AUTHOR INFORMATION

Section 1 of 12

Authored by Darius J Adams, MD, Assistant Professor, Department of Pediatrics, Section of Genetics and Metabolism, Albany Medical Center

Coauthored by Melissa Wasserstein, MD, Assistant Professor, Departments of Human Genetics and Pediatrics, Mount Sinai School of Medicine

Darius J Adams, MD, is a member of the following medical societies: American Academy of Pediatrics

Edited by Robert D Steiner, MD, Professor, Departments of Pediatrics and Molecular and Medical Genetics, Vice Chair for Research, Head of Division of Metabolism, Department of Pediatrics, Oregon Health & Science University; Director, Consulting Staff, Metabolic Bone Disease Clinic, Shriner's Hospital; Robert Konop, PharmD, Director, Clinical Account Management, Ancillary Care Management, Inc; Leonard G Feld, MD, PhD, MMM, Chairman of Pediatrics, Carolinas Medical Center; Chief Medical Officer, Levine Children's Hospital, Carolinas Healthcare System; Paul D Petry, DO, FACOP, FAAP, Clinical Assistant Professor of Pediatrics, University of North Dakota, School of Medicine and Health Sciences; Consulting Staff, Altru Health System; and Bruce A Buehler, MD, Professor, Department of Pathology and Microbiology, Chairman, Department of Pediatrics, Director, Hattie B Munroe Center for Human Genetics, University of Nebraska Medical Center

Author's Email:	Darius J Adams, MD
Editor's Email:	Robert D Steiner, MD

eMedicine Journal, February 24 2005, VOLUME 6, Number 2

INTRODUCTION

Section 2 of 12

Background: Glutathione synthetase (GS) deficiency, first described in 1970, is a rare inborn error of glutathione metabolism characterized by severe metabolic acidosis, hemolytic anemia, and neurological problems. Biochemical findings include massive excretion of 5-oxoproline in the urine. In mild GS deficiency, which is characterized by hemolytic anemia, enzyme deficiency occurs primarily in erythrocytes.

Pathophysiology: Glutathione is involved in several important biologic functions, including membrane transport, detoxification of xenobiotics, and protection of cells from free radicals. Glutathione is produced from the amino acids cysteine, glycine, and glutamine via the consecutive actions of gamma-glutamylcysteine synthetase and GS. It is also widely used by red blood cells, which are vulnerable to oxidative damage caused by peroxides. Reduced glutathione is required as an antioxidant in these cases.

Multiple mutations causing GS deficiency have been described for the GS gene, GSS. The erythrocyte variant has been linked to a homozygous missense mutation causing enzyme instability; thus, enzyme deficiency is most significant in erythrocytes and manifests as hemolytic anemia. Thirteen different missense mutations in GSS have been identified in individuals with severe GS deficiency. The mutations were found in 9 unrelated patients from different geographic areas. Two of these mutations were in individuals who were found to have central nervous system (CNS) involvement. In all cases, residual enzyme activity was noted, indicating that a complete loss of enzyme function is probably lethal.

Frequency:

• In the US: Frequency is unknown.

• Internationally: This condition is very rare. Worldwide, only approximately 40-50 cases in which the patient survived the newborn period have been published. Overall frequency is unknown.

Mortality/Morbidity: Recently, authors have recommended that 3 forms of GS deficiency be identified: mild, moderate, and severe (see History). In the severe systemic form, chronic metabolic acidosis must be managed. Long-term prognosis is guarded. With careful treatment during infancy, many patients survive, and the metabolic acidosis may become more manageable after infancy. The lack of glutathione in erythrocytes alone is apparently tolerable, as has been noted with the mild form of this condition; however, in severe GS deficiency, a progressive loss of function occurs, leading to severe mental retardation, ataxia, and seizure disorders. According to one review, the oldest reported survivor with the severe from was aged 24 years and had experienced significant neurological deterioration over the previous few years. Reports also exist of patients with the moderate or mild forms with long-term survival and little or no neurological sequelae.

Race: No race predilection exists.

