**Abstract**

Inborn errors of metabolism (IEM) represent a group of inherited diseases in which genetic defect leads to the block on a metabolic pathway, resulting in a single enzyme dysfunction. As a downstream consequence of the residual or full loss of the enzymatic activity, there is an accumulation of toxic metabolites in the proximity of the metabolic block and/or a deficiency of an essential metabolic product which leads to the clinical presentation of the disease. While individually IEMs are rare, a collectively estimated incidence of metabolic inherited disorders is 1:800. The genetic basis of IEMs can involve abnormalities such as point mutations, deletions or insertions, or more complex genomic rearrangements. Categorization of IEM can be simply made on the basis of the affected metabolic network: fatty acids oxidation disorders, protein/amino acids metabolism disorders, disorders of carbohydrate metabolism, lysosomal storage diseases, peroxisomal disorders, and mitochondrial diseases. This chapter will overview amino acid metabolism-related inherited disorders and amino acid analysis for the diagnosis and routine monitoring of this category of IEMs.

**Keywords:** inborn error of metabolism, amino acids disorders, quantitative amino acids analysis, ion exchange chromatography, mass spectrometry

#### **1. Introduction**

Amino acids (**Figure 1**) play multiple important roles in our body: they are basic structural protein units and precursors of neurotransmitters, porphyrins, and nitric oxide. Furthermore, amino acids derived from the dietary proteins serve as energy source since while catabolized in our body, amino acids form organic acids that can replenish Krebs cycle and ammonia that eliminates through urea cycle [1].

Amino acids disorders (also called aminoacidopathies) are a group of inborn errors of metabolism diseases, caused by the inherited defects in pathways involved in amino acids metabolism. All primary amino acids disorders (**Table 1**) follow an autosomal recessive mode of inheritance which means that the mutation caused a metabolic block is present in the genetic material of both parents. As a result of mutation, the inherited defect is reflected downstream as a lack or a partial biological activity of enzymes involved in amino acids metabolism. Consequently, some substrates in these pathways accumulate or are diverted into alternative pathways. Therefore, amino acids disorders are biochemically characterized by abnormal levels of single or several amino acids and their downstream plasma and/or urine metabolites (**Tables 2–6**). Amino acid disorders are presented with variable and often nonspecific clinical symptoms. In conjunction with medical support, these disorders are managed by nutritional restrictions, supplements and medical

**112**

*Biochemical Testing - Clinical correlation and Diagnosis*

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complement pathway hemolytic assays reveal incomplete complement blockade in patients treated with eculizumab. Clinical Immunology. 2017;**183**:1-7

S, Garnier A, et al. Alternative

#### **Figure 1.**

*The general structure of amino acids consist of an amino group, a carboxylic group and a variable R side chain that has a major effect on solubility and polarity.*

foods that limit consumption of an offending amino acid or in some cases protein consumption. It is important therefore routinely perform amino acids' analysis to monitor dietary treatment outcomes in already diagnosed individuals. In the next chapters, primary amino acids disorders are reviewed and quantitative amino acid analysis in clinical settings is discussed.

#### **1.1 Phenylketonuria**

Phenylketonuria (commonly known as PKU, incidence 1 in 13,500–19,000 births in the United States [2]) is an inherited disorder of phenylalanine metabolism characterized by phenylalanine hydroxylase deficiency (**Figure 1**). The enzyme catalyzes the conversion of phenylalanine to tyrosine in the presence of tetrahydrobiopterin (BH4) as a cofactor. Based on plasma phenylalanine level, PKU is categorized by severe (Phe > 1200 μmol/L), mild (Phe = 600–1200 μmol/L) and hyperphenylalaninemia (above the normal cut off but below 600 μmol/L) phenotypes. Clinically PKU can be presented with growth failure, global developmental delay, severe intellectual disabilities and other severe symptoms. During pregnancy, elevated levels of phenylalanine have teratogenic effects on the developing fetus [3] and the condition is recognized as maternal PKU. Phenylalanine accumulation is also seen in defects of biopterin cofactor biosynthesis and regeneration [4] (**Table 1**). Nutritional management of PKU targets excessive accumulation of phenylalanine by restriction of natural protein intake in combination with the use of phenylalanine-free protein substitutes.

