Pyruvate carboxylase deficiency type C; variable presentation and beneficial effect of triheptanoin.

Pyruvate carboxylase is a mitochondrial enzyme essential for the tricarboxylic acid cycle (TCA), gluconeogenesis and fatty-acid synthesis. Pyruvate carboxylase deficiency (PCD) mostly presents with life-limiting encephalopathy (types A/B). A milder type C presentation is rare, with a comparatively favourable prognosis. Therapies remain essentially supportive. Triheptanoin is an odd-chain triglyceride, with the potential to replenish TCA intermediates (anaplerosis), and its metabolites cross the blood-brain-barrier. Outcomes of triheptanoin treatment in PCD types A/B have been disappointing, but have not been reported in type C. Here, we present two new patients with PCD type C, and report the response to treatment with triheptanoin in one. Patient 1 (P1) presented with neonatal-onset lactic acidosis and recurrent symptomatic lactic acidosis following exercise and during illnesses, with frequent hospitalisations. Speech development was delayed. MRI-brain showed delayed cerebral myelination. Patient 2 (P2) presented with episodic ketoacidosis, hyperlactataemia and hypoglycaemia at 2 years of age, with gross motor delay and mild global volume loss on MRI brain. Treatment with triheptanoin was commenced in P1 at 3 years of age with up-titration to 35 mL/day (25% of daily energy intake) over 6 months, due to transient diarrhoea. Dietary long-chain triglycerides were restricted, with fat-soluble vitamin supplementation. Subsequently, hospitalisations during intercurrent illnesses decreased, post-exertional hyperlactataemia resolved and exercise tolerance improved. Continued developmental progress was observed, and repeat MRI 18 months after initiation showed improved myelination. Triheptanoin was well-tolerated and appeared efficacious during 2 years' follow-up, and has potential to restore energy homeostasis and myelin synthesis in PCD type C.

Inter- and intra-observer agreement of Prechtl's method on the qualitative assessment of general movements in preterm, term and young infants.

BACKGROUND: Prechtl's method on the qualitative assessment of general movements (GMs) has been shown to be a good predictor of neurological outcome. There is substantial evidence that this method has good inter- and intra-observer agreement. AIMS: We wanted to find out whether this high agreement is reproducible in the clinical setting. STUDY DESIGN: Reliability study (inter- and intra-observer agreement). SUBJECTS: Twenty video-sequences of children at the age of preterm and writhing movements (31-41 weeks postmenstrual age) and 10 video-sequences of children at the fidgety movements age (49-54 weeks postmenstrual age) were rated by five physiotherapists. OUTCOME MEASURES: Intra- and inter-observer agreements were analyzed with percentage agreement and with nominal kappa statistic with bootstrapped bias corrected 95% confidence intervals. RESULTS: We found fair to substantial inter-observer reliability for the six response categories (time-point 1(t1): median kappa 0.44, range 0.27 to 0.59, time-point 2 (t2): median kappa 0.55, range 0.41 to 0.77) and fair to almost perfect for the normal/abnormal ratings (t1: median kappa 0.53, range 0.29 to 0.61, t2: median kappa 0.63, range 0.29 to 0.85). There was statistically significant improvement from t1 to t2 for the six response categories. The intra-observer reliability for the 9-week interval was moderate to almost perfect (median kappa 0.68, range 0.41 to 0.86). CONCLUSIONS: We were not able to exactly reproduce the generally very good results. In our clinical setting now videos are evaluated by at least two trained therapists and the results are discussed, if necessary, to reach a consensus.

[Virtual tissue. Quaoaring]. / Virtuelles Gewebe. Quaoaring.

Virtual tissue can be generated by employing various methods. First steps en route to virtual tissue may encompass the generation of virtual cells. One such approach termed Quaoaring was applied to produce artificial erythrocytes and these were both discocyte and echinocyte in shape. The results were subsequently compared with data gleaned from scanning electron microscopy and atomic force microscopy. Quaoaring has, however, proved to be unsuccessful in creating convincing objects, particularly those which should be echinocytic in appearance.

Quantum mechanical scattering cross sections and permeability coefficients for ions crossing the human red cell and resting squid axon membranes.