Sex: No sex predilection exists.

Age: Most individuals with systemic GS deficiency are diagnosed in the newborn period. However, with the isolated erythrocyte form, the diagnosis may not be made until adulthood, even though hemolytic anemia is present at birth.

CLINICAL

Section 3 of 12

History: The phenotypic manifestations that have been described in association with GS deficiency include hemolytic anemia (occurring in mild GS deficiency) and 5-oxoprolinuria (pyroglutamicaciduria) and variable degrees of secondary neurological involvement (occurring in systemic GS deficiency). As stated in Mortality/Morbidity, authors have recently suggested GS deficiency be described as mild, moderate, and severe. These categories represent a continuum of disease severity that is dependent on the degree of enzyme function; therefore, patients can have manifestations anywhere along the continuum of mild to severe GS deficiency.

• The severe phenotypic manifestation, 5-oxoprolinuria (pyroglutamicaciduria), resulting from systemic GS deficiency, is an autosomal recessive disorder. It has been characterized by very large peaks of 5-oxoproline on urine organic acid analysis, metabolic acidosis, hemolytic anemia, and eventual CNS damage. Because of deficient enzyme activity, a decreased quantity of glutathione results, which likely causes promoter activation of the GSS gene. The large amounts of gamma-glutamylcysteine synthetase that are produced increase the pool of gamma-glutamylcysteine, which is then converted to 5-oxproline because of the inability of the defective GSS gene to produce glutathione.



• In moderate GS deficiency, neonatal acidosis and hemolytic anemia are present, but neurological involvement is not. The prognosis for the moderate form is intermediate.



• In mild GS deficiency, hemolytic anemia is the primary finding with apparently no effects outside of erythrocytes. Individuals with this form do well clinically.

Physical: Patients with GS deficiency appear healthy and do not have unusual dysmorphic features.

• In severe GS deficiency, neurological findings are the most prevalent and may include the following:


• • Spasticity (spastic tetraparesis)
  
  
  
  • Ataxia and other cerebellar findings
  
  
  
  • Intention tremors
  
  
  
  • Dysarthria
  
  
  
  • Mental retardation
  
  
  
  • Psychosis
  
  
  
  • Seizure disorders
  
  
  
  • Eye abnormalities, which tend to be peripheral retinal pigmentation abnormalities
  
  
  
• Individuals with moderate GS deficiency may have apparent respiratory distress as their bodies try to correct metabolic acidosis; however, other signs are usually not present.



• In mild GS deficiency, physical findings are not present.

Causes: Southern blot hybridizations that have been performed with a GS complementary DNA (cDNA) have revealed that only 1 GSS gene is present in the human genome. It is located at band 20q11.2. These findings suggest that the different phenotypic types observed in GS deficiency are part of a spectrum of disease that is dependent on the degree of GS function.

DIFFERENTIALS

Section 4 of 12

Acidosis, Metabolic
Galactose-1-Phosphate Uridyltransferase Deficiency (Galactosemia)
Methylmalonic Acidemia
Propionic Acidemia (Propionyl CoA Carboxylase Deficiency)

Other Problems to be Considered:

Distal renal tubular acidosis, autosomal dominant
Cobalamin C disease
Cobalamin D disease

WORKUP

Section 5 of 12

Lab Studies:

• Electrolyte and blood gas determinations indicate high anion gap metabolic acidosis. The CBC count demonstrates hemolytic anemia. Diagnosis of GS deficiency is confirmed by the presence of a large peak of 5-oxoproline in the urine. Concentrations of this metabolite are also increased in the cerebrospinal fluid (CSF) and blood. Diagnosis has been confirmed through enzyme analysis of GS (which is found to be deficient) in cultured skin fibroblasts. On routine testing, in addition to a hemolytic anemia, episodic neutropenia may occur. Additional findings include the following:


• • Neutrophil bactericidal and iodination defects, which are corrected with vitamin E therapy
  
  
  
  • Pyroglutamic aciduria
  
  
  
  • Pyroglutamic acidemia
  
  
  
  • Decreased glutathione in erythrocytes
  
  
  
  • Increased gamma-glutamylcysteine synthetase
  
  
  

TREATMENT

Section 6 of 12

Medical Care: Treatment of individuals who have been diagnosed with GS deficiency involves providing supplements to correct the metabolic acidosis and supplying antioxidants such as vitamin E and vitamin C. A combination of sodium citrate and citric acid (Bicitra) may be used as an oral medication and can maintain plasma bicarbonate levels within the reference range. Alternatively, bicarbonate may be used; however, very large doses may be needed.