#### **1.2 Disorders of tyrosine metabolism**

Tyrosine metabolic pathway consists of five enzymatic reactions taking place mainly in hepatocytes and renal proximal tubules. Tyrosinemia I is the most severe inherited disorder of tyrosine metabolism caused by a deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. The disorder has a high incidence in French Canadian ethnicity [5] and involves hepatorenal dysfunction. Tyrosinemia II is caused by a deficiency of the hepatic tyrosine aminotransferase and manifested by mental retardation and other severe symptoms [6]. A deficiency in the activity of 4-hydroxyphenylpyruvate dioxygenase leads to tyrosinemia III, a rare disorder characterized by mild mental retardation and/or convulsions [7, 8]. All three disorders biochemically characterized by high levels of plasma tyrosine (hypertyrosinemia) and urine excretion of downstream tyrosine metabolites (**Table 1**). Elevated plasma tyrosine can also be seen due to vitamin-C responsive transient tyrosinemia during the neonatal period (**Figure 2**).

**115**

**Figure 2.**

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

**involved**

**Enzyme or transport defect**

Tyrosinemia I Tyr (B) high FAH Succinylacetone (DBS, U), 4-hydroxy-

Tyrosinemia II Tyr (B) high TAT 4-hydroxyphenylpyruvic, 4-hydroxy-

*PAH, phenylalanine hydroxylase; GTPCH, GTP cyclohydrolase; PTPS, 6-pyrovoyltetrahydropterin synthase; PCBD1, pterin-4a-carbinolamine dehydratase; DHPR, dihydropterin reductase; FAH, fumarylacetoacetate hydrolase; TAT,* 

*Reaction catalyzed by phenylalanine hydroxylase. Tetrahydrobiopterin (BH4) is a co-factor of PAH. DHPR,* 

*dihydropteridine reductase; PCBD1, pterin-4-α-carbinolamine dehydratase.*

Tyrosinemia III Tyr (B) high HPPD 4-hydroxyphenylpyruvic,

*tyrosine aminotransferase; HPPD, 4-hydroxyphenylpyruvate dioxygenase.*

*Laboratory findings in aromatic amino acids disorders.*

PKU classical Phe (B) high PAH Phe: Tyr ratio (B), phenylpyruvic, phenyllactic

Phe (B) high GTPCH Low biopterin, neopterin (U)

Phe (B) high PTPS Low biopterin, high neopterin (U)

Phe (B) high PCBD1 High neopterin and primapterin (U)

Phe (B) high DHPR High biopterin (U) and low DHPR activity in dried blood spots

(U)

phenyllactic acids (U)

4-hydroxyphenyllactic acids (U)

**Additional biomarkers**

and 2-hydroxyphenylacetic acids (U)

phenylpyruvic, 4-hydroxy-phenyllactic acids

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

**Aromatic amino acids disorders**

Defect of biopterin cofactor biosynthesis

Defect of biopterin cofactor biosynthesis

Defect of biopterin cofactor regeneration

Defect of biopterin cofactor regeneration

**Table 1.**

**Disorder name Amino acid** 


*PAH, phenylalanine hydroxylase; GTPCH, GTP cyclohydrolase; PTPS, 6-pyrovoyltetrahydropterin synthase; PCBD1, pterin-4a-carbinolamine dehydratase; DHPR, dihydropterin reductase; FAH, fumarylacetoacetate hydrolase; TAT, tyrosine aminotransferase; HPPD, 4-hydroxyphenylpyruvate dioxygenase.*

#### **Table 1.**

*Biochemical Testing - Clinical correlation and Diagnosis*

analysis in clinical settings is discussed.

*that has a major effect on solubility and polarity.*

**1.2 Disorders of tyrosine metabolism**

**1.1 Phenylketonuria**

**Figure 1.**

foods that limit consumption of an offending amino acid or in some cases protein consumption. It is important therefore routinely perform amino acids' analysis to monitor dietary treatment outcomes in already diagnosed individuals. In the next chapters, primary amino acids disorders are reviewed and quantitative amino acid

*The general structure of amino acids consist of an amino group, a carboxylic group and a variable R side chain* 

