Comparison of biotransformation of inorganic selenium by Lactobacillus and Saccharomyces in lactic fermentation process of yogurt and kefir.

The aim of the present study was to characterize, quantify, and compare the different selenium species that are produced when lactic fermentation with two different types of microorganisms, bacteria (Lactobacillus) and yeast (Saccharomyces), take place to produce yogurt and kefir, respectively, and to study the transformation process of these species as a function of time. These two dairy products were chosen for the study because they are highly consumed in different cultures. Moreover, the microorganisms present in the fermentation processes are different. While the bacteria Lactobacillus is the one responsible for yogurt fermentation, a partnership between bacteria and the yeast Saccharomyces causes kefir fermentation. A comparative study has been carried out by fermenting Se(IV) enriched milk in the presence of both types of microorganisms, where the concentration range studied was from 0.5 to 20 microg g (-1). Enzymatic extraction enabled selenium speciation profiles, obtained by anionic exchange and ion-pairing reversed phase high performance liquid chromatography (IP-RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) detection. Scanning electron microscopy (SEM) applied to the enriched samples showed segregated Se (0), at added concentrations higher than 5 microg g (-1). The main Se species formed depended on the type of microorganism involved in the fermentation process, SeCys 2 and MeSeCys being the main species generated in yogurt and SeMet in kefir. The results obtained are different for both kinds of samples. Lactic fermentation for yogurt produced an increment in selenocystine (SeCys 2) and Se-methylselenocysteine (MeSeCys), while fermentation to produce kefir also incremented the selenomethionine (SeMet) concentration. The Se species are stable for at least 10 and 15 days for kefir and yogurt, respectively. After this period, selenocystine concentration decreased, and the concentration of Se-methylselenocysteine was found to significantly increase.

Neurophysiological monitoring of lumbosacral spinal roots during spinal surgery: continuous intraoperative electromyography (EMG).

INTRODUCTION: Neurophysiological monitoring during spinal surgery reduces the associated neurological complications. Continuous EMG recording has developed an useful technique for spinal root monitoring PATIENTS AND METHODS: Fifty four patients who underwent surgery for several lumbosacral spinal lesions (low and high degree spondylolisthesis, spinal stenosis, degenerated or herniated lumbar discs) were studied to evaluate the effectiveness of continuous EMG recording in monitoring spinal root function during surgery. Electrical root or screw stimulation was also performed in nine of them. To correlate surgical spinal root lesion with a precise EMG injury activity an animal study with 5 pigs was performed; lesion was produced by prolonged spinal root traction. RESULTS: In the porcine group EMG discharges lasting longer than one minute after cessation of root traction was noted in 74% of spinal root levels (neurotonic discharges or pseudo-rhythmic activity in 70% of the cases). Spinal root lesion was demonstrated through EMG three weeks after surgery. In the patient group pathological-significant EMG activity was not recorded in any case during monitoring. Mechanical or chemical root stimulation during surgery produced brief lasting EMG bursts of no pathological significance. Only a patient developed a mild acute L5-S1 radiculopathy after surgery (1 false negative) and post-operative deficit was not observed in the rest. Electrical stimulation of spinal roots and screws allowed to identify root level and prove the adequate placement of screws. CONCLUSION: Spontaneous and evoked EMG recordings are simple techniques that provide continuous information about lumbosacral spinal roots function throughout surgery.

[A neurophysiological study of the alterations to the central and peripheral nervous systems in Friedreich's ataxia]. / Estudio neurofisiológico de las alteraciones del sistema nervioso central y periférico en la ataxia de Friedreich.