• The prognosis for GS deficiency is quite variable. In some cases, early use of sodium citrate and citric acid (Bicitra) or other buffers, vitamin C, and vitamin E may allow normal development to occur.



• Experiments using lipoic acid as an intracerebral antioxidant have been performed in animal models. Lipoic acid penetrates the blood-brain barrier well and may prevent the onset of learning disabilities in children with GS deficiency. However, individuals may have an absolute requirement of glutathione for the production of certain leukotrienes and possibly even neurotransmitters. If this is the case, lipoic acid may not effectively correct the problem.



• N-acetylcysteine (NAC) has been used in patients with GS deficiency because it is thought to increase the low intracellular glutathione concentrations and cysteine availability in the leukocytes of these patients. Whether these findings in leukocytes may result in similar changes in neurons is not yet known.

Consultations: Consultations with a clinical biochemical geneticist and/or metabolic-disease specialist and hematologist may be indicated.

MEDICATION

Section 7 of 12

Treatment of individuals who have been diagnosed with GS deficiency involves providing supplements to correct the metabolic acidosis and supplying antioxidants such as vitamin E and vitamin C. NAC has been used in patients with GS deficiency because it is thought to increase the low intracellular glutathione concentrations and cysteine availability in the leukocytes of patients with this disorder. Use of sodium citrate and citric acid (Bicitra), vitamin C and vitamin E, thioctic acid (ie, lipoic acid), and NAC are included here.

Drug Category: Alkalinizing agents -- Sodium bicarbonate is used as a gastric, systemic, and urinary alkalinizer and has been used in the treatment of acidosis resulting from metabolic and respiratory causes, including diabetic coma, diarrhea, kidney disturbances, and shock. Sodium bicarbonate also increases renal clearance of acidic drugs. Citric acid mixtures may also be used. With normal hepatic function, 1 mEq of citrate is converted to 1 mEq of bicarbonate.

Drug Name	Sodium citrate and citric acid (Bicitra) -- An oral medication useful in outpatient treatment of individuals with persistent acidosis. Each mL contains 1 mEq sodium ion and is equivalent to 1 mEq of bicarbonate. Also contains butylparaben, flavoring, and sodium saccharin. In certain situations, potassium citrate (as contained in Polycitra-K) may be preferable. Palatability enhanced if chilled before swallowing.
Adult Dose	2-6 tsp (10-30 mL) diluted in 1-3 oz water, followed by additional water if desired, PO pc and qhs or as directed
Pediatric Dose	<2 years: Based on consultation with physician >2 years: 1-3 tsp (5-15 mL) diluted in 1-3 oz water, followed by additional water if desired, PO pc and qhs or as directed
Contraindications	Renal insufficiency and patients in sodium-restricted diet
Interactions	Urine alkalinization may decrease serum levels of lithium, chlorpropamide, methenamine, methotrexate, salicylates, or tetracyclines; urine alkalinization may increase serum levels of flecainide, quinidine, or sympathomimetics; coadministration with aluminum-containing antacids may increase serum aluminum levels
Pregnancy	C - Safety for use during pregnancy has not been established.
Precautions	Caution with low urinary output unless under the supervision of a physician; dilute adequately with water and ingest each dose pc; caution in patients with cardiac failure, hypertension, impaired renal function, peripheral and pulmonary edema, and toxemia of pregnancy; periodic examinations and determinations of serum electrolytes, particularly serum bicarbonate level, should be done in those patients with renal disease Conversion to bicarbonate may be impaired in hepatic failure, shock, and in severely ill patients

Drug Category: Vitamins and antioxidants -- These are organic substances required by the body in small amounts for various metabolic processes. Vitamins may be synthesized in small or insufficient amounts in the body or not synthesized at all, thus requiring supplementation. They are used clinically for the prevention and treatment of specific vitamin deficiency states.

