Transient 5-oxoprolinuria: unusually high anion gap acidosis in an infant.

Transient 5-oxoprolinuria is a phenomenon that is well recognised in adults. We illustrate an unusual paediatric case of transient 5-oxoprolinuria presenting during an episode of severe sepsis with concomitant paracetamol use. The 15-month-old patient had an extremely high anion gap metabolic acidosis. Adequate resuscitation failed to correct the biochemical disturbance, and high levels of 5-oxoproline were identified. A combination of haemofiltration, replenishment of glutathione stores with N-acetylcysteine and cessation of paracetamol administration resulted in the resolution of the acidosis. Subsequent testing following treatment of the sepsis revealed no ongoing 5-oxoprolinuria. CONCLUSION: Transient 5-oxoprolinuria has been previously reported in the adult population during episodes of severe sepsis and various pharmaceutical interventions. This case illustrates that it is a phenomenon that should be considered in paediatric patients where a very high anion gap metabolic acidosis exists that cannot be explained by the biochemical indices. WHAT IS KNOWN: • 5-oxoprolinuria in the paediatric population is usually secondary to an inborn error of metabolism. • Transient 5-oxoprolinuria is well recognised in adults during episodes of severe glutathione depletion. WHAT IS NEW: • Transient 5-oxoprolinuria is a phenomenon rarely reported in the paediatric population. • It highlights the importance of investigating a high anion gap such that unusual diagnoses are not missed.

New insights into the genetics of 5-oxoprolinase deficiency and further evidence that it is a benign biochemical condition.

UNLABELLED: Inherited 5-oxoprolinase (OPLAH) deficiency is a rare inborn condition characterised by 5-oxoprolinuria. To date, three OPLAH mutations have been described: p.H870Pfs in a homozygous state, which results in a truncated protein, was reported in two siblings, and two heterozygous missense changes, p.S323R and p.V1089I, were independently identified in two unrelated patients. We describe the clinical context of a young girl who manifested 5-oxoprolinuria together with dusky episodes and who is compound heterozygote for two novel OPLAH variations: p.G860R and p.D1241V. To gain insight into the aetiology of the 5-oxoprolinase deficiency, we investigated the pathogenicity of all the reported missense mutations in the OPLAH gene. A yeast in vivo growth assay revealed that only p.S323R, p.G860R and p.D1241V affected the activity of the enzyme. CONCLUSION: Taken together, this report further suggests that hereditary 5-oxoprolinase deficiency is a benign biochemical condition caused by mutations in the OPLAH gene, which are transmitted in an autosomal recessive manner, but 5-oxoprolinuria may be a chance association in other disorders.

An unusual phenotype of X-linked developmental delay and extreme behavioral difficulties associated with a mutation in the EBP gene.

We report on a family in which four males over three generations are affected with X-linked recessive developmental delay, learning difficulties, severe behavioral difficulties and mild dysmorphic features. Plasma sterol analysis in three of the four affected males demonstrated increased concentrations of 8-dehydrocholesterol (8-DHC) and cholest-8(9)-enol. All four affected males had a novel hemizygous missense mutation, p.W47R (c.139T>C), in EBP. Functional studies showed raised levels of cholest-8(9)-enol in patient's cultured fibroblast cells, which were suppressed when the cells were incubated with simvastatin. EBP encodes 3ß-hydroxysteroid-delta8, delta7-isomerase, a key enzyme involved in the cholesterol biosynthesis pathway. Mutations in EBP have previously been associated with Conradi-Hunermann-Happle syndrome (CHH), an X-linked dominant disorder characterized by skeletal dysplasia, skin, and ocular abnormalities, which is usually lethal in males. Four previous reports describe X-linked recessive multiple anomaly syndromes associated with non-mosaic EBP mutations in males, two at the same amino acid position, p.W47C. This phenotype has previously been described as "MEND" syndrome (male EBP disorder with neurological defects). The family reported herein represent either a novel phenotype, or an expansion of the MEND phenotype, characterized by extreme behavioral difficulties and a scarcity of structural anomalies. Simvastatin therapy is being evaluated in two males from this family.

A vitamin B-12 supplement of 500 µg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 µg/d, 100 µg/d, and 10 µg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (µmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 µg/d, but even 500 µg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 µg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Glutaric aciduria type 1 in South Africa-high incidence of glutaryl-CoA dehydrogenase deficiency in black South Africans.

Glutaric Aciduria type 1 (GA 1) is an inherited disorder of lysine and tryptophan catabolism that typically manifests in infants with acute cerebral injury associated with intercurrent illness. We investigated the clinical, biochemical and molecular features in 14 known GA 1 patients in South Africa, most of whom were recently confirmed following the implementation of sensitive urine organic acid screening at our laboratory. Age at diagnosis ranged from 3days to 5years and poor clinical outcome reflected the delay in diagnosis in all but one patient. Twelve patients were unrelated black South Africans of whom all those tested (n=11) were found homozygous for the same A293T mutation in the glutaryl-CoA dehydrogenase (GCDH) gene. Excretion of 3-hydroxyglutarate (3-OHGA) was >30.1µmol/mmol creatinine (reference range <2.5) in all cases but glutarate excretion varied with 5 patients considered low excretors (glutarate <50µmol/mmol creatinine). Fibroblast GCDH activity was very low or absent in all of five cases tested. Heterozygosity for the A293T mutation was found 1 in 36 (95% CI; 1/54 - 1/24) unrelated black South African newborns (n=750) giving a predicted prevalence rate for GA 1 of 1 in 5184 (95% CI; 1/11664 - 1/2304) in this population. GA 1 is a treatable but often missed inherited disorder with a previously unrecognised high carrier frequency of a single mutation in the South African black population.