Phenylketonuria (commonly known as PKU, incidence 1 in 13,500–19,000 births in the United States [2]) is an inherited disorder of phenylalanine metabolism characterized by phenylalanine hydroxylase deficiency (**Figure 1**). The enzyme catalyzes the conversion of phenylalanine to tyrosine in the presence of tetrahydrobiopterin (BH4) as a cofactor. Based on plasma phenylalanine level, PKU is categorized by severe (Phe > 1200 μmol/L), mild (Phe = 600–1200 μmol/L) and hyperphenylalaninemia (above the normal cut off but below 600 μmol/L) phenotypes. Clinically PKU can be presented with growth failure, global developmental delay, severe intellectual disabilities and other severe symptoms. During pregnancy, elevated levels of phenylalanine have teratogenic effects on the developing fetus [3] and the condition is recognized as maternal PKU. Phenylalanine accumulation is also seen in defects of biopterin cofactor biosynthesis and regeneration [4] (**Table 1**). Nutritional management of PKU targets excessive accumulation of phenylalanine by restriction of natural protein

intake in combination with the use of phenylalanine-free protein substitutes.

Tyrosine metabolic pathway consists of five enzymatic reactions taking place mainly in hepatocytes and renal proximal tubules. Tyrosinemia I is the most severe inherited disorder of tyrosine metabolism caused by a deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. The disorder has a high incidence in French Canadian ethnicity [5] and involves hepatorenal dysfunction. Tyrosinemia II is caused by a deficiency of the hepatic tyrosine

aminotransferase and manifested by mental retardation and other severe symptoms [6]. A deficiency in the activity of 4-hydroxyphenylpyruvate dioxygenase leads to tyrosinemia III, a rare disorder characterized by mild mental retardation and/or convulsions [7, 8]. All three disorders biochemically characterized by high levels of plasma tyrosine (hypertyrosinemia) and urine excretion of downstream tyrosine metabolites (**Table 1**). Elevated plasma tyrosine can also be seen due to vitamin-C

responsive transient tyrosinemia during the neonatal period (**Figure 2**).

**114**

*Laboratory findings in aromatic amino acids disorders.*

#### **Figure 2.**

*Reaction catalyzed by phenylalanine hydroxylase. Tetrahydrobiopterin (BH4) is a co-factor of PAH. DHPR, dihydropteridine reductase; PCBD1, pterin-4-α-carbinolamine dehydratase.*

#### **1.3 Maple syrup urine disease**

Maple syrup urine disease is a disorder of branch chain amino acids metabolism caused by a deficiency of branched-chain α-keto acid dehydrogenase complex. MSUD is presented with five clinical phenotypes on the basis of the age at onset, the severity of symptoms and response to thiamine supplementation [9]. MSUD characterized biochemically by elevated plasma branched-chain amino acids (leucine, isoleucine, valine, allo-isoleucine) and their abnormal ratio (normal ratio is valine:is oleucine:leucine/3.5:1:2). The disease is managed by dietary leucine restriction, thus all branch chain amino acids and allo-isoleucine are routinely monitored. The classic MSUD is the most severe form of the disease characterized by no or very low residual enzyme activity and clinically manifested by developmental and neurological delays, encephalopathy, feeding problems, and a characteristic maple syrup odor in urine.


#### **Table 2.**

*Laboratory findings in MSUD.*

#### **1.4 Urea cycle disorders**

During protein catabolism, amino acids' carbon skeleton is metabolized to gluconeogenic and/or ketogenic precursors whereas nitrogen group is converted to ammonia through the deamination process. Toxic ammonia derived from amino acids and other metabolic sources is entering the urea cycle and further is converted to the readily excreted and nontoxic urea. The cycle takes place in the liver and a deficiency of any enzymes or transporters involved in the urea cycle can cause ammonia accumulation (hyperammonemia) which has a highly toxic effect on the central nervous system. The overall estimated incidence of urea cycle disorders is 1:8000. All urea cycle disorders have an autosomal recessive inheritance, with the exception of ornithine-transcarbamylase deficiency (OTCD), which is X-linked. Plasma citrulline is a key amino acid in the biochemical diagnosis of urea cycle defects (**Table 3**).

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by the mutations in the SLC25A15 or ORNT1 gene which result in the deficiency of ornithine translocase. The protein transports ornithine, lysine, and arginine across the inner mitochondrial membrane in peripheral tissues and pericentral hepatocytes. ORC1 deficiency reduces the availability of mitochondrial ornithine, which leads to the ornithine increase in the cytosol (hyperornithinemia). In the liver, since the mitochondrial ornithine is a required substrate for ornithine transcarbamylase (OTC), the reduced level of mitochondrial ornithine slows down flux through the urea cycle (**Figure 3**). As a result of the reduced capacity of the urea cycle, ammonia and carbamoyl-phosphate levels increase (hyperammonemia). At the same time, an excess of carbamoyl-phosphate is diverted to react with lysine to form homocitrulline (homocitrullinuria) or enters in the pyrimidine pathway, to form orotic acid which is later excreted in urine. Similarly, as for other urea cycle disorders, early diagnosis in infancy may improve the clinical outcome of HHH.

