Udo Engelke

Proton nuclear magnetic resonance spectroscopy of body fluids in the field of inborn errors of metabolism

Proton nuclear magnetic resonance (NMR) spectroscopy of body fluids has been successfully applied to the field of inborn errors of metabolism. This technique has the advantage of minimal sample pretreatment not requiring extraction or derivatization steps. Moreover, the spectrum provides a comprehensive metabolic profile of proton-containing, low-molecular-weight metabolites. The sensitivity limit is in the low micromolar range. This allows diagnosis of many inborn errors of metabolism. This review explains the key features of the NMR spectrum and reviews the available literature on metabolic diseases. Three novel diseases have been delineated with the technique. Relevant parts of the spectra from the urine samples of patients with these diseases are shown. NMR spectroscopy may develop to become a key tool in a metabonomics approach in clinical biochemistry.

Biochemical and genetic analysis of 3-methylglutaconic aciduria type IV: a diagnostic strategy.

The heterogeneous group of 3-methylglutaconic aciduria type IV consists of patients with various organ involvement and mostly progressive neurological impairment in combination with 3-methylglutaconic aciduria and biochemical features of dysfunctional oxidative phosphorylation. Here we describe the clinical and biochemical phenotype in 18 children and define 4 clinical subgroups (encephalomyopathic, hepatocerebral, cardiomyopathic, myopathic). In the encephalomyopathic group with neurodegenerative symptoms and respiratory chain complex I deficiency, two of the children, presenting with mild Methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness, harboured SUCLA2 mutations. In children with a hepatocerebral phenotype most patients presented with complex I deficiency and mtDNA-depletion, three of which carried POLG1-mutations. In the cardiomyopathic subgroup most patients had complex V deficiency and an overlapping phenotype with that previously described in isolated complex V deficiency, in three patients a TMEM70 mutation was confirmed. In one male with a pure myopathic form and severe combined respiratory chain disorder, based on the pathogenomic histology of central core disease, RYR1 mutations were detected. In our patient group the presence of the biochemical marker 3-methylglutaconic acid was indicative for nuclear coded respiratory chain disorders. By delineating patient-groups we elucidated the genetic defect in 10 out of 18 children. Depending on the clinical and biochemical phenotype we suggest POLG1, SUCLA2, TMEM70 and RYR1 sequence analysis and mtDNA-depletion studies in children with 3-methylglutaconic aciduria type IV.

High-resolution proton nuclear magnetic resonance spectroscopy of ovarian cyst fluid.

Most ovarian tumors are cystic structures containing variable amounts of fluid. Several studies of ovarian cyst fluid focus on one specific metabolite using conventional assay systems. We examined the potential of 1H‐nuclear magnetic resonance spectroscopy in evaluation of the overall metabolic composition of cyst fluid from different ovarian tumors. Ovarian cyst fluid samples obtained from 40 patients with a primary ovarian tumor (12 malignant and 28 benign) were examined. After deproteinization and pD standardization, we performed 1H‐NMR spectroscopy on a 600 MHz instrument. With 1H‐NMR spectroscopy we found detectable concentrations of 36 metabolites with high intersample variation. A number of unassigned resonances as well as unexpected metabolites were found. We introduce an overall inventory of the low‐molecular‐weight metabolites in ovarian cyst fluid with corresponding resonances. Significant differences in concentration (p < 0.01) were found for several metabolites (including an unknown metabolite) between malignant and benign ovarian cysts. Furthermore, higher concentrations in malignant‐ and lower in benign fluids were found compared to normal serum values, indicating local cyst wall metabolic processes in case of malignant transformation. We conclude that 1H‐nuclear magnetic resonance spectroscopy can give an overview of low‐molecular‐weight proton‐containing metabolities present in ovarian cyst fluid samples. The metabolic composition of cyst fluid differs significantly between benign and malignant ovarian tumors. Furthermore, differences between benign subgroups possibly related to histopathological behaviour can be detected. The presence of N‐acetyl aspartic acid and 5‐oxoproline exclusively in serous cystadenoma samples is remarkable. Future studies will concentrate on these findings and explore the possibilities of extrapolating information from the in vitro studies to in vivo practice, in which metabolic differences between malignant and benign subtypes can be of great importance in a pre‐operative phase. Copyright

Cloning of dimethylglycine dehydrogenase and a new human inborn error of metabolism, dimethylglycine dehydrogenase deficiency

