Antonio Vivi

Guanidinoacetate methyltransferase (GAMT) deficiency diagnosed by proton NMR spectroscopy of body fluids

In patients with guanidinoacetate methyltransferase (GAMT) deficiency several parameters may point towards the diagnosis of GAMT deficiency. These include the low levels of creatine and creatinine in urine, the high concentration of guanidinoacetic acid (GAA) in urine and the low levels of creatine and creatinine in the cerebrospinal fluid (CSF). In this study, body fluids from 10 GAMT deficient patients were analysed using 1H NMR spectroscopy. The urine 1D 1H NMR spectra of all the patients showed a doublet resonance at 3.98 ppm (pH 2.50) derived from GAA present in high concentration. For this compound, a good recovery and good correlation was found between an LC‐MS/MS method and 1H NMR spectroscopy. In CSF NMR spectra of these patients, the singlet resonances of creatine and creatinine (3.05 and 3.13 ppm, respectively) were absent (normally always present in 1H NMR spectra of CSF). Due to overlap by other resonances, the doublet of GAA could not be observed. Our data demonstrate that 1H NMR spectroscopy of urine and CSF can be used to diagnose patients with GAMT deficiency. Copyright

The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck.

The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.

Comparison of action of the anti-neoplastic drug lonidamine on drug-sensitive and drug-resistant human breast cancer cells: 31P and 13C nuclear magnetic resonance studies.

Lonidamine (LND) is a relatively new anti-cancer drug,and several clinical trials have indicated that it may be effectivein combinations with other therapeutic modalities. LND isclassified within the metabolic inhibitor agents. Multidrugresistance (MDR) phenomenon is often associated with increasedenergy requirements, and enhanced glycolysis rate. These studieswere performed to delineate the mechanism of action of LNDon MDR human breast cancer cells, and to investigate whetherLND as a single agent, or in combination with anotheranti-metabolism drug, 2-deoxyglucose (2-DG), may be usefulagainst MDR tumors. The effects of LND on intact perfuseddrug-sensitive (WT) and 33-fold resistant to Adriamycin(Adr) MCF-7 cells, embedded in alginate micro capsules, were continuouslymonitored by 31P and 13C nuclearmagnetic resonance (NMR) spectroscopy. 31PNMR studies showed that LND induced intracellular acidificationand depletion of NTP in both WT and Adr cells. However, pH and NTPlevels decreased less in the Adr cells than in the WT cells(p < 0.05 for both parameters). 13CNMR demonstrated that LND inhibited lactate transport,and lactate signals were elevated in both cell lines. However, theintracellular lactate levels increased to a greater extentin the WT than in the Adr cells (p < 0.05).There were major differences in the effects of LND onmetabolism between sensitive and resistant cells.While LND enhanced glucose uptake in the WT cells, and itsadministration was followed by continuous increase oflactate signal, both processes were not affected by LNDin the Adr cells. 2-DG is a glucose analogue that inhibitsboth cellular uptake and utilization of glucose, leading to cell starvation. Combined treatment with LND and2-DG yielded at best additive, but not synergistic,cellular toxicity, and the metabolic effects of LNDwere attenuated by 2-DG. These results showed that the principalmechanism of action of LND is inhibition of lactate transportleading to intracellular lactate accumulation and acidificationin both WT and Adr cells. The Adr cells were only 2-fold resistantto LND (compared to the WT cells), and since cellular uptakeof alkaloid chemotherapy is improved in acidic environment,LND may have a role in the treatment protocols of MDR tumors,especially when given as the initial means for inductionof intracellular acidification.

A metabolomic study of preterm and term human and formula milk by proton MRS analysis: preliminary results

Abstract Objective: To investigate changes in global metabolic profile between: 1 – breast milk and formula milk, 2 – breast milk from mothers delivering at different gestational age (GA) collected within one week from delivery, and then week by week until term equivalent age. Methods: Proton magnetic resonance spectroscopy (MRS) was used to analyze the water-soluble and lipid fractions extracted from 50 milk samples, 46 human milk at different GA, from 23 weeks of gestation until term equivalent age and four different formula milks. Results: The formula milk for premature infants was the most similar to breast milk of preterm babies. Breast milk showed higher lactose concentrations than formula milk, that conversely presented higher galactose 1-phosphate and maltose concentrations. Mother’s milk of very preterm babies (23–25 wks of GA) showed a different metabolic profile from preterm infants ≥29 wks of GA with a subsequent trend to similarity around the 30th week of post-natal age. Breast milk from preterm infants of 29–34 wks, collected up to 40 wks of post-natal age showed a temporal change over the first three weeks of lactation, approaching to zero with the achievement of term age. Conclusions: Metabolome is a promising tool to study human and artificial milk global metabolic profile.