**117**

**Figure 3.**

*transcarbamylase.*

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

**involved**

elevated

Cit (B) moderate high, Met, Lys (B)

ASA (B), (U) elevated

**Enzyme or transport defect**

Asp/Glu mitochondrial exchanger

Cit (B) low OTC Nonspecific amino

Gln (B) high NAGS Cit (B) low, alanine high

CPS-I deficiency Cit (B) low CPS1 Can be accompanied

Argininemia Arg (B) high Arginase Orotic acid (U), Normal or

HHH Orn (B, U), high ORC1 Homocitrulline (U) high

*Urea cycle. ARG, arginase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; CPS1, carbamoylphosphate I synthase; ORNT 1, mitochondrial ornithine transporter; OTC, ornithine* 

**Additional biomarkers**

orotic acid (U), can be accompanied by high glutamine and alanine (B)

Hyperammonemia, orotic acid (U). Citrulline is moderately elevated

by high glutamine and

acid profile: increased glutamine, alanine and decreased ornithine, arginine (B). Orotic acid (U) markedly increased

reduced citrulline (B)

alanine (B)

arginine (B)

(B)

ASS Hyperammonemia,

ASL Low citrulline, low

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

**Disorder name Amino acid** 

Citrullinimia I Cit (B) markedly

**Urea cycle disorders**

Citrullinimia II/citrin

deficiency

Ornithine transcarbamylase deficiency

Arginino-succinic acidemia

Co-factor producing *N*-acetyl glutamate synthetase deficiency

*Laboratory findings in urea cycle disorders.*

*B, blood; U, urine.*

**Table 3.**


#### **Table 3.**

*Biochemical Testing - Clinical correlation and Diagnosis*

Maple syrup urine disease is a disorder of branch chain amino acids metabolism

caused by a deficiency of branched-chain α-keto acid dehydrogenase complex. MSUD is presented with five clinical phenotypes on the basis of the age at onset, the severity of symptoms and response to thiamine supplementation [9]. MSUD characterized biochemically by elevated plasma branched-chain amino acids (leucine, isoleucine, valine, allo-isoleucine) and their abnormal ratio (normal ratio is valine:is oleucine:leucine/3.5:1:2). The disease is managed by dietary leucine restriction, thus all branch chain amino acids and allo-isoleucine are routinely monitored. The classic MSUD is the most severe form of the disease characterized by no or very low residual enzyme activity and clinically manifested by developmental and neurological delays, encephalopathy, feeding problems, and a characteristic maple syrup odor in urine.

> **Enzyme or transport defect**

**Additional biomarkers**

BCKDC Plasma ratio of Val:Ile:Leu (3.5:1:2), branch chain 2-ketoacids and 2-hydroxyacids (U)

During protein catabolism, amino acids' carbon skeleton is metabolized to gluconeogenic and/or ketogenic precursors whereas nitrogen group is converted to ammonia through the deamination process. Toxic ammonia derived from amino acids and other metabolic sources is entering the urea cycle and further is converted to the readily excreted and nontoxic urea. The cycle takes place in the liver and a deficiency of any enzymes or transporters involved in the urea cycle can cause ammonia accumulation (hyperammonemia) which has a highly toxic effect on the central nervous system. The overall estimated incidence of urea cycle disorders is 1:8000. All urea cycle disorders have an autosomal recessive inheritance, with the exception of ornithine-transcarbamylase deficiency (OTCD), which is X-linked. Plasma citrulline is a key amino acid in the biochemical diagnosis of urea cycle defects (**Table 3**). Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by the mutations in the SLC25A15 or ORNT1 gene which result in the deficiency of ornithine translocase. The protein transports ornithine, lysine, and arginine across the inner mitochondrial membrane in peripheral tissues and pericentral hepatocytes. ORC1 deficiency reduces the availability of mitochondrial ornithine, which leads to the ornithine increase in the cytosol (hyperornithinemia). In the liver, since the mitochondrial ornithine is a required substrate for ornithine transcarbamylase (OTC), the reduced level of mitochondrial ornithine slows down flux through the urea cycle (**Figure 3**). As a result of the reduced capacity of the urea cycle, ammonia and carbamoyl-phosphate levels increase (hyperammonemia). At the same time, an excess of carbamoyl-phosphate is diverted to react with lysine to form homocitrulline (homocitrullinuria) or enters in the pyrimidine pathway, to form orotic acid which is later excreted in urine. Similarly, as for other urea cycle disorders, early diagnosis in infancy may improve the clinical outcome of HHH.

