + H4folate + NAD+ ↔ 5,10-methylene-H4folate + CO2 + NH3 + NADH + H+

To be continued….

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Polyamines

Polyamines are low molecular weight aliphatic polycations, highly charged and ubiquitously present in all living cells. The naturally abundant polyamines are –

1)  Putrescine – H2N–(CH2)4–NH2 (diamine)

 2) Spermidine- H2N–(CH2)4–NH–(CH2)3–NH2 (triamine)

 3) Spermine H2N–(CH2)3–NH–(CH2)4–NH–(CH2)3–NH2 (tetramine)

Significance of Polyamines

The polyamines are involved in a large number of cellular processes. They  exert their role through ionic interactions, owing to their unique structural feature of regularly spaced positive charges. Some of the important functions are as follows-

1) Modulation of chromatin structure

2) Gene transcription and translation

3) DNA stabilization

4) Signal transduction

5) Cell growth and proliferation

6) Membrane stability

7) Functioning of ion channels 

8) Receptor-ligand interactions

9) Pharmacologic doses of polyamines are hypothermic and hypotensive

Since their primary and secondary amino groups are all protonated at physiological pH, putrescine is divalent, spermidine trivalent and spermine tetravalent organic cation. In the cells polyamines interact electrostatically with negatively charged moieties such as DNA, RNA, proteins and phospholipids.The unique feature of polyamine structure compared to inorganic cations like Mg2+ or Ca2+ is that they have positive charges at defined distances and between them methylene groups that can participate in hydrophobic interactions. Thus polyamines form stronger and more specific interactions than inorganic cations.

There is equilibrium between polyamines that are bound to different polyanionic molecules (mainly DNA and RNA) and free polyamines. The free polyamine pool represents 7-10% of the total cellular polyamine content. Only the free intracellular polyamines are available for immediate cellular needs and therefore are subject to strict regulation. Polyamines are maintained within very narrow range because decrease in their concentrations inhibits cell proliferation while excess appears to be toxic.Therefore, the free polyamine pools are regulated in a very fast, sensitive and precise manner.

Synthesis of Polyamines

Polyamine synthesis occurs in the cytoplasm of cells .Polyamines are synthesized from two amino acids: L-Methionine and L-Ornithine (an amino acid that is not incorporated  into tissue proteins, but is an intermediate  of urea cycle).

In mammalian cells, putrescine is formed by decarboxylation of ornithine, a reaction catalyzed by the enzyme ornithine decarboxylase (ODC). Ornithine is available from the plasma and can also be formed within the cell from arginine by the action of arginase. It is possible that arginase, which is much more widely distributed than other enzymes of the urea cycle, is present in extrahepatic tissues to ensure the availability of ornithine for polyamine production. Arginase can, therefore, be thought of as an initial step in polyamine biosynthesis.

For the synthesis of Putrescine , the amino propyl group must be added. This amino propyl moiety is derived from methionine, which is first converted into S-adenosylmethionine and is then decarboxylated. The resulting decarboxylated S-adenosylmethionine is used as an aminopropyl donor in an analogous manner to the use of S-adenosylmethionine itself as a methyl donor. Once it has been decarboxylated, S-adenosylmethionine is committed to Polyamine synthesis. Therefore the concentration of decarboxylated S- adenosylmethionine is kept low and constitutes the rate-limiting factor in spermidine formation. 

Synthesis of spermidine and spermine require the action of two enzymes: first, the S-adenosyl-methionine decarboxylase (AdoMetDC) for the synthesis of decarboxylated S-Adenosyl Methionine, the aminopropyl donor; and second, a transferase enzyme (spermidine synthase or spermine synthase) which catalyze the transfer of the aminopropyl group to the primary amine groups of putrescine or spermidine, respectively (decarboxylated S-Adenosyl Methionine reacts with Putrescine in the presence of Spermidine synthase forming Spermidine and that reacts with another molecule of decarboxylated S-Adenosyl Methionine in the presence of Spemine synthase forming Spermine) (Figure-1)

Ornithine decarboxylase is a B6-P dependent enzyme. It is present in very small amounts in quiescent cells, and its activity can be increased many fold within a few hours of exposure to  hormones, drugs, tissue regeneration, and growth factors. Spermidine  synthase and spermine synthase are discrete enzymes each specific for its own particular substrate.