Creatine transporter defect diagnosed by proton NMR spectroscopy in males with intellectual disability.

Creatine deficiency syndrome due to mutations in X‐linked SLC6A8 gene results in nonspecific intellectual disability (ID). Diagnosis cannot be established on clinical grounds and is often based on the assessment of brain creatine levels by magnetic resonance spectroscopy (MRS). Considering high costs of MRS and necessity of sedation, this technique cannot be used as a first level‐screening test. Likewise, gene test analysis is time consuming and not easily accessible to all laboratories. In this article feasibility of urine analysis (creatine/creatinine (Cr/Crn) ratio) performed by nuclear magnetic resonance (NMR) as a first level‐screening test is explored. Before running a systematic selection of cases a preliminary study for further molecular analysis is shown. NMR urine spectra (n = 1,347) of male patients with an ID without a clinically recognizable syndrome were measured. On the basis of abnormal Cr/Crn ratio, three patients with the highest values were selected for molecular analysis. A confirmatory second urine test was positive in two patients and diagnosis was further confirmed by a decreased brain creatine level and by SLC6A8 gene analysis. A de novo mutation was identified in one. Another patient inherited a novel mutation from the mother who also has a mild ID. A repeat urine test was negative in the third patient and accordingly creatine level in the brain and SLC6A8 gene analysis both gave a normal result. We conclude that Cr/Crn ratio measured by NMR for male patients represents a rapid and useful first level screening test preceding molecular analysis.

Proton nuclear magnetic resonance spectroscopy of urine samples in preterm asphyctic newborn: a metabolomic approach.

In order to highlight differences in the metabolic profile of healthy (control) compared with asphyxiated newborns, by using untargeted metabolomic approach coupled with (1)H NMR spectroscopy, we evaluated the effects of asphyxia on newborn urine metabolites. Our results showed that lactate, glucose and TMAO, together with threonine plus 3-hydroxyisovalerate are the metabolites more characterizing the asphyxiated group; lower contribute to discrimination is related to other metabolites such as dimethylglycine, dimethylamine, creatine, succinate, formate, urea and aconitate. After 24-48h from resuscitation preterm asphyctic neonates showed their recovery pattern that still can be differentiated by the controls.

Using Metabolomics to Monitor Kidney Transplantation Patients by Means of Clustering to Spot Anomalous Patient Behavior

BACKGROUND NMR spectroscopy-based metabolomics is a system approach used to investigate the metabolic profile of biological fluids with multivariate data analysis tools. The aim of this study was to examine the kidney graft recovery process noninvasively through the examinations of urine samples using (1)H NMR spectroscopy combined with chemometric tools. METHODS Urine samples were treated as the source of metabolites reflecting the pathological and clinical conditions of patients with transplanted kidneys. We observed 15 subjects (9 males and 6 females) during the graft recovery process and initial days thereafter. The patients provided at least 9 samples each, applying advanced statistical methods of analysis: Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis PLS-DA). RESULTS The PCA model (for all subjects exp. var. PC1 13.96% and PC2 9.88%) allowed us to clearly designate 3 stages of recovery: initially the kidney is not working; in the second stage, it regains functions, and the third stage includes follow-up during hospitalization. PCA analysis of a single patient follows graft recovery based on biochemical (metabolites) information, assigning the appropriate recuperation stage. CONCLUSIONS NMR spectroscopy together with chemometric analysis allow monitoring of kidney graft recovery to identify patients who are not progressing within the normal range.

Silent increase of urinary ethylmalonic acid is an indicator of nonspecific brain dysfunction.