INTRODUCTION: Friedreich's ataxia (FA) is the most common type of early-onset hereditary ataxia, starting during childhood and adolescence. Both the central and peripheral nervous systems are compromised. AIM. To describe the alterations found in the neurophysiological examination and their relation with the degree of disability and the time the disease lasts. PATIENTS AND METHODS: We examined 26 patients diagnosed with FA by means of a genetic study with multimode evoked potentials, transcranial magnetic stimulation, peripheral nerve conductions and a study of small myelinated-unmyelinated sensory fibres using quantitative sensory tests (thermotest). RESULTS: Peripheral sensory potentials, somatosensory potentials and motor responses with transcranial stimulus were pathological in all patients. From the early stages of the disease there were both peripheral sensory neuropathy and alterations of the somatosensory potentials and motor responses with transcranial stimulus in the lower limbs. Alterations in the somatosensory potentials in the upper limbs and the motor responses obtained by transcranial stimulus were related with the degree of disability. Brainstem and visual potentials were altered in 16 and 28% of cases, respectively; only reduced amplitude of the P100 was related with the degree of disability. In the thermotest, 50% of the patients showed alterations. CONCLUSIONS: Increased disability in patients with FA is related with the progressive involvement of the somatosensory and pyramidal pathways. Although traditionally reports have claimed that the thick myelinated fibres are the ones involved in FA, we found alterations in the small calibre fibres in half the patients.

[Mitochondrial respiratory chain diseases. Evaluation and variability in 52 patients]. / Enfermedades de la cadena respiratoria mitocondrial. Evaluación y variabilidad en 52 pacientes.

INTRODUCTION: Clinical, electrophysiological, genetic and biochemical deficiencies variability were evaluated in 52 patients diagnosed of mitochondrial respiratory chain diseases (MRCD). PATIENTS AND METHODS: 26 men and 26 women, aged 19 to 79 years, were tested by clinical examination, electrophysiological techniques, muscle biopsy and genetic and biochemical studies. RESULTS: The patients were classified into seven phenotypes: myopathy, chronic progressive external ophthalmoplegia, progressive ophthalmoplegia plus ataxia, Kearns-Sayre syndrome, mitochondrial encephalomyopathy with lactic acidosis and stroke episodes (MELAS), myoclonic encephalopathy with ragged-red fibers (MERRF), and encephalopathies. Each phenotype may begin by different ways. The electromiography showed myopathy in 39 cases and various types of neuropathy in 10. Ragged-red COX negative fibers or widespread electron microscopic abnormalities were found in 47 cases. Simple deletions, multiple deletions and three different point mutations were observed. Deficiency of complexes I, II, III and IV were found alone or in different associations. CONCLUSIONS: MRCD shows wide variations in clinical, genetic and biochemical studies. Some patients with nonspecific manifestations, mainly of central nervous system, need careful attention and to be on account of diagnostic suspicion.

Stability of total selenium and selenium species in lyophilised oysters and in their enzymatic extracts.

To obtain reliable information on speciation analysis it is necessary to previously evaluate the stability of the species in the sample of interest. Furthermore, in those cases in which sample treatment to extract the species is time-consuming, an evaluation of how to maintain species integrity in the extracts is paramount. Thus, the present paper reports the stability of total Se, SeMet and TMSe+ in freeze-dried oyster and in the enzymatic extracts stored in Pyrex and polyethylene containers at different temperatures (-18, 4 and 20 degrees C). Total selenium determinations and Se speciation were carried out by HG-AAS after acid digestion in a microwave oven and by on-line coupling of cation exchange HPLC-ICP-MS after enzymatic hydrolysis, respectively. The results obtained for the freeze-dried sample showed that total Se and the selenium species evaluated are stable for at least 12 months, under all the conditions tested. However, Se species in the enzymatic extracts are only stable for 10 days if stored at 4 degrees C in Pyrex containers. These results show that the extracts do not necessarily have to be analysed just after sample treatment.

[Hypokalemic periodic paralysis: exercise test]. / Paralisis periódica hipopotasémica: prueba de ejercicio.

INTRODUCTION: Periodic paralyses are illnesses related with the dysfunction of the ionic pathways of the muscle fibre membrane. Neurophysiological diagnosis by means of a conventional electromyogram (EMG) is rather unsatisfactory when exploring patients in the asymptomatic phase, since they often display unspecific changes. Furthermore, it does not identify the alterations in muscular excitability. The exercise test consists in determining the variation in the area or the amplitude of the muscular action potential, which is obtained through electrical stimuli that are repeated periodically for several minutes after having carried out local exercise with the explored muscles. It is a simple technique that enables us to achieve a significant reduction in the area or amplitude of the action potential (more than 40% or 50% with regard to the basal value) in certain diseases produced by ionic pathway dysfunction, while the patient is in the asymptomatic phase. CLINICAL CASES: We report the cases of three patients diagnosed as suffering from hypokalemic periodic paralysis. During the asymptomatic phase, the EMG revealed characteristic myopathic changes in only one case and unspecific alterations in the other two. Nevertheless, the exercise test that was also performed during the asymptomatic phase showed a characteristic pathological response in the three patients. CONCLUSION: The exercise test is a technique that indirectly shows up the progressive loss of muscle fibre membrane excitability and helps diagnosis in asymptomatic patients.