Drug Name	Ascorbic acid (Vita-C, Cecon) -- An antioxidant; one of the water-soluble vitamins.
Adult Dose	200-4000 mg/d or 100 mg/kg/d PO
Pediatric Dose	100 mg/kg/d PO
Contraindications	Documented hypersensitivity; patients with renal failure have difficulty clearing vitamin C, which can result in acidosis
Interactions	Decreases effects of warfarin and fluphenazine; increases aspirin levels
Pregnancy	A - Safe in pregnancy
Precautions	Prolonged high doses may cause renal calculi, especially in patients with diabetes

Drug Name	Vitamin E (Vita-Plus E Softgels, Aquasol E) -- An antioxidant; one of the fat-soluble vitamins.
Adult Dose	10 mg/kg/d PO; up to 3000 mg/d has been used and is probably safe
Pediatric Dose	Administer as in adults
Contraindications	Documented hypersensitivity
Interactions	Mineral oil decreases absorption of vitamin E; vitamin E delays absorption of iron and increases effects of anticoagulants
Pregnancy	A - Safe in pregnancy
Precautions	Pregnancy category C with doses exceeding the RDA; may induce vitamin K deficiency; necrotizing enterocolitis may occur with large doses

Drug Name	Thioctic acid (Thiocid) -- Also called alpha-lipoic acid. An antioxidant considered to be more effective than vitamin E or C in crossing the blood-brain barrier.
Adult Dose	100 mg/d PO; administer on empty stomach
Pediatric Dose	Administer as in adults
Contraindications	Documented hypersensitivity
Interactions	Ethanol may antagonize actions; additive effect with insulin or oral hypoglycemic agents; antagonizes cisplatin effects
Pregnancy	C - Safety for use during pregnancy has not been established.
Precautions	May decrease blood glucose; temporary worsening of neuropathy following initiation of treatment has been observed

Drug Category: Amino acids -- NAC is the N-acetyl derivative of the amino acid cysteine. NAC enhances the levels of glutathione in the liver, plasma, and bronchioalveolar lavage fluid. It is used to treat various diseases with the underlying etiology of decreased glutathione.

Drug Name	N-acetylcysteine (Mucomyst) -- Has been used with GS deficiency because it is thought to increase low intracellular glutathione concentrations and cysteine availability in leukocytes.
Adult Dose	Nebulization into a face mask, mouth piece, or tracheostomy: 1-10 mL of the 20% solution or 2-20 mL of the 10% solution may be given q2-6h; recommended dose for most patients is 3-5 mL of the 20% solution or 6-10 mL of the 10% solution tid/qid
Pediatric Dose	Administer as in adults
Contraindications	Documented hypersensitivity
Interactions	None reported
Pregnancy	B - Usually safe but benefits must outweigh the risks.
Precautions	Possible transient disagreeable odor upon initiation of treatment but soon not noticeable; with face mask, stickiness on face may occur after nebulization, which is easily removed with water Under certain conditions, color change may take place in the solution of acetylcysteine in opened bottle; light purple color is result of chemical reaction that does not significantly impair the safety or mucolytic effectiveness of acetylcysteine Continued nebulization of acetylcysteine solution with a dry gas results in increased concentration of drug in nebulizer because of evaporation of solvent; extreme concentration may impede nebulization and efficient delivery of drug; dilution of nebulizing solutions with sterile water for injection, USP as concentration occurs, obviates this problem

FOLLOW-UP

Section 8 of 12

Further Outpatient Care:

• GS deficiency is a chronic life-threatening disorder. Regular follow-up with a metabolic-disease specialist may be indicated. All patients should be encouraged to wear a medical alert bracelet or necklace.