The other product of the aminopropyl transferase reactions is 5’-methylthioadenosine. Although this nucleoside is produced in stoichiometric amounts with the polyamines, its concentration in the cell is kept low and is rapidly degraded.

Regulation of polyamine biosynthesis

Ornithine decarboxylase and S-Adenosyl decarboxylase are inducible enzymes with short half lives. Hormones like Growth hormone, corticosteroids, testosterone and growth factors increase the activity of Ornithine decarboxylase. Spermidine synthase and spermine synthase are non inducible enzymes.

The activity of S-Adenosyl methionine decarboxylase is inhibited by decarboxylated S-adenosyl Methionine and activated by Putrescine.

Interconversion

An interconversion or recycling pathway converts spermidine and spermine back to putrescine. Spermidine synthase and spermine synthase reactions are effectively irreversible, but it has been known for many years that conversion of spermine into spermidine and spermidine into putrescine can occur in vivo. This interconversion takes place by the action of two enzymes, spermidine-N1-acetyltransferase and polyamine oxidase. The former enzyme uses acetyl CoA to convert spermidine to N1-acetylspermidine and Spermine to N1-acetylspermine. The N1-acetylspermidine or N1-acetylspermine is then oxidized by polyamine oxidase , which cleaves the polyamine at a secondary amino nitrogen to release 3- acetamidopropion aldehyde  and putrescine or spermidine,  depending upon the substrate (Figure-1)

Degradation of Polyamines              

The enzyme polyamine oxidase present in liver peroxisomes oxidizes Spermine to Spermidine, that undergoes oxidation by the same enzyme to form Putrescine. Diamino propane is released, both these are converted to β-amino propane aldehyde. Putrescine is finally oxidized to NH+ and CO2. Major portions of polyamines are excreted in urine as acetylated derivatives (Figure-2)

Clinical Significance

The drug DFMO (Difluoromethyl ornithine) is a powerful inhibitor of polyamine biosynthesis. It inhibits ornithine decarboxylase enzyme and is used for the treatment of African sleeping sickness.

polyamine1

Figure 1. Biosynthesis and interconversion of polyamines. The enzymes catalyzing reactions are: 1.L-methionine S-adenosyltransferase, 2. S-adenosylmethionine decarboxylase , 3. Ornithine decarboxylase , 4. Spermidine synthase , 5. Spermine synthase, 6. Spermidine/spermine N1-acetyltransferase , and 7. Polyamine oxidase.

poly2

 

Figure-2 Showing the degradation of polyamines

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Alkaptonuria

Alkaptonuria (AKU) is a rare inherited genetic disorder of tyrosine metabolism characterized by the triad of Homogentisic aciduria, ochronosis and arthritis. It is one of the conditions in which Mendelian recessive inheritance was proposed.  It was also one of the four inborn errors of metabolism described by Garrod.

The most obvious sign in adults is a thickening and blue-black discoloration of the ear cartilage. This blue-black discoloration of connective tissue (including bone, cartilage, and skin) is caused by deposits of yellow or ochre-colored pigment, and is called Ochronosis.

Frequency

The condition is rare, affecting one in 250,000 to one million people worldwide. In US, the incidence is 1 case per 4 million populations.

Biochemical Defect

AKU is an autosomal recessive disorder, caused due to deficiency of Homogentisic acid oxidase (HGAO) which catalyzes the conversion of HGA (also called alkaptone) to maleyl acetoacetate (Figure-1). Inability to convert homogentisic acid to maleylacetoacetic acid results in accumulation of the former. Homogentisic acid is subsequently converted to benzoquinone acetic acid and spontaneously polymerized (Figure-1).