**1.3 Maple syrup urine disease**

**Disorders branched-chain amino acids**

MSUD BCAA (B) high,

**Amino acid involved**

allo-Ile (B) high

*BCKDC, branched-chain ketoacid dehydrogenase complex.*

**1.4 Urea cycle disorders**

*Laboratory findings in MSUD.*

**Disorder name**

**Table 2.**

**116**

*Laboratory findings in urea cycle disorders.*

#### **Figure 3.**

*Urea cycle. ARG, arginase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; CPS1, carbamoylphosphate I synthase; ORNT 1, mitochondrial ornithine transporter; OTC, ornithine transcarbamylase.*

### **1.5 Disorders of sulfur amino acids metabolism**

Homocystinuria is a disorder of methionine metabolism (**Figure 4**). The main biochemical finding in homocystinuria is accumulation of a sulfur-containing amino acid homocysteine and its metabolites in the blood and urine. Homocysteine is formed from methionine via transmethylation. Once generated homocysteine can be irreversibly degraded via transsulfuration pathway to cysteine or remethylated back to methionine by methionine synthase. Remethylation involves a transfer of methyl group from 5-methyltetrahydrofolate to homocysteine via cobalamin (Cbl) dependent methionine synthase (MT) and links folate cycle and homocysteine pathway. Homocysteine can also be remethylated through an additional pathway which involves liver and kidney betaine-homocysteine methyltransferase. Defects in any of these steps can result in homocystinuria. The classic homocystinuria is caused by cystathionine β-synthase (CBS) deficiency [10], a key enzyme in the trans-sulfuration pathway that converts homocysteine into cystathionine. A block at cystathionine β-synthase limits transsulfuration to the cysteine and results in both increased homocysteine and methionine, the latter caused by enhanced remethylation. The remethylation homocystinuria disorders include methylenetetrahydrofolate reductase deficiency (MTHFR) and defects of cobalamin (Cbl) metabolism [11]. It has to be noted that methionine and not homocysteine is analyzed through the newborn screening, thus, MTHFR disorder and the cobalamin defects may not be detected because methionine level in these disorders can be normal. To increase the detection rate in cobalamin related disorders and MTHFR, some studies report a benefit of adding total homocysteine analysis to the diagnostic workflow [12]. Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and protein-bound forms. The measurement of total homocysteine requires an immediate separation and freezing of the collected plasma.

#### **Figure 4.**

*Sulfur amino acids metabolism. CBS, cystathionine β synthase; Cbl, cobalamin; SAM, S-adenosyl methionine; SAH, S-adenosylhomocysteine; MAT, methionine adenosyltransferase; MS, methionine synthase; MTHFR, methylene tetrahydrofolate reductase; THF, tetrahydrofolate.*

**119**

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

**involved**

homocystine (B, U) high

SSC\*

SSC\*

Hyper-methioninemia Met (B) high MAT

*xanthine dehydrogenase; AO, aldehyde oxidase; GNMT, glycine-N-methyltransferase.*

(B, U) high SUOX,

**Enzyme or transport defect**

XDH, AO

Met (B) high AdoHcyas Mildly elevated total plasma Hcy,

(B, U) high SUOX Taurine, low cystine (B, U)

Met (B) high GNMT S-adenosylmethionine (B)

**Additional biomarkers**

S-adenosylhomocysteine, (B), S-adenosylmethionine (B)

Taurine, low cystine (B, U), elevated hypoxanthine and xanthine (U), low

CBS Total Hcy and methionine (B, U)

uric acid (B)