Are the enzymatic methods currently being used to measure bronchoalveolar lavage bile salt levels fit for purpose?

BACKGROUND: Microaspiration after gastroesophageal reflux has been implicated in the chronic loss of allograft function in lung transplant patients. Bronchoalveolar lavage fluid (BALF) assessment for pepsin and bile salts is a common method to document reflux and aspiration. Clinically used methods for bile salt analysis include tandem mass spectrometry and diagnostic enzymatic kits designed to measure bile salts in serum. In clinical research, the enzymatic kits have been commonly used for BALF assays in lung transplant recipients, with reports of detection limits of 0.2 µmol/liter, and the levels used to inform clinical decisions. This study assessed the sensitivity of detection by 2 enzymatic assay kits compared with tandem mass spectrometry. METHODS: These 2 kits were used to measure (1) the absorbance changes for 0 to 50 µmol/liter bile salts, (2) levels in gastric juice (10-10,010 µmol/liter), and (3) bile salt levels of 40 BALF samples that were also measured using tandem mass spectrometry (0.01-1.19 µmol/liter). Measurements of pH/impedance were done in 14 of 15 patients. RESULTS: Neither kit had detection limits as low as claimed in previous BALF studies. The kits could be made more sensitive with a longer incubation time, (5 µmol/liter). All patients had detectable lavage bile acids using mass spectroscopy, 71% had pathologic distal gastroesophageal reflux, and 43% had pathologic proximal reflux. CONCLUSIONS: The enzymatic kits are not sensitive enough for use in situations where bile salt levels are much below 5 µmol/liter, which is the case in BALF. In addition, reports in the literature of levels significantly below 5 µmol/liter need reassessing. Tandem mass spectrometry with a lower limit of detection of 0.01 µmol/liter should be the method of choice.

Riboflavin-responsive trimethylaminuria in a patient with homocystinuria on betaine therapy.

A 17-year-old female patient with pyridoxine non-responsive homocystinuria, treated with 20 g of betaine per day, developed a strong body odour, which was described as fish-like. Urinary trimethylamine (TMA) was measured and found to be markedly increased. DNA mutation analysis revealed homozygosity for a common allelic variant in the gene coding for the TMA oxidising enzyme FMO3. Without changing diet or betaine therapy, riboflavin was given at a dose of 200 mg per day. An immediate improvement in her odour was noticed by her friends and family and urinary TMA was noted to be greatly reduced, although still above the normal range.Gradual further reductions in TMA (and odour) have followed whilst receiving riboflavin. Throughout this period, betaine compliance has been demonstrated by the measurement of dimethylglycine (DMG) excretion, which has been consistently increased. Marked excretions of DMG when the odour had subsided also demonstrate that DMG was not the source of the odour.This patient study raises the possibility that betaine may be converted to TMA by intestinal flora to some degree, resulting in a significant fish odour when oxidation of TMA is compromised by FMO3 variants. The possibility exists that the body odour occasionally associated with betaine therapy for homocystinuria may not be related to increased circulating betaine or DMG, but due to a common FMO3 mutation resulting in TMAU. Benefits of riboflavin therapy for TMAU for such patients would allow the maintenance of betaine therapy without problematic body odour.

Determinants of urinary methylmalonic acid concentration in an elderly population in the United Kingdom.

BACKGROUND: An age-related deterioration of vitamin B-12 status has been well documented. The early detection of deficiency may prevent the development of serious clinical symptoms, but plasma vitamin B-12 concentration is known to be an imperfect measure of vitamin B-12 status. Urinary methylmalonic acid (MMA) may be a more informative biomarker of vitamin B-12 status; however, biochemical, dietary, and other lifestyle determinants are not known. OBJECTIVE: We identified determinants of urinary MMA concentrations in free-living men and women aged ≥65 y in the United Kingdom. DESIGN: A cross-sectional study in 591 men and women aged 65-85 y, with no clinical evidence of vitamin B-12 deficiency, was conducted to determine the demographic, clinical, and lifestyle determinants of urinary MMA concentration expressed as the ratio of micromoles of MMA to millimoles of creatinine (uMMA ratio). RESULTS: Twenty percent of subjects had plasma vitamin B-12 concentrations <200 pmol/L. Seventeen percent of the variation in the uMMA ratio could be explained by plasma holotranscobalamin and sex; total vitamin B-12 intake and measures of renal function and gastric function made only a small contribution to the model. The uMMA ratio was lower in people with moderately impaired renal function. CONCLUSIONS: Plasma holotranscobalamin and sex were the most important determinants of uMMA ratio in elderly people with no clinical diagnosis of renal impairment. This biomarker might underestimate vitamin B-12 deficiency in a population in which renal impairment is prevalent. This trial was registered at www.controlled-trials.com as ISRCJN83921062.

