Glutathione Synthetase Deficiency Research Articles

Severe Hemolytic Anemia and Metabolic Acidosis at Birth with Glutathione Synthetase Deficiency and Progressive Neurological Symptoms on Follow-Up.

BACKGROUND Glutathione synthetase deficiency (GSD) is a rare autosomal recessive disorder caused by glutathione synthetase (GSS) gene variants that occur in 1 in 1 million individuals. The severe form of GSD is characterized by hemolytic anemia, metabolic acidosis with 5-oxoprolinuria, progressive neurological symptoms, and recurrent bacterial infections. This case report presents a male Japanese infant with severe hemolytic anemia and metabolic acidosis at birth caused by GSD, who developed progressive neurological symptoms on follow-up. CASE REPORT A Japanese male term infant developed severe hemolytic anemia and metabolic acidosis in the early neonatal period. We suspected GSD based on his symptoms and a high 5-oxoproline urine concentration. We began correcting his metabolic acidosis and administering vitamins C and E supplements. The patient required blood transfusion twice during the acute phase for hemolytic anemia. After age 1 month, he maintained good control of metabolic acidosis and hemolytic anemia. A definitive diagnosis of GSD was made based on high concentrations of 5-oxoproline in urine, low concentrations of glutathione and GSS activity in erythrocytes, and genetic testing. Several episodes of febrile convulsions were started at age 11 months, but none occurred after 2 years. At the last follow-up at age 25 months, metabolic acidosis and hemolytic anemia were well controlled, but he had mild neurodevelopmental delay. CONCLUSIONS This case report shows that GSD can present with severe hemolytic anemia and metabolic acidosis at birth, and manifest with subsequent neurological impairment despite early diagnosis and treatment. Therefore, a careful long-term follow-up that includes neurological evaluation is essential for patients with GSD.

Genetic Spectrum of Inherited/Congenital Hemolytic Anemias in Indian Patients

IntroductionHemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling.ObjectivesThis study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests.MethodsSeventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members.ResultsAfter targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing.Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found.Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4).We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1.We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns.A total of seven patients underwent splenectomy and are transfusion free.ConclusionsOur cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments. DisclosuresNo relevant conflicts of interest to declare.

Hyperammonemia and Pyroglutamic Acidemia in a Sepsis Patient with Gamma-Glutamyl-Cyclus Failure- A Case and Literature Review

We report the rare case of an adult sepsis patient with very low urea blood values, a high anion gap metabolic acidosis, and high ammonia levels. After exclusion of a primary disturbance of the urea cycle, we found a severe pyroglutamic (also called 5-oxoproline) acidemia, 841 µmol l-1 (norm: 5-150), indicating a disrupture of the gamma-glutamyl cycle, a cycle which is responsible for amino acid transportation, redox metabolism, and detoxification of medications, e.g., analgesics and antibiotics. The patient initially was treated with long-term, high-dosage rifampicine, linezolid, and piperacillin/tazobactam. Such cycle defects are rarely reported and are mostly found as an inborn disease in early childhood. In adults, diseases of the liver or renal failure, sepsis or diabetes, and certain medications may trigger a secondary cycle disturbance by depletion of vitally important glutathione stores or enzyme inhibition. We recommend that physicians remain aware of extremely low blood urea values that may indicate the beginning of gamma-glutamyl-cycle failure. Countermeasures such as discontinuation of causative medication and nutrition adaption must follow.

Genotype and clinical phenotype in four patients with glutathione synthetase deficiency

Background and aimsGlutathione synthetase deficiency (GSSD) is an autosomal recessive disorder described in the literature in roughly 80 patients. Currently, there is little known about genotype-phenotype correlations in GSSD though severity can be predicted to a limited extent via mutation analysis. Here, we describe four patients with GSSD and assess their genotypes and phenotypes. Additionally, we have provided an updated review of the literature. MethodsWe retrospectively reviewed all medical charts for patients with GSSD in the last ten years at Al-Makassed Hospital in Jerusalem, Palestine. We reviewed the literature for medical management and latest research in treatment and discussed the phenotype-genotype correlations. ResultsWe describe four patients with confirmed GSSD. Clinical presentation varied in severity, but patients typically presented with hemolytic anemia and lactic acidosis. Urine organic acid analysis demonstrated massive excretion of lactic acid and pyroglutamic acid. All patients were treated with N-acetylcysteine, vitamin E, vitamin C, and sodium hydrogen carbonate. All improved significantly following treatment, apart from one patient who died at two months of age. ConclusionGSSD presents similarly to many other diseases, at times causing a delay in diagnosis. Early initiation of treatment can improve clinical outcomes and overall development. If there is a high suspicion for GSSD, it is important to consider mRNA sequencing in an effort to prevent a delay in diagnosis when a splice site mutation is present.