Pathophysiology

In the absence of the enzyme HGAO, Homogentisic acid and benzoquinone acetic acid (BQA) build up in the body (Figure-1).  Homogentisic acid is rapidly cleared in the kidney and excreted.

Although homogentisic acid blood levels are kept very low through rapid kidney clearance, over time homogentisic acid is deposited in cartilage throughout the body and is converted to the pigment like polymer through an enzyme-mediated reaction that occurs chiefly in collagenous tissues. As the polymer accumulates within cartilage, a process that takes many years, the normally transparent tissues become slate blue, an effect ordinarily not seen until adulthood.

The earliest sign of the disorder is the tendency for diapers to stain black. Throughout childhood and most of early adulthood, an asymptomatic, slowly progressive deposition of pigment like polymer material into collagenous tissues occurs.

In the fourth decade of life, external signs of pigment deposition, called ochronosis, begin to appear.

The slate blue, gray, or black discoloration of sclerae and ear cartilage is indicative of widespread staining of the body tissues, particularly cartilage. The hips, knees, and intervertebral joints are affected most commonly and show clinical symptoms resembling rheumatoid arthritis. Although unproven, the deposition of polymer is assumed to also cause an inflammatory response that results in calcium deposition in affected joints.

 

Clinical Manifestations

Most patients don’t have any symptoms throughout childhood or early adult life and it is not until they reach their 40’s that other signs of the disease start appearing.

  • One of the earliest signs is thickening of the ear cartilage (the pinna feels noticeably thickened and flexible). In addition the skin turns a blue-black color (Figure-2)

a2

 

 

Figure-2- Showing blackening of the ear cartilage

  • Earwax is often reddish-brown or jet-black.
  • Bones and cartilage of the lower back, knees, shoulders and hips are most affected. Firstly patients suffer low back pain with stiffness, followed by knee, shoulder and hip pain over the next 10 years. Cartilage becomes brittle and can break apart easily. In some cases this leads to spinal injuries such as prolapsed intervertebral discs.
  • Deposits around the trachea, larynx and bronchi may cause shortness of breath and difficulty breathing.
  • Deposits around the heart and blood vessels can calcify and lead to atherosclerotic plaques.
  • Pigmentation of the sclera of the eye usually occurs early on. This does not affect vision but appears as brown or grey deposits on the surface of the eye (Figure-3)

a3

 Figure-3- Showing black spots on the sclera

  • Skin color changes are most apparent on areas exposed to the sun and where sweat glands are found (Figure-4)

a4

 Figure-4- Showing blackening of the skin

  • Urine exposed to air can become dark; this is useful for diagnosing young children using diapers. The urine is malodorous.

 

 a5

 Figure-5- Showing the darkening of urine on standing.

Diagnosis

Presumptive diagnosis can be made by adding sodium or potassium hydroxide to urine and observing the formation of a dark brown to black pigment on the surface layer of urine within 30 minutes to 1 hour.

The fresh urine of an alkaptonuric appears normal but starts darkening on exposure to the air. This is caused by oxidation and polymerization of the HGA that speeds up on alkalization. Hence, (strongly) acidic urine may not darken for many hours on standing. This may be one of the reasons why darkening of the urine may not be noted in an affected child and the diagnosis is delayed until adulthood when arthritis or ochronosis appears.

HGA is a strong reducing substance that produces a positive reaction with Benedict’s and Fehling’s reagent. With Fehling’s (FeCl3) reagent, it gives transient blue-green Color. 

The diagnosis of alkaptonuria is confirmed by measurement of HGA concentration in the urine by paper and thin layer chromatography and photometry.

HGA is not elevated in the blood but excreted in the urine in heavy amounts – as much as 4-8gm / day.

Treatment

Alkaptonuria is a life long disease. There is no cure for the condition.Prevention is not possible and the treatment is aimed at ameliorating symptoms. Reducing intake of the amino acids phenylalanine and tyrosine to the minimum required to sustain health (phenylalanine is an essential amino acid) can help slow the progression of the disease. Vitamin C has been found to slow down the conversion of homogentisic acid to the polymeric deposits in cartilage and bone. A dose of up to 1g/day is recommended for older children and adults.