Transfer:

• Transfer to a center where metabolic-disease specialists are available may be indicated.

Deterrence/Prevention:

• Because accumulation of 5-oxoproline occurs in all body fluids, especially the urine, of affected individuals, the possibility of prenatal diagnosis with amniotic fluid was realized; most of this fluid is from fetal urine after the first trimester. In 1994, 2 pregnancies of 2 at-risk couples were studied at 16 weeks' gestation. In both cases, the levels of 5-oxoproline were 25-30 times reference range values. Both pregnancies were terminated, and the diagnosis was confirmed in 1 case by cultured fetal fibroblast enzyme analysis.

Prognosis:

• In the systemic form, chronic metabolic acidosis must be treated, but long-term prognosis is guarded. The lack of glutathione in erythrocytes alone is apparently tolerable, as has been noted with the peripheral form of this condition; however, in severe GS, a progressive loss of function occurs, leading to severe mental retardation, ataxia, and seizure disorders. The oldest reported survivor of severe GS was aged 24 years and had experienced significant neurological deterioration over the previous few years. Reports exist of older children with mild and moderate forms who are doing well.

MISCELLANEOUS

Section 9 of 12

Medical/Legal Pitfalls:

• Severe high anion gap metabolic acidosis may be attributed to poisoning. The laboratory diagnosis of this condition is nontrivial and requires ordering the correct test (ie, urine organic acids). Laboratory personnel with experience in detecting 5-oxoproline should perform this test.

TEST QUESTIONS

Section 10 of 12

CME Question 1: A newborn presents with hemolytic anemia and respiratory distress. Which of the following would be the most appropriate screening test for glutathione synthetase deficiency?

A: Plasma amino acid analysis
B: Urine organic acid analysis
C: Electrolyte level test
D: Urine amino acid analysis
E: Liver function test

The correct answer is B: Urine organic acid analysis reveals a large peak of 5-oxoproline, which occurs when a small glutathione pool is present. Findings from the other tests would not show the presence of this metabolite.

CME Question 2: A child who has been diagnosed with glutathione synthetase deficiency presents with frequent bacterial infections. Which of the following therapies may provide improvement?

A: Lipoic acid
B: Vitamin D
C: Vitamin E
D: Coenzyme Q10
E: Vitamin K

The correct answer is C: Individuals with the severe form of glutathione synthetase deficiency have neutrophil bactericidal and iodination defects, which can be corrected with vitamin E therapy.

Pearl Question 1 (T/F): Most of the mutations in glutathione synthetase deficiency are of the missense type.

The correct answer is True: Most cases reported to date in which molecular analysis has been performed have revealed missense mutations. Multiple mutations causing glutathione synthetase deficiency have been described for the glutathione synthetase gene, GSS. The erythrocyte variant has been linked to a homozygous missense mutation causing enzyme instability; thus, enzyme deficiency is most significant in erythrocytes and manifests as hemolytic anemia. Thirteen different missense mutations in the GSS gene have been identified in individuals with severe glutathione synthetase deficiency.

Pearl Question 2 (T/F): All forms of glutathione synthetase deficiency are usually diagnosed in the newborn period.

The correct answer is False: The peripheral form of glutathione synthetase may not be diagnosed until adulthood because individuals with this form of the disorder do not have any other findings aside from hemolytic anemia.

Pearl Question 3 (T/F): Reduced glutathione is required as an antioxidant in red blood cells in order to prevent oxidative damage caused by peroxides.

The correct answer is True: The hemolytic anemia in individuals with glutathione synthetase deficiency is caused by the lack of available glutathione.

Pearl Question 4 (T/F): Two separate genes have been found in glutathione synthetase deficiency, and mutations in these genes result in either the mild or severe form.

The correct answer is False: Only 1 glutathione synthetase gene, GSS, located at band 20q11.2, is present in the human genome. This suggests that the different phenotypic types observed are part of a spectrum of disease, which is dependent on the degree of glutathione synthetase function.