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

**Disorder name Amino acid** 

**Disorders of sulfur amino acids**

Homocystenuria Free

S-Adenosylhomocysteinehydroxylase deficiency

Sulfate oxydase deficiency

Glycine-*N*methyltransferase deficiency

*\**

*deficiencies.*

**Table 4.**

Molybden cofactor deficiency

**1.6 Nonketotic hyperglycemia**

*Laboratory findings in disorders of sulfur amino acids.*

**Disorders of amino acids transport Disorder name Amino acid** 

Cystinuria Cystine (U)

*Laboratory findings in renal aminoacidurias.*

Lysinuric protein intolerance

Fanconi syndrome

*U, urine.*

**Table 5.**

**involved**

elevated

Lys (U) markedly elevated

All amino acids elevated (U)

Nonketotic hyperglycemia (NKH) is a severe disorder of glycine metabolism. Glycine is catabolized through the four-peptide cleavage complex. P-protein, a pyridoxal phosphate-containing protein, T-protein, a protein required for the tetrahydrofolate-dependent reaction, H-protein, a protein that carries the aminomethyl intermediate and then hydrogen through the prosthetic lipoyl moiety, and L-protein, a lipoamide dehydrogenase. The disorder is so severe, that most of the affected individuals die within few months of life or survive with significant intellectual disabilities. Main laboratory findings in NKH is plasma and CSF elevated glycine.

*CBS, cystathionine beta synthase; AdoHcyas, S-adenosylhomocysteine hydroxylase; SUOX, sulfate oxidase; XDH,* 

*S-sulphocysteine may not be detectable in plasma using routine methods in sulfite oxidase and molybdenum co-factor* 

**Enzyme/transport defect Additional biomarkers**

Lysine, ornithine, arginine

increase (U)

Arginine, ornithine moderate increase (U), orotic acid (U)

Cystine and dibasic amino acids in GI tract and renal tubule

Defects in proximal renal tubule

Cationic amino acids transporter SLC7A7


*CBS, cystathionine beta synthase; AdoHcyas, S-adenosylhomocysteine hydroxylase; SUOX, sulfate oxidase; XDH, xanthine dehydrogenase; AO, aldehyde oxidase; GNMT, glycine-N-methyltransferase. \**

*S-sulphocysteine may not be detectable in plasma using routine methods in sulfite oxidase and molybdenum co-factor deficiencies.*

#### **Table 4.**

*Biochemical Testing - Clinical correlation and Diagnosis*

**1.5 Disorders of sulfur amino acids metabolism**

Homocystinuria is a disorder of methionine metabolism (**Figure 4**). The main biochemical finding in homocystinuria is accumulation of a sulfur-containing amino acid homocysteine and its metabolites in the blood and urine. Homocysteine is formed from methionine via transmethylation. Once generated homocysteine can be irreversibly degraded via transsulfuration pathway to cysteine or remethylated back to methionine by methionine synthase. Remethylation involves a transfer of methyl group from 5-methyltetrahydrofolate to homocysteine via cobalamin (Cbl) dependent methionine synthase (MT) and links folate cycle and homocysteine pathway. Homocysteine can also be remethylated through an additional pathway which involves liver and kidney betaine-homocysteine methyltransferase. Defects in any of these steps can result in homocystinuria. The classic homocystinuria is caused by cystathionine β-synthase (CBS) deficiency [10], a key enzyme in the trans-sulfuration pathway that converts homocysteine into cystathionine. A block at cystathionine β-synthase limits transsulfuration to the cysteine and results in both increased homocysteine and methionine, the latter caused by enhanced remethylation. The remethylation homocystinuria disorders include methylenetetrahydrofolate reductase deficiency (MTHFR) and defects of cobalamin (Cbl) metabolism [11]. It has to be noted that methionine and not homocysteine is analyzed through the newborn screening, thus, MTHFR disorder and the cobalamin defects may not be detected because methionine level in these disorders can be normal. To increase the detection rate in cobalamin related disorders and MTHFR, some studies report a benefit of adding total homocysteine analysis to the diagnostic workflow [12]. Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and protein-bound forms. The measurement of total homocysteine requires an immediate separation and freezing of the

*Sulfur amino acids metabolism. CBS, cystathionine β synthase; Cbl, cobalamin; SAM, S-adenosyl methionine; SAH, S-adenosylhomocysteine; MAT, methionine adenosyltransferase; MS, methionine synthase; MTHFR,* 

*methylene tetrahydrofolate reductase; THF, tetrahydrofolate.*

**118**

**Figure 4.**

collected plasma.

*Laboratory findings in disorders of sulfur amino acids.*