Medical therapy is used to ameliorate the rate of pigment deposition. This minimizes articular and cardiovascular complications in later life.

Reduction of phenylalanine and tyrosine has reportedly reduced homogentisic acid excretion. Whether a mild dietary restriction from early in life would avoid or minimize later complications is not known, but such an approach is reasonable.

Prognosis

Life expectancy is normal although patients may be at increased risk of heart conditions and may require surgical treatments for spine, hip, knee and shoulder joint problems. Exogenous cutaneous Ochronosis has been successfully treated by laser.

 

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Disease Biochemical defect and pathogenesis Laboratory findings Clinical Manifestations Treatment
Alkaptonuria It is caused by a defect in the enzyme Homogentisic acid oxidase.Due to deficiency Homogentisic acid remains in the body and slowly and progressively get deposited in bones and cartilages where it turns into a pigmented polymeric material. 

These polymerised Homogentisic acid products are excreted in large amounts in urine and impart the black color to urine.

 

This blue-black discoloration of connective tissue (including bone, cartilage, and skin) is caused by deposits of yellow or ochre-colored pigment and  is called Ochronosis.

 

 

Diagnosis can be confirmed by demonstrating the presence of Homogentisic acid in the urine.This may be done by paper chromatography  or Thin-layer chromatography.  The earliest signs is thickening of the ear cartilage, skin turns a blue-black colour,Bones and cartilage of the lower back, knees, shoulders and hips are most affected, Deposits around the trachea, larynx and bronchi may cause shortness of breath and difficulty in breathing deposits around the heart and blood vessels can calcify and lead to atherosclerotic plaques,  

Urine exposed to air can become dark; this is useful for diagnosing young children using diapers.

 

Sclera becomes pigmented but this does not affect the vision.

 

Reducing intake of the amino acids phenylalanine and tyrosine to the minimum .Vitamin C has been found to slow down the conversion of Homogentisic acid to the polymeric deposits in cartilage and bone.
Albinism Albinism is a defect of melanin production that results in little or no color (pigment) in the skin, hair, and eyes.There are three  main categories of albinism in humans:In oculo cutaneous albinism pigment is lacking in the eyes, skin and hair.In ocular albinism, only the eyes lack pigment.

 People who have ocular albinism have generally normal skin and hair color.

In patchy albinism– patches of depigmentation  are there on skin and hair

 

The diagnosis of the condition is based on the appearance of skin, hair, and eyes.Genetic testing offers the most accurate way to diagnose albinism and its type. Absence of coloring from the hair, skin, or iris of the eye, Long-term sun exposure greatly increases the risk of skin damage and skin cancers, including an aggressive form of skin cancer called melanoma, in people with this condition. Symptomatic treatment. Sun screen lotions and sun glasses are very helpful.Genetic counseling is  recommended  if the disease runs in families.
Phenylketonuria (PKU) Deficiency of the enzyme phenylalanine hydroxylase or of its cofactor tetrahydrobiopterin causes accumulation of phenylalanine in body fluids and the central nervous system (CNS).

 

In classic PKU, levels may range from 6 to 80mg/dl, but are usually greater than 30mg/dl. Identification and measurement of phenylketones in the urine has no place in any screening program.

 

Identification of phenylketones in the urine by ferric chloride may offer a simple test for diagnosis of infants with developmental and neurologic abnormalities.

CLASSICAL    PKU- The infants are normal at birth, Mental retardation may develop gradually, Vomiting, sometimes severe enough to be misdiagnosed as pyloric stenosis, may be an early symptom. Older untreated children become hyperactive with purposeless movements. On physical examination these infants are fairer in their complexion than unaffected siblings. About 25% of children have seizures, and more than 50% have electroencephalographic abnormalities.

Microcephaly, prominent maxilla with widely spaced teeth, enamel hypoplasia, and growth retardation are other common findings in untreated children.