Metabolic Acidosis Due To Pyroglutamic Acid.

Metabolic acidosis is a common clinical disturbance due to increased plasma acidity caused by a primary decrease in serum HCO3- concentration. It is classified as normal or high anion gap metabolic acidosis. High anion gap metabolic acidosis can result from either a decrease in unmeasured cations (K+, Ca2+, Mg2+) or an increase in unmeasured anions (PO43−, albumin). However, other anions such as lactic acid or keto acids may cause this acid-base disorder. It can also result from renal failure and intoxication (salicylate, methanol, ethylene glycol), or more rarely, from massive rhabdomyolysis and pyroglutamic (5-oxoproline) acidemia. Acidemia due to pyroglutamic acid should be considered when no other aetiology is found. High anion gap metabolic acidosis is diagnosed in children with inherited defects in enzymes of the γ-glutamyl cycle. In adults, this disorder from pyroglutamic acid has been described in association with chronic acetaminophen misuse. We report a case of pyroglutamic acidosis in a woman with acute misuse of acetaminophen concurrent with chronic use.LEARNING POINTParacetamol is an easily available drug with potentially harmful consequences.Accumulation of pyroglutamic acid (5-oxoproline) may be a cause of high anion gap metabolic acidosis.Reporting cases of 5-oxoprolinemia acidosis contributes to better understanding of the condition.

Treatment of cystoid macular edema in homozygous twins with glutathione synthetase deficiency and retinal dystrophy

Monozygotic twins with glutathione synthetase deficiency, progressive retinal dystrophy and cystoid macular edema were followed for foveal changes on optical coherence tomography under different treatment modalities. The purpose of the study is to show the effect of topical dorzolamide in conjunction with systemic acetazolamide in terms of decreasing macular edema in this specific disease. The results showed that systemic acetazolamide alone or in combination with topical dorzolamide decreased CME in both patients for a certain period of time. The result can be temporary sustained after treatment discontinuation. In conclusion, topical dorzolamide, in conjunction with systemic acetazolamide, could reduce cystoid macular edema in GSSD.

Sibling Screening of a Case of Pyroglutamic Aciduria Resulting in Normal Development- A Case Report

Pyroglutamic aciduria is a condition usually associated with metabolic acidosis and increased excretion of pyroglutamic acid in urine. It occurs due to metabolic defect in γ-glutamyl cycle. Here we report a case of 5-month-old male baby presented with respiratory symptoms who had a history of two months age sibling death due to pyroglutamic aciduria which was diagnosed by Gas Chromatography Mass Spectrometry (GCMS). Analysis of urine for organic acid by GCMS in the present baby also showed increased excretion of pyroglutamic acid. Hence in the present baby history of sibling death due to pyroglutamic aciduria led to the early diagnosis. Proper medication and timely management during fever and cold resulted in normal development.

Unravelling 5-oxoprolinuria (pyroglutamic aciduria) due to bi-allelic OPLAH mutations: 20 new mutations in 14 families.

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

Five Chinese patients with 5-oxoprolinuria due to glutathione synthetase and 5-oxoprolinase deficiencies

5-Oxoprolinuria is a rare inherited metabolic disorder caused by a defective gamma-glutamyl cycle resulting from mutations in the genes encoding 5-oxoprolinase (OPLAH) and glutathione synthetase (GSS). No inherited 5-oxoprolinuria case has been reported in mainland China until now. In this study, clinical, biochemical, and genetic aspects of five Chinese 5-oxoprolinuria patients with OPLAH or GSS gene mutations were investigated. Three boys and two girls from five unrelated Chinese families with symptomatic 5-oxoprolinuria were identified within the past 3years in Peking University First Hospital. OPLAH and GSS genes were analyzed. Patients were hospitalized between the age of 13days to 1year and 3months for hypersomnia, developmental retardation, feeding deficiency, vomiting, icterus and recurrent pneumonia. All patients had significantly elevated urine 5-oxoproline. Three novel mutations (c.1904G>A and c.2813_2815delGGG in Patient 1, c.2978G>T in Patient 2) on OPLAH, on GSS, one novel mutation (c.1252C>T in Patient 3) and a reported mutation (c.491G>A in Patients 3-5) were detected. Patient 4 has homozygous mutation c.491G>A, the others are heterozygous. After treatment by l-carnitine, vitamin E, B1, B2 and coenzyme Q10, three patients with GSS deficiency improved, but the two 5-oxoprolinase-deficient patients did not respond to treatment. 5-Oxoprolinase deficiency and GSS deficiency share some clinical and biochemical features. Genetic analysis is important for the deferential diagnosis. In this study, five Chinese patients had severe central nervous system damage. Antioxidant treatments were proved effective for the three patients with GSS deficiency but not for the two patients with 5-oxoprolinase deficiency.