Selenium speciation in animal tissues after enzymatic digestion by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry.

A procedure is described for the enzymatic digestion of tuna and mussel samples that allows the determination of selenium species by high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. The species were extracted by two-step enzymatic hydrolysis with a non-specific protease (subtilisin). The selenium species were separated on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions, namely phosphate buffers at pH 2.8 and pH 6.0 as mobile phases. The method determines organic (trimethylselenonium, selenocystine, selenomethionine and selenoethionine) and inorganic selenium species (selenite and selenate), but only organic selenium species were found in the samples. The sum of identified selenium species in the sample was about 30% of the total selenium present in the enzymatic extract despite the fact that recoveries of total hydrolysed selenium were 93-102%. Trimethylselenonium ion and selenomethionine were found in both tuna and mussel samples and an unknown selenium species was also found in tuna samples.

A flow-through fluorescent sensor to determine Fe(III) and total inorganic iron.

A flow-through fluorescent sensor for the consecutive determination of Fe(III) and total iron is described. The reactive phase of the proposed sensor, which has a high affinity for complexed Fe(III), consists of pyoverdin immobilized on controlled pore glass (CPG) by covalent bonding. This pigment selectively reacts with Fe(III) decreasing its fluorescence emission. Total inorganic iron is determined as Fe(III) after on-line oxidation in a mini-column containing persulphate immobilized on an ion exchange resin. The developed method allows the determination of Fe(III) in the 3-200 (g l(-1) range. The relative standard deviations of 10 determinations of 60 (g l(-1) of Fe(III) and 20 (g l(-1) of Fe(III)+Fe(II) are 3 and 5%, respectively. The sensor has been satisfactorily applied to speciate iron in synthetic, tap and well waters and wines. There were no significant differences for total inorganic iron determination between this new method and the atomic absorption spectroscopy reference method at the 95% confidence level. The sensor allows the concentration of Fe(II) to be calculated as the difference between total inorganic iron and Fe(III). The lifetime of the sensor is at least 3 months in continuous use or the equivalent of 1000 determinations.

[Small fiber dysfunction in peripheral neuropathies]. / Afectación de fibras finas en la patología del nervio periférico.

INTRODUCTION: Disfunction of thin myelinated and unmyelinated fibers may appear isolated or in association with large-myelinated fibers lesion. Small-fiber neuropathy includes autonomic and sensory symptoms, most prominent of them thermo-algesic deficits. DEVELOPMENT AND CONCLUSION: In some acute neuropathies, small-fiber lesion is relatively pure, as in pandysautonomia, but it also appears in disorders with prominent somatic involvement, such as the Guillain-Barre syndrome, in which case autonomic symptoms worsens the prognosis. Small-fiber dysfunction is important in certain diseases that involve different components of the nervous system, like paraneoplastic syndromes and porphyria. Some drugs and toxic substances may damage thin myelinated and unmyelinated fibers. Nowadays, chronic idiopathic small-fiber neuropathy is diagnosed more frequently, because of the recent development of techniques that selectively evaluate this peripheral nerve component. Hereditary sensory and autonomic neuropathies can also be studied. Small-fiber dysfunction is very prominent in some diseases, e.g. diabetes mellitus and amyloidosis. In the pure autonomic failure, only the peripheral component of the autonomic nervous system is affected, and this feature is the key to make diagnosis versus multisystem atrophy. There are situations in which there is no clear deviation from normality, namely old age autonomic failure and orthostatic intolerance syndrome in which autonomic evaluation is mandatory.

Determination of selenium species in human urine by high performance liquid chromatography and inductively coupled plasma mass spectrometry.

A method developed to determine organic and inorganic selenium species in human urine samples is presented in detail. After a simple sample treatment based on elimination of non-charged organic compounds, selenium species were separated by high performance liquid chromatography (HPLC) on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions: phosphate buffers at pH 2.8 and 6.0. Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS). Trimethylselenonium ion and two unknown selenium species in urine samples were found. Selenium species were shown to have stability problems, with the maximum allowed storage time of 1 week.