 

 

The goal of PKU treatment is to maintain the blood level of phenylalanine between 2 and 10 mg/dl.Some phenylalanine is needed for normal growth.This requires a diet that has some phenylalanine but in much lower amounts than normal.Because phenylalanine is not synthesized by the body, “over treatment” may lead to phenylalanine deficiency manifested by lethargy, failure to thrive, anorexia, anemia, rashes, diarrhea, and even death; moreover, tyrosine becomes an essential amino acid in this disorder and its adequate intake must be ensured.

Oral administration of the cofactor (BH4 ) to patients with milder forms of hyperphenylalaninemia from phenylalanine hydroxylase deficiency may produce significant reductions.

 

 

 

Hart Nup Disease It is caused by impaired neutral (i.e., monoaminomonocarboxylic) amino acid transport in the apical brush border membrane of the small intestine and the proximal tubules of the kidney. Excessive amounts of amino acids, such as Tryptophan, are excreted in the urine.The body is thus left with inadequate amounts of amino acids. Urine Analysis –   Neutral amino acids, Tryptophan, Indole derivatives and Tryptophan degradation products are present in urine. Patients present with pellagra like skin eruptions, cerebellar ataxia,, nystagmus , tremor, failure to thrive, photosensitivity,  and gross aminoaciduria.Symptoms may be triggered by sunlight, fever, drugs, emotional or physical stress. A high-protein diet can overcome the deficient transport of neutral amino acids in most patients. Avoiding excessive exposure to sunlight, wearing protective clothing, and using physical and chemical sunscreens are mandatory, daily supplementation with nicotinic acid or nicotinamide reduces both number and severity of attacks.
Maple syrup urine disease MSUD is caused by a deficiency of the branched-chain alpha-Keto acid dehydrogenase complex (BCKDH), leading to a buildup of the branched-chain amino acids (leucine, Isoleucine, and Valine) and their toxic by-products in the blood and urine. Blood and urine in this disease have elevated levels of branched amino acids and their keto derivatives. The keto derivatives cause acidosis. Poor feeding, vomiting, dehydration, lethargy, hypotonia, seizures, Ketoacidosis, opisthotonus, pancreatitis, coma and neurological decline. The disease is characterized in an infant by the presence of sweet-smelling urine, with an odor similar to that of maple syrup. Infants with this disease seem healthy at birth but if left untreated suffer severe brain damage, and eventually die.  A diet with minimal levels of the amino acids leucine, Isoleucine, and Valine must be maintained in order to prevent neurological damage.As these three amino acids are required for proper metabolic functions, specialized protein preparations containing substitutes and adjusted levels of the amino acids have been synthesized and tested, allowing MSUD patients to meet normal nutritional requirements without causing harm.
Cystinuria The amino acid transporter for cystine and other amino acids like Arginine, ornithine and Lysine, is defective both in the intestine and in PCT of nephron. This leads to retention of cystine and other amino acids in urine. Cystine gets precipitated in acidic pH 1)Sodium cyanide nitroprusside test. in the presence of cystine a purple-red colour is observed.2) Flat x-ray of the kidney, ureters and bladder (KUB), is undertaken to visualize the stones.3) Microscopic examination  of urine  reveals flat hexagonal crystals of cystine.  Recurrent nephrolithiasis. Possible complications include obstruction of the urinary tract, which can predispose to infection of the urine ,fever may be apparent, and white blood cells are noted in the urine. Unrelieved obstruction leads to renal dysfunction, renal failure and the need for dialysis are quite rare. Medical therapy is directed toward dissolution of existing calculi and prevention of new stone formation.Increasing urine volume by generous oral fluid intake is beneficial.Cystine solubility can be improved by urinary alkalinization and where necessary by the administration of thiol chelators, particularly D-penicillamine or mercaptopropionyl glycine.