G382(P) Mind the gap! elevated anions secondary to paracetamol and sepsis

Aim Metabolic acidosis is a common finding in children presenting with sepsis. Hypovolaemia and hypoxia are the common causes for this derangement but sometimes there are other culprits. We aim to highlight the significance of correlating the anion gap with the biochemical picture and, when there are discrepancies, look for alterative diagnoses. An unusual case of transient pyroglutamic aciduria, presenting during an episode of severe sepsis and paracetamol use, will be used to outline the importance of examining the anion gap. Methods We illustrate the case of a 15 month old girl who presented with an 11 day history of diarrhoea and vomiting. She presented to the emergency department in a state of decreased consciousness. She was found to be hypotensive, hypoglycaemic and have a profound metabolic acidosis. She required mechanical ventilation and fluid resuscitation. Despite these interventions, she continued to have a profound metabolic acidosis with a very high anion gap (30.5). The levels of lactate and ketones were insufficient to explain the clinical picture. Results Metabolic investigations for the child were instigated. Whilst a majority of these were normal, examination of the patient’s organic acid profile revealed large peaks of pyroglutamic acid (5-oxoproline) and paracetamol. Termination of paracetamol use, administration of N-acetylcysteine to replenish the glutathione stores and haemofiltration caused resolution of the patient’s acidaemia. Subsequent testing of the infants urine revealed no further evidence of pyroglutamate. Conclusions Pyroglutamic aciduria (5-oxoprolinuria) is usually reported in children in the context of inherited errors of metabolism. The transient form that we describe here, whilst reported in the adult population, has rarely been described in children. Various pharmaceutical interventions, most commonly paracetamol, in combination with severe sepsis have been linked with this transient phenomenon. When the lactate and ketone levels don’t correlate with the anion gap, it is important to pursue further diagnostic testing as illustrated by this case. In addition, scrutiny of the child’s medication may give a clue to the diagnosis.

Fundus autofluorescence and optical coherence tomography findings in glutathione synthetase deficiency

We report the autofluorescence pattern and optical coherence tomography (OCT) characteristics in a 6-year-old boy with glutathione synthetase deficiency. The patient underwent complete ophthalmological examination, including full-field electroretinography, visual evoked potentials, fundus autofluorescence, and OCT imaging. Electrophysiological studies showed time-delay and subnormal responses. Fundus autofluorescence imaging revealed increased parafoveal autofluorescence compared to normal, and OCT showed alteration of photoreceptor and retinal pigment epithelium layers in the parafoveal region.

Case Report of Acquired Pyroglutamic Acidemia in a Pediatric Patient

Pyroglutamic acid (also known as 5-oxoproline) is an organic acid intermediate of the gamma-glutamyl cycle. Accumulation of pyroglutamic acid is a rare cause of high anion gap metabolic acidosis. In the pediatric population, the congenital form of pyroglutamic acidemia has been extensively described. However, there are scarce reports of the acquired form of this condition in children. The urine test for organic acids confirms the diagnosis of pyroglutamic acidemia. We report the case of a 16-month-old girl who developed transient 5-oxoprolinemia associated with malnutrition and the use of acetaminophen and ampicillin for the treatment of acute otitis media and abdominal pain. The patient received 21-hour course of n-acetylcysteine with improvement of metabolic acidosis. This report highligts the need of considering pyroglutamic acidemia in the differencial diagnosis for high anion gap metabolic acidosis in pediatric patients with malnutrition and other risk factors.

Hemolytic Anemia and Metabolic Acidosis: Think about Glutathione Synthetase Deficiency

Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

Chronic Low Dose Rate Ionizing Radiation Exposure Induces Premature Senescence in Human Fibroblasts that Correlates with Up Regulation of Proteins Involved in Protection against Oxidative Stress.