PICTURES

Section 11 of 12

Caption: Picture 1. Biochemical pathway of Glutathione Synthetase.
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BIBLIOGRAPHY

Section 12 of 12

• Boxer LA, Oliver JM, Spielberg SP, et al: Protection of granulocytes by vitamin E in glutathione synthetase deficiency. N Engl J Med 1979 Oct 25; 301(17): 901-5[Medline].
• Brüggemann LW, Groenendaal F, Ristoff E: Glutathione synthetase deficiency associated with antenatal cerebral bleeding. J Inherit Metab Dis 2004; 27(2): 275-6[Medline].
• Dahl N, Pigg M, Ristoff E, et al: Missense mutations in the human glutathione synthetase gene result in severe metabolic acidosis, 5-oxoprolinuria, hemolytic anemia and neurological dysfunction. Hum Mol Genet 1997 Jul; 6(7): 1147-52[Medline].
• Divry P, Roulaud-Parrot F, Dorche C, et al: 5-Oxoprolinuria (glutathione synthetase deficiency): a case with neonatal presentation and rapid fatal outcome. J Inherit Metab Dis 1991; 14(3): 341-4[Medline].
• Erasmus E, Mienie LJ, de Vries WN, et al: Prenatal analysis in two suspected cases of glutathione synthetase deficiency. J Inherit Metab Dis 1993; 16(5): 837-43[Medline].
• Fily A, Vaillant C, Truffert P: [Gluthathion synthetase deficit in a newborn infant.]. Arch Pediatr 2004 Nov; 11(11): 1339-41[Medline].
• Jellum E, Kluge T, Borresen HC, et al: Pyroglutamic aciduria--a new inborn error of metabolism. Scand J Clin Lab Invest 1970 Dec; 26(4): 327-35[Medline].
• Larsson A, Zetterstrom R, Hagenfeldt L, et al: Pyroglutamic aciduria (5-oxoprolinuria), an inborn error in glutathione metabolism. Pediatr Res 1974 Oct; 8(10): 852-6[Medline].
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• Martensson J, Gustafsson J, Larsson A: A therapeutic trial with N-acetylcysteine in subjects with hereditary glutathione synthetase deficiency (5-oxoprolinuria). J Inherit Metab Dis 1989; 12(2): 120-30[Medline].
• Meister A, Anderson ME: Glutathione. Annu Rev Biochem 1983; 52: 711-60[Medline].
• Mohler DN, Majerus PW, Minnich V, et al: Glutathione synthetase deficiency as a cause of hereditary hemolytic disease. N Engl J Med 1970 Dec 3; 283(23): 1253-7[Medline].
• Njalsson R, Carlsson K, Winkler A, et al: Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene. Hum Mutat 2003 Dec; 22(6): 497[Medline].
• Ristoff E, Mayatepek E, Larsson A: Long-term clinical outcome in patients with glutathione synthetase deficiency. J Pediatr 2001 Jul; 139(1): 79-84[Medline].
• Robertson PL, Buchanan DN, Muenzer J: 5-Oxoprolinuria in an adolescent with chronic metabolic acidosis, mental retardation, and psychosis. J Pediatr 1991 Jan; 118(1): 92-5[Medline].
• Shi ZZ, Habib GM, Rhead WJ, et al: Mutations in the glutathione synthetase gene cause 5-oxoprolinuria. Nat Genet 1996 Nov; 14(3): 361-5[Medline].
• Spielberg SP, Boxer LA, Oliver JM, et al: Oxidative damage to neutrophils in glutathione synthetase deficiency. Br J Haematol 1979 Jun; 42(2): 215-23[Medline].
• Uhlig S, Wendel A: The physiological consequences of glutathione variations. Life Sci 1992; 51(14): 1083-94[Medline].
• Webb GC, Vaska VL, Gali RR, et al: The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2. Genomics 1995 Dec 10; 30(3): 617-9[Medline].
• Yapicioğlu H, Satar M, Tutak E: A newborn infant with generalized glutathione synthetase deficiency. Turk J Pediatr 2004 Jan-Mar; 46(1): 72-5[Medline].

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