Search

Current dietary therapy for long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency consists of fasting avoidance, and limiting long-chain fatty acid (LCFA) intake. This study reports the relationship of dietary intake and metabolic control as measured by plasma acylcarnitine and organic acid profiles in 10 children with LCHAD or TFP deficiency followed for 1 year. Subjects consumed an average of 11% of caloric intake as dietary LCFA, 11% as MCT, 12% as protein, and 66% as carbohydrate. Plasma levels of hydroxypalmitoleic acid, hydroxyoleic, and hydroxylinoleic carnitine esters positively correlated with total LCFA intake and negatively correlated with MCT intake suggesting that as dietary intake of LCFA decreases and MCT intake increases, there is a corresponding decrease in plasma hydroxyacylcarnitines. There was no correlation between plasma acylcarnitines and level of carnitine supplementation. Dietary intake of fat-soluble vitamins E and K was deficient. Dietary intake and plasma levels of essential fatty acids, linoleic and linolenic acid, were deficient. On this dietary regimen, the majority of subjects were healthy with no episodes of metabolic decompensation. Our data suggest that an LCHAD or TFP-deficient patient should adhere to a diet providing age-appropriate protein and limited LCFA intake (10% of total energy) while providing 10-20% of energy as MCT and a daily multi-vitamin and mineral (MVM) supplement that includes all of the fat-soluble vitamins. The diet should be supplemented with vegetable oils as part of the 10% total LCFA intake to provide essential fatty acids.

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1–/–) deficient mice previously revealed elevated γ-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified β-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of β-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

Smith–Lemli–Opitz syndrome (SLOS), an autosomal recessive condition with multiple malformations, mental retardation, and growth failure, results from markedly reduced activity of the final enzyme in the cholesterol biosynthetic pathway, 7-dehydrocholesterol Δ7-reductase (DHCR7). We diagnosed SLOS in a fetus following intrauterine demise at 32 weeks' gestation. Chorionic villus (CV) sampling had been performed at 30 weeks because oligohydramnios and atrioventricular septal defect were noted on fetal ultrasound. On fetal post-mortem examination, a midline U-shaped soft palate cleft, micrognathia, postaxial polydactyly of the fingers with single transverse palmar creases bilaterally, and cutaneous syndactyly of toes two–three bilaterally suggested SLOS. We hypothesized that SLOS could be confirmed by analysis of tissue sterols despite extensive autolysis, and by measurement of enzyme activity in CV cells. Measurement of DHCR7 activity in CV cells was undertaken using ergosterol as a substrate. CV cells were unable to convert any ergosterol to brassicasterol after a 72 h incubation period while control CV cells reduced 12.6–71.8% of ergosterol to brassciasterol in a 72 h period. SLOS was confirmed by measurement of elevated 7-dehydrocholesterol (7-DHC) in the CV cells. Measurements of sterols were made in multiple fetal tissues. All tissues analysed showed elevated 7-DHC with markedly increased 7-DHC/cholesterol ratios. Copyright © 2000 John Wiley & Sons, Ltd.

Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive condition with multiple malformations, mental retardation, and growth failure, results from markedly reduced activity of the final enzyme in the cholesterol biosynthetic pathway, 7-dehydrocholesterol Delta(7)-reductase (DHCR7). We diagnosed SLOS in a fetus following intrauterine demise at 32 weeks' gestation. Chorionic villus (CV) sampling had been performed at 30 weeks because oligohydramnios and atrioventricular septal defect were noted on fetal ultrasound. On fetal post-mortem examination, a midline U-shaped soft palate cleft, micrognathia, postaxial polydactyly of the fingers with single transverse palmar creases bilaterally, and cutaneous syndactyly of toes two-three bilaterally suggested SLOS. We hypothesized that SLOS could be confirmed by analysis of tissue sterols despite extensive autolysis, and by measurement of enzyme activity in CV cells. Measurement of DHCR7 activity in CV cells was undertaken using ergosterol as a substrate. CV cells were unable to convert any ergosterol to brassicasterol after a 72 h incubation period while control CV cells reduced 12.6-71.8% of ergosterol to brassciasterol in a 72 h period. SLOS was confirmed by measurement of elevated 7-dehydrocholesterol (7-DHC) in the CV cells. Measurements of sterols were made in multiple fetal tissues. All tissues analysed showed elevated 7-DHC with markedly increased 7-DHC/cholesterol ratios.