The risks of non-cancerous diseases associated with exposure to low doses of radiation are at present not validated by epidemiological data, and pose a great challenge to the scientific community of radiation protection research. Here, we show that premature senescence is induced in human fibroblasts when exposed to chronic low dose rate (LDR) exposure (5 or 15 mGy/h) of gamma rays from a 137Cs source. Using a proteomic approach we determined differentially expressed proteins in cells after chronic LDR radiation compared to control cells. We identified numerous proteins involved in protection against oxidative stress, suggesting that these pathways protect against premature senescence. In order to further study the role of oxidative stress for radiation induced premature senescence, we also used human fibroblasts, isolated from a patient with a congenital deficiency in glutathione synthetase (GS). We found that these GS deficient cells entered premature senescence after a significantly shorter time of chronic LDR exposure as compared to the GS proficient cells. In conclusion, we show that chronic LDR exposure induces premature senescence in human fibroblasts, and propose that a stress induced increase in reactive oxygen species (ROS) is mechanistically involved.

Rare Case of an Infant with Glutathione Synthetase Deficiency

An infant has generalized Glutathione Synthetase Deficiency. Pyroglutamic Aciduria (5-Oxoprolinuria) is a rare autosomal recessive disorder caused by either Glutathione Synthetase Deficiency or 5-Oxoprolinase Deficiency. The severe form of the disease, generalized GSSD, is characterized by acute Metabolic Acidosis, usually present in the neonatal period with Hemolytic Anemia and Progressive Encephalopathy. We report a male infant who had a severe Metabolic Acidosis with high Anion Gap, Hemolytic Anemia, and Hyperbilirubinemia. High level of Pyruglutamic Acids was detected in his urine. He passed away of severe Metabolic Acidosis and Sepsis.

An Unusual Cause of High Anion Gap Metabolic Acidosis

Pyroglutamic acidemia is an uncommon metabolic disorder, which is usually diagnosed at early ages. The mechanism of action is thought to be glutathione depletion, and its clinical manifestations consist of hemolytic anemia, mental retardation, ataxia, and chronic metabolic acidosis. However, an acquired form has been described in adult patients, who usually present with confusion, respiratory distress, and high anion gap metabolic acidosis (HAGMA). It is also associated with many conditions, including chronic acetaminophen consumption. A 68-year-old white male, with chronic acetaminophen use presented to our service on multiple occasions with severe HAGMA. The patient was admitted to the intensive care unit and required mechanical ventilation and aggressive supportive measures. After ruling out the most frequent etiologies for his acid-base disorder and considering the long history of Tylenol ingestion, his 5-oxiproline (pyroglutamic acid) levels were sent to diagnose pyroglutamic acidemia. Clinicians need to be aware of this cause for metabolic acidosis since it might be a more common metabolic disturbance in compromised patients than would be expected. Subjects with HAGMA that cannot be explained by common causes should be tested for the presence of 5-oxoproline. Discontinuation of the offending drug is therapeutic.

Paracetamol prevents hyperglycinemia in vervet monkeys treated with valproate

Valproate administration increases the level of the inhibitory transmitter, glycine, in the urine and plasma of patients and experimental animals. Nonketotic hyperglycinemia (NKH), an autosomal recessive disorder of glycine metabolism, causes increased glycine concentrations in blood, urine, and cerebrospinal fluid (CSF), most likely due to a defect in the glycine cleavage enzyme or possibly deficits in glycine transport across cell membranes. We investigated the relationship between the hyperglycinemic effect of valproate and induced pyroglutamic aciduria via paracetamol in the vervet monkey. Firstly it was determined if valproate could induce hyperglycinemia in the monkey. The second aim was to increase glutamic acid (oxoproline) urine excretion using paracetamol as a pre-treatment and to assess whether valproate has an influence on the γ-glutamyl cycle. Hyperglycinemia was induced in healthy vervet monkeys when treated with a single oral dose of 50 mg/kg valproate. An acute dose of 50 mg/kg paracetamol increased oxoproline in the urine. Pre-treatment with paracetamol opposed the hyperglycinemic effect of valproate. However, the CSF:serum glycine ratio in a nonketotic monkey increased markedly after paracetamol treatment and remained high following valproate treatment. These results indicate that the γ-glutamyl cycle does indeed play a role in the hyperglycinemic effect of valproate treatment, and that paracetamol may have value in preventing and/or treating valproate-induced NKH.

5‐Oxoprolinuria as a cause of high anion gap metabolic acidosis

5‐Oxoprolinuria as a cause of high anion gap metabolic acidosis

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.

Glutathione is essential for early embryogenesis – Analysis of a glutathione synthetase knockout mouse

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene ( Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.