 

Carcinoid syndrome Carcinoid syndrome refers to the array of symptoms that occur secondary to Carcinoid tumors. Many of the symptoms of carcinoid syndrome are produced by serotonin or its metabolites. 24 hour urine levels of 5-HIAA (5-hydroxyindoleacetic acid), a breakdown product of serotonin. Patients with carcinoid syndrome usually excrete >25 mg of 5-HIAA per day. For localization of both primary lesions and metastasis, the initial imaging method is Octreoscan,  Flushing ,pellagra like rash, Secretory diarrhea and abdominal cramps are also characteristic features of the syndrome. When the diarrhea is intensive it may lead to electrolyte disturbance and dehydration. Other associated symptoms are nausea, and vomiting. About 50% of patients have cardiac abnormalities, caused by serotonin-induced fibrosis of the tricuspid and pulmonary valves. Symptomatic treatment,.octreotide (a somatostatin analogue that neutralizes serotonin and decreases urinary 5-HIAA),methysergide maleate (antiserotonin agent but not used because of serious side effect of retroperitoneal fibrosis),cyproheptadine (an antihistamine drug).Surgical resection of tumor and chemotherapy (5-FU and doxorubicin) are the alternative treatments. 
Homocystinuria (Homocysteinemia).

 

 

Three major forms of homocysteinemia and Homocystinuria have been identified :1) Homocystinuria due to Cystathionine beta Synthetase deficiency,(Classical Homocystinuria),2)  The other forms can be caused by defective Methionine synthase or reduced availability of two essential cofactors, 5-methyltetrahydrofolate and Methylcobalamine (methyl-vitamin B12).

 

Elevations of both Methionine and homocystine in body fluids are the diagnostic laboratory findings.Total plasma homocysteine is extremely elevated (usually >100 M). 

Cystine is low or absent in plasma.

 

The diagnosis may be established by assay of the enzyme in liver biopsy specimens, cultured fibroblasts.

 

Failure to thrive and developmental delay. Subluxation of the ocular lens (ectopia lentis) occurs. This causes severe myopia and iridodonesis (quivering of the iris), Astigmatism, glaucoma, cataracts, retinal detachment, and optic atrophy may develop later in life.  

Progressive mental retardation is common, skeletal abnormalities resemble those of Marfan syndrome;

Patients usually have fair complexions, blue eyes, and a peculiar malar flush.

Generalized osteoporosis, especially of the spine, is the main radiographic finding.

 

Thromboembolic episodes involving both large and small vessels, especially those of the brain, are common and may occur at any age.

 

 

 

Supplementation with pyridoxine, folic acid, B12 or trimethylglycine (betaine) reduces the concentration of homocysteine considerably in the blood stream. Those who do not respond require a low Methionine diet.
Cystinosis Cystinosis occurs due to a mutation in the gene CTNS, located on chromosome 17, which codes for cystinosin, the lysosomal cystine transporter. The accumulation is caused by abnormal transport of Cystine from lysosomes, resulting in a massive intra-lysosomal cystine accumulation in tissues. Via an as yet unknown mechanism, lysosomal cystine appears to amplify and alter apoptosis in such a way that cells die inappropriately, leading to loss of renal epithelial cells. This results in renal Fanconi syndrome and similar loss in other tissues can account for the short stature, retinopathy, and other features of the disease.  Definitive diagnosis and treatment monitoring are most often performed through measurement of white blood cell cystine level using tandem mass spectrometry.

 

 

Symptoms are first seen at about 3 to 18 months of age with profound polyuria (excessive urination), followed by poor growth, photophobia, and ultimately kidney failure by age 6 years in the nephropathic form. The other forms are Intermediate cystinosis (Juvenile) and Non-nephropathic or ocular cystinosis (Adult) forms. The drug cysteamine slows the progression of cystinosis by removing the cystine from cells, but for the drug treatment to be effective, it must be taken every six hours. Without specific treatment, these children progress to end-stage renal failure by an average age of nine years. Cystinosis is a common cause of the Fanconi Syndrome, a renal tubular disease.  

 

 

 

 

 

 

                                                    

 

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