Gene dosage effect in one family with myoclonic epilepsy and ragged-red fibers (MERRF).

OBJECTIVES: We analyzed the percentage of mitochondrial DNA (mtDNA) heteroplasmy in blood samples of 13 individuals belonging to a three family generation of myoclonic epilepsy with ragged-red fibers (MERRF) and compared the 5 affected patients and the 8 unaffected relatives. MATERIAL AND METHODS: DNA was extracted from blood and muscle of the proband and from blood of 12 maternal relatives. A PCR restriction analysis method was used to detect the mutation. RESULTS: The proband had the complete MERRF phenotype. The phenotype in three other individuals in the maternal lineage was consistent with the MERRF syndrome. The remaining were asymptomatic. The np 8344 mutation was observed in muscle and blood of the proband, and in blood from every one of 12 maternal relatives, ranging from 44% to 83% of mutated genomes. Symptomatic individuals had higher levels (P < 0.001) of mutated mtDNA than asymptomatic maternal relatives. However, high proportions of mutant genomes (up to 63%) were found in asymptomatic relatives. CONCLUSIONS: Although there seems to be a gene dosage effect in MERRF, we found no absolute relationship between the relative proportion of mutant genomes in blood and clinical severity. Factors other than gene dosage in blood may account for the differences in clinical phenotype.

A biosensor for ferric ion.

A new biosensor for monitoring iron has been developed. The active solid phase is pyoverdin, a natural fluorescent pigment biosynthesized by Pseudomonas fluorescens immobilized on controlled pore glass (CPG) and packed in a quartz flow-through cell. The biosensor is very selective for iron(III) and can be easily regenerated in about 2 min by passing 1M HCl through the cell. The optimum conditions and analytical characteristics (detection limit, precision and linear range) for the new sensor in solution (DL = 10 ng/ml) and in immobilized form (DL = 3 ng/ml) are reported. The biosensor has good stability and can be used continuously over a period for at least 3 months (over 1000 determinations). The sensor was successfully applied to determine iron in different water samples. There were no significant differences between the new method and the Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) reference method at the 95% confidence level.

[Multifocal polyneuropathy with persistent conduction blockage. A new subset of chronic inflammatory polyneuropathies]. / Polineuropatía multifocal con bloqueos de conducción persistentes. Un nuevo subgrupo dentro de las polineuropatías inflamatorias crónicas.

Recently a subset of chronic demyelinating inflammatory polyneuropathies with asymmetrical involvement limited to upper limbs, at least at the onset, resembling a multifocal mononeuropathy has been described. Electrodiagnostic testing disclosed multifocal CB outside the common entrapment sites has been described. We report a 55 years old man with a 4 years history of paresis, numbness, fasciculations, myokymia, cramps and mild amyotrophy. Electrophysiological evaluation showed proximal multifocal conduction block and abundant spontaneous activity as fasciculations, myokymia and scarce denervation activity. The importance of taking into account this entity in the differential diagnosis of patients with suspected mononeuritis multiplex or motoneuron disease is emphasized. The nosologic place of this entity is also discussed.

Electrophysiologic study in benign human botulism type B.

Conventional electromyography (EMG) and single fiber EMG (SFEMG) were performed in a 64-year-old diabetic woman with mild type B botulism. The main clinical signs were autonomic dysfunction and cranial nerves paresis. Conventional EMG was normal, except for small changes that were consistent with mild mixed peripheral neuropathy in the lower limbs and were related to diabetes. Repetitive stimulation and results of single stimulus before and after full effort were normal. SFEMG showed increased jitter and impulse blocking in clinically normal muscles. The jitter was frequency dependent and improved at a higher innervation rate. Impulse blocking in potentials with only slightly increased jitter was found. The follow-up showed improvement of the jitter in agreement with clinical recovery. Jitter abnormalities were recorded after 16 weeks, when clinical signs of botulism had been reversed to normal. Motor unit fiber density increased progressively, and on examination at 8 weeks, some potentials showed very high jitter values. Both findings might suggest new endplate formation, perhaps due to ultraterminal sprouting development.