Lanosterol biosynthesis in the prokaryote Methylococcus capsulatus: insight into the evolution of sterol biosynthesis.

A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general.

Stomatocytic haemolysis and macrothrombocytopenia (Mediterranean stomatocytosis/macrothrombocytopenia) is the haematological presentation of phytosterolaemia.

Phytosterolaemia (sitosterolaemia) is a recessively inherited metabolic condition in which the absorption of both cholesterol and plant-derived cholesterol-like molecules at the gut is unselective and unrestricted. In haematology, Mediterranean stomatocytosis or Mediterranean macrothrombocytopenia is a poorly understood haematological condition that combines stomatocytic haemolysis with the presence of very large platelets. Five pedigrees showing this haematology were identified. Gas chromatography mass spectrometry (GC-MS) showed that all of the patients with this highly specific haematology had grossly elevated levels of phytosterols in the blood, diagnostic of phytosterolaemia. All showed mutations in the ABCG5 and ABCG8 previously linked to phytosterolaemia. Three pedigrees showed five new mutations, while two pedigrees showed the common W361X mutation in ABCG8. We draw the following four conclusions: (i) that Mediterranean stomatocytosis/macrothrombocytopenia is caused by an excess of phytosterols in the blood; (ii) that phytosterolaemia, which does not respond to standard statin treatment, can be diagnosed via the distinctive haematology described here, even when the cholesterol is normal; (iii) that phytosterolaemia should be considered in the differential diagnosis of all patients with large platelets; and (iv) that the platelet size should be noted in patients with hypercholesterolaemia.

Mutations in Saccharomyces cerevisiae sterol C5-desaturase conferring resistance to the CYP51 inhibitor fluconazole.

Understanding fluconazole resistance is important as it emerged as a serious clinical problem for this CYP51, sterol 14alpha-demethylase, inhibitor. One mechanism, observed first in Saccharomyces cerevisiae, was through defective sterol C5-desaturase (Erg3p) required to form the fungistatic sterol end-product resulting from CYP51 inhibition, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol. Here, we report molecular changes resulting in both blocked mutants and also leaky mutants in which reduced ergosterol levels were detected. Blocked mutants exhibited nonsense and frameshift mutations, while leaky mutants contained missense mutations that were generally in conserved positions based on the alignment of sterol C5-desaturases and located mainly between residues 250 and 282.

Sterol 14alpha-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family.

The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14alpha-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from Mycobacterium smegmatis.

The genetic locus encoding cytochrome P450 51 (CYP51; P450(14DM)) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14 alpha-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.

A novel sterol 14alpha-demethylase/ferredoxin fusion protein (MCCYP51FX) from Methylococcus capsulatus represents a new class of the cytochrome P450 superfamily.

Sterol 14alpha-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. This M. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass approximately 62 kDa on SDS/PAGE and bound lanosterol as a putative substrate. Sterol 14alpha-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.

Candida dubliniensis in a Brazilian family with an HIV 1- infected child: identification, antifungal susceptibility, drug accumulation and sterol composition

This study investigated the prevalence of C. dubliniensis in a Brazilian family with an HIV - infected child. A total of 42 oral isolates were obtained from eight family members. The identification of C. dubliniensis was performed by polymerase chain reactions (PCR) using primers against a specific sequence of the C. dubliniensis cytochrome b gene. Only the HIV-infected child and his grandmother were colonized by C. dubliniensis. In this study C. dubliniensis isolated from the HIV-infected child exhibited high susceptibility for azoles tested with MICs of 0.125 and 0.5 æg/mL for voriconazole and fluconazole, respectively. Accumulation of [³H] fluconazole in C. dubliniensis isolated from the HIV-infected child was slightly reduced in comparison to the reference susceptible strain. C. dubliniensis isolates had significantly lower ergosterol levels in comparison to C. albicans reference strains.

O presente estudo investigou a prevalência de C. dubliniensis em uma família brasileira com uma criança infectada pelo vírus HIV. Um total de 42 isolados orais foram obtidos de 8 membros da família. A identificação de C. dubliniensis foi realizada por polymerase chain reactions (PCR) usando primers contra a sequência específica para o gene C. dubliniensis cytochrome b. Apenas a criança infectada pelo vírus HIV e a avó estavam colonizados por C. dubliniensis. Neste estudo C. dubliniensis isolado da criança infectada pelo vírus HIV exibiu alta susceptibilidade para azoles com concentração mínima inibitória de 0.125 and 0.5 æg/mL para voriconazole and fluconazole respectivamente. Acúmulo de [³H] fluconazol intra-celular foi ligeiramente reduzido em C. dubliniensis isolado da criança infectada pelo vírus HIV em comparação com a cepa referência sensível ao fluconazole. Isolados de C. dubliniensis neste estudo apresentaram níveis significantemente reduzidos de ergosterol da membrane celular em comparação com C. albicans.