Our aim was to compare urinary ethylmalonic acid (EMA) levels in subjects who had no apparent clinical reason to have increased levels of this substance but were suffering from non‐specific CNS impairment, and healthy controls. Urinary EMA concentrations detected by 1H‐NMR spectroscopy were studied in 130 subjects with CNS impairment of unknown origin (with no definite diagnosis, no specific symptoms or signs, and normal common biochemical and metabolic screening results) and 130 age‐ and sex‐matched healthy subjects. EMA levels exceeding two standard deviations (SD) above normal (i.e. 8.1 mmol/molCn) were found in a subgroup of CNS‐impaired patients and healthy controls. EMA levels exceeding 2 SD above normal were fourfold prevalent in the urine of patients with non‐specific CNS impairment compared to from the EMA levels in healthy controls. Moreover, we found that the level exceeding > 8.1 mmol/molCn (i.e. > + 2 SD) had sufficient discrimination accuracy in identifying subjects with non‐specific CNS impairment; the level exceeding 12 mmol/molCn (i.e. > + 6 SD) reaches suitable accuracy (i.e. 100% specificity and 78.6% sensitivity). These observations are of importance, as we found that subtle increases in urinary EMA levels are frequent in patients with non‐specific CNS impairment. The reasons for this association remain unknown. Copyright

New neurocutaneous syndrome with defect in cell trafficking and melanosome pathway: The future challenge

OBJECTIVE Case study of a CNS impairment lacking in presumptive cause; case presents with a clinical phenotype encompassing multiple differently expressed and combined symptoms, as well as a subtle skin defect. MATERIALS AND METHODS A 6-year-old male with apparently isolated mental delay, speech delay, attention deficit/hyperactivity disorder, epilepsy, and subtle and insignificant skin dyschromias. The patient underwent a systematic evaluation, including clinical history; medical, neurological and ophthalmologic examinations. Skin, teeth, nails, hair and sudation were examined for defects. Routine laboratory tests for blood, urine, were performed. The proband had thyroid function tests, electrocardiography, genitourinary system and abdominal examinations. Special examinations pertaining to mental performance, biochemistry, chromosome studies, imaging and electrodiagnostic studies, and skin biopsy were also performed. RESULTS Investigators ruled out genetic syndromes, congenital infections, fetal deprivation, perinatal insults, intrauterine exposure to drug abuse, and postnatal events such as CNS infections as possible common causes of brain impairment. Being all further test negative, the patient exhibited an ultrastructural defect of the skin, identical to that previously described [Buoni S, Zannolli R, de Santi MM, Macucci F, Hayek J, Orsi A et al. Neurocutaneous syndrome with mental delay, autism, blockage in intracellular vesicular trafficking and melanosome defects. Eur J Neurol 2006;13:842-51], suggesting that some cell compartments, such as rough endoplasmic reticulum, lysosomes, Golgi apparatus, and the vesicular zone (racket) of Birbeck granules, sharing similar components, can be altered, resulting in a common defect in cell trafficking, associated to melanosome defects. CONCLUSIONS This new devasting, ultrastructural phenotype accompanied by apparently unspecific and mixed neurological symptoms should represent a future challenge to finally discover the pathogenesis of many childhood CNS symptoms, that currently seem to lack any apparent cause.

[(1)H] magnetic resonance spectroscopy of urine: diagnosis of a guanidinoacetate methyl transferase deficiency case.

For the first time, the use of urine [1H] magnetic resonance spectroscopy has allowed the detection of 1 case of guanidinoacetate methyl transferase in a database sample of 1500 pediatric patients with a diagnosis of central nervous system impairment of unknown origin. The urine [1H] magnetic resonance spectroscopy of a 9-year-old child, having severe epilepsy and nonprogressive mental and motor retardation with no apparent cause, revealed a possible guanidinoacetic acid increase. The definitive assignment of guanidinoacetic acid was checked by addition of pure substance to the urine sample and by measuring [1H]-[1H] correlation spectroscopy. Diagnosis of guanidinoacetate methyl transferase deficiency was further confirmed by liquid chromatography—mass spectrometry, brain [1H] magnetic resonance spectroscopy, and mutational analysis of the guanidinoacetate methyl transferase gene. The replacement therapy was promptly started and, after 1 year, the child was seizure free. We conclude that for this case, urine [1H] magnetic resonance spectroscopy screening was able to diagnose guanidinoacetate methyl transferase deficiency.