With the exception of beta-kelothiolase (BKT) deficiency, there are no reported inborn errors of human metabolism specific to L-isoleucine degradation. EA presented at three days of age with lethargy, hypoglycemia and apnea. MRI demonstrated ischemic changes bilaterally in parietal and occipital lobes and abnormal EEG. Acylcarnitines in plasma were increased (range 8-22 uM, n=7; nl, 3-10). Tandem mass spectrometry (MS/MS) analysis revealed elevated plasma C5-acylcarnitine (1.4-2.4 μM, nl < 0.4), verified as 2-methylbutyrylcarnitine by gas chromatography-mass speclrometry. Urine 2-methylbutyrylglycine (2-MBG) was consistently elevated (range 8-30 mg/g creatinine, n=6; nl < 8). Intact fibroblast conversion of U-14C-isoleucine to 14CO2 was decreased to 26% of control, comparable to that of BKT deficient fibroblasts. Conversion of 13C6-leucine and 13C5-valine in patient's fibroblasts incubated with L-carnitine revealed no abnormal accumulation of acylcarnitines; in the same cells, there was a 10-fold accumulation of C5-acylcarnitine in comparison to control (consistent with impaired 2-MBCDase activity) when incubated with 13C6-isoleucine and L-carnitine. Western-blot analysis of fibroblasts from EA revealed greatly reduced 2-MBCDase cross-reactive material. Mutation screening in patient fibroblasts revealed a C778T substitution in the 2-MBCDase gene, substituting leucine for phenylalanine at amino acid 222 (L222F); the patient's mother also carried this mutation. In a subsequent pregnancy, the concentration of 2-MBG in amniotic fluid obtained at 15 weeks gestation was 0.27 umol/L (nl < 0.04, n=5), while total C5-acylcarnitine concentration was 1.9 umol/L (nl < 0.7, n=27), suggesting an affected fetus. Cultured amniocytes accumulated excess (10-fold) C5-acylcarnitine when incubated with 13C6-isoleucine and L-carnitine. At 1 year of age EA carries the diagnosis of athetoid cerebral palsy, and continues to manifest impaired visual, motor and cognitive skills. Our studies reveal 1) the first documented case of isolated 2-MBCDase deficiency; 2) a novel mutation in the 2-MBCDase gene; and 3) the first prenatal diagnosis of an affected fetus with 2-MBCDase deficiency, a new inborn error of L-isoleucine metabolism in humans.

The cobalamin C (cbl C) complementation group of combined MMA/HCYS is the most common o f the inborn errors of vitamin B12 metabolism, affecting both methylmalonyl-CoA and homocysteine (Hcys) degradation. We have recently diagnosed 2 patients with this disorder. AH. a 3 week old male, presented with lethargy, decreased oral intake, dehydration and pancytopenia. There had been a preceding upper respiratory infection. Parents were first-cousin Saudi Arabians. VM, a Hispanic female, presented at 19 days of life with lethargy. hyptonicity, dehydration, cough, decreased oral intake, significant thrombocytopenia, and mild pancytopenia. For both, worsening neurologic status was accompanied by seizures, and sepsis was suspected followed by appropriate intervention. Subsequent metabolic work-up revealed MMAIHCYS; fibroblast analysis for AH verified the cbl C complementation subgroup (Dr. D. Rosenblatt, Montreal, Canada; studies in VM are pending, although she likely falls within the same subgroup). Plasma total Hcys remains persistently elevated in AH (26-39 uM, n=8; nl < 10) and was 216 uM for VM (n=l). CSF total Hcys was 35 uM for VM (nl < 0.08). We conclude that I) diagnosis of patients with MMA/HCYS of the cbl C complementation subgroup may be hampered by absence of acute metabolic derangement (acidosis, hyperammonemia, hypoglycemia, ketosis) combined with features (lethargy, obtundation) suggesting sepsis; and 2) significantly increased CSF Hcys may be the underlying cause of altered neurologic status in these patients.

Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder which results from a defect in cholesterol biosynthesis. The finding of elevated levels of cholesterol precursors is diagnostic of SLO. There have been few reported cases of the prenatal diagnosis of SLO in the presence of a non-contributory family history. Abnormal triple screen, sex discrepancy, and polydactyly led to such a diagnosis in this case. L.M. was a 29 year old G2 P1 ABO who presented at 17.1 weeks gestation due to a positive maternal serum triple screen which estimated a midtrimester risk for fetal trisomy 18 of 1/15. The triple screen was reported as follows: MSAFP 0.44 MOM; ESTRIOL 0.43 MOM; HCG 0.35 MOM at 16.1 weeks. The couple opted for amniocentesis which resulted in a normal 46,XY karyotype. Due to a suspicion of early IUGR, a repeat ultrasound examination was performed at 20.1 weeks gestation, revealing a discrepancy between observed fetal sex and karyotype, post-axial polydactyly of both feet and the left hand, and growth lag. A repeat amniocentesis was performed to confirm fetal sex. L.M. was counseled regarding the sonographic and biochemical evidence that was suggestive of SLO. She elected to terminate the pregnancy. Amniotic fluid analysis subsequently revealed that 7-dehydrocholesterol (7-DHC) was greater than 800-fold increased in comparison to control; conversion of ergosterol to brassicasterol (estimating 7-DHC reductase activity) was 0.5% in fetal fibroblasts (control 15-35%, n=2), verifying SLO. Features consistent with SLO reported on autopsy included ambiguous genitalia with micropenis, severe hypospadias, and bifid scrotum. There was left hand post-axial polydactyly, bilateral post-axial polydactyly of the feet, and partial syndactyly of the second and third toes bilaterally. Facial dysmorphia included a broad nasal tip and anteverted nares, broad maxillary alveolar ridges, with a high arched hard palate and a soft palate cleft. This case demonstrates a possible role for triple screen, particularly uE3 analysis, in the detection of fetuses at risk for SLO.

Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals. We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcarnitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.