Dimethylglycine dehydrogenase (DMGDH) (E.C. number 1.5.99.2) is a mitochondrial matrix enzyme involved in the metabolism of choline, converting dimethylglycine to sarcosine. Sarcosine is then transformed to glycine by sarcosine dehydrogenase (E.C. number 1.5.99.1). Both enzymes use flavin adenine dinucleotide and folate in their reaction mechanisms. We have identified a 38-year-old man who has a lifelong condition of fishlike body odor and chronic muscle fatigue, accompanied by elevated levels of the muscle form of creatine kinase in serum. Biochemical analysis of the patients serum and urine, using (1)H-nuclear magnetic resonance NMR spectroscopy, revealed that his levels of dimethylglycine were much higher than control values. The cDNA and the genomic DNA for human DMGDH (hDMGDH) were then cloned, and a homozygous A-->G substitution (326 A-->G) was identified in both the cDNA and genomic DNA of the patient. This mutation changes a His to an Arg (H109R). Expression analysis of the mutant cDNA indicates that this mutation inactivates the enzyme. We therefore confirm that the patient described here represents the first reported case of a new inborn error of metabolism, DMGDH deficiency.

1H-magnetic resonance spectroscopy

Currently used techniques such as ultrasound, computed tomography, and magnetic resonance imaging are not fully capable of differentiating benign from malignant ovarian tumors. Magnetic resonance spectroscopy (MRS) may help solve this clinical problem by analyzing the composition of fluid from ovarian cysts.

Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSF

Background: Two unrelated girls had early onset of nystagmus and epilepsy, absent psychomotor development, and almost complete absence of myelin on cerebral MRI. The clinical features and MR images of both patients resembled the connatal form of Pelizaeus-Merzbacher disease (PMD), which is an X-linked recessive disorder caused by duplications or mutations of the proteolipid protein gene (PLP). Objective: To define a unique neurometabolic disorder with failure of myelination. Methods and Results: 1H-NMR of CSF in both girls was performed repeatedly, and both showed highly elevated concentrations of N-acetylaspartylglutamate (NAAG). The coding sequence of the gene coding for glutamate carboxypeptidase II, which converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced but revealed no mutations. Even though both patients are girls, the authors sequenced the PLP gene and found no abnormality. Conclusions: NAAG is an abundant peptide neurotransmitter whose exact role is unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its elevated concentration in CSF may be the biochemical hallmark for a novel neurometabolic disorder. The cause of its accumulation is still unclear.

The 3-methylglutaconic acidurias: what's new?

The heterogeneous group of 3-methylglutaconic aciduria (3-MGA-uria) syndromes includes several inborn errors of metabolism biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. Five distinct types have been recognized: 3-methylglutaconic aciduria type I is an inborn error of leucine catabolism; the additional four types all affect mitochondrial function through different pathomechanisms. We provide an overview of the expanding clinical spectrum of the 3-MGA-uria types and provide the newest insights into the underlying pathomechanisms. A diagnostic approach to the patient with 3-MGA-uria is presented, and we search for the connection between urinary 3-MGA excretion and mitochondrial dysfunction.

β-Ureidopropionase deficiency: A novel inborn error of metabolism discovered using NMR spectroscopy on urine

In this work, NMR investigations that led to the discovery of a new inborn error of metabolism, β‐ureidopropionase (UP) deficiency, are reported. 1D 1H‐NMR experiments were performed using a patients urine. 3‐Ureidopropionic acid was observed in elevated concentrations in the urine spectrum. A 1D 1H‐1H total correlation spectroscopy (TOCSY) and two heteronuclear 2D NMR techniques (heteronuclear multiple bond correlation (HMBC) and heteronuclear single‐quantum correlation (HSQC)) were used to identify the molecular structure of the compound that caused an unknown doublet resonance at 1.13 ppm. Combining the information from the various NMR spectra, this resonance could be assigned to 3‐ureidoisobutyric acid. These observations suggested a deficiency of UP. With 1D 1H‐NMR spectroscopy, UP deficiency can be easily diagnosed. The 1H‐NMR spectrum can also be used to diagnose patients suffering from other inborn errors of metabolism in the pyrimidine degradation pathway. Magn Reson Med 46:1014–1017, 2001.

Optimized metabolomic approach to identify uremic solutes in plasma of stage 3-4 chronic kidney disease patients

Background Chronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes. Methods In this study, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3–4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality. Results Protein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO2) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0–9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = −0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity. Conclusions This study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using 1H-NMR spectroscopy, and provide insight into the negative impact of DMSO2 and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.

NANS-mediated synthesis of sialic acid is required for brain and skeletal development

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.