Quantum mechanical calculations of elastic scattering cross sections for some permeant ions crossing the human red blood cell and resting axolemma squid axon membranes have been carried out using the three-dimensional spherically symmetric square potential well. Making the assumption that the permeability coefficient is inversely proportional to scattering cross section, we obtain the order of membrane selectivity for the ions as well as values for the permeability coefficients. Despite the relatively simple method used, good agreement between calculated permeability coefficients and data available in the literature is obtained. We suggest that elastic scattering cross section measurements for ions in various membranes would be valuable not only because they give a precise idea about the permeability ratios between ions but they also determine the form of the potential the ions are moving in.

The study of Ca2+ influx in human erythrocytes in isotonic polyethylene (glycol) 1500 (PEG-1500) and sucrose media.

Effects of isotonic solutions of polyethylene (glycol) 1500 (PEG-1500) and sucrose on Ca2+ influx into ATP-depleted red blood cells were studied using the Ca2+ -sensitive fluorescent dye fura-2AM. When incubated in isotonic low ionic strength media (containing 2 mM CaCl2 in addition to sucrose and PEG-1500), the initial rate of Ca2+ influx was higher than that for the cells in physiological (normal ionic strength) medium. After 20 minutes of incubation in the PEG-1500-containing solution, a 10-fold increase of Ca2+ influx was observed, whereas in the sucrose medium the rate of Ca2+ influx decreased compared to that in physiological medium. 1H-NMR data provided no evidence of direct interaction between PEG-1500 and the erythrocyte membrane. Moreover, PEG-1500 did not affect lipid peroxidation (LPO) induction in erythrocyte membranes. We propose that a change in the hydrogen environment of Ca2+ -ATPase of the erythrocytes suspended in the PEG-1500 solution is the primary cause of altered Ca2+ homeostasis in these cells. The activation of the Ca2+ -ATP-ase in sucrose medium may result in an incomplete suppression of the Ca2+-pump activity in ATP-depleted cells, which is accelerated when calmodulin binds with the Ca2+-ATP-ase under the conditions of rapid Ca2+ accumulation.

The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation.

Using the patch-clamp technique, the non-selective, voltage-activated cation channel in the human red blood cell (RBC) membrane was further characterised. Activity of the cation channel could be demonstrated at a range of salt concentrations with the current-voltage characteristics for monovalent cations going from linear to superlinear functions, depending on the cation concentration in the range of 100-500 mM. The non-selective voltage-activated cation channel was demonstrated to be permeable to the divalent cations Ca2+ and Ba2+, and even Mg2+. The current-voltage relations for the divalent cations were superlinear even at 75 mM salt concentration, but indicated outward rectification in contrast to the I-V curve for monovalent cations. The degree of activation at a given membrane potential depended strongly on the prehistory of the channel. The gating exhibited hysteretic-like behaviour, since the quasi steady-state deactivation and activation curves were displaced by approximately 25 mV. This result fully explains apparent discrepancies between V0.5-values previously obtained by slightly different experimental protocols. The possible physiological/pathophysiological role of the channel is discussed in the context of the demonstrated permeability for divalent cations.

Adiabatic compressibility of red blood cell membrane: influence of skeleton.

Measurements of ultrasound velocity and density were used for determination of the adiabatic compressibility of red blood cells (RBC) during detachment of the membrane skeleton. Skeleton detachment was induced by addition of nystatin into a low ionic strength RBC suspension resulting in an increase (10%) of the ultrasound velocity concentration increment, [u], while the specific volume of cells, phi(v) did not change significantly. Changes of the concentration increment had rather long kinetics and were not completed even after 60 min. Both [u] and phiV values were used for calculation of the specific apparent adiabatic compressibility of RBC, phiK/beta0. The value of the specific apparent compressibility decreases following addition of nystatin. This corresponds to an increase in the volume elastic modulus of RBC membranes during detachment of the membrane skeleton. Control experiments with large unilamellar liposomes at conditions similar to that performed with the RBC did not reveal significant changes of [u] after the addition of nystatin. Our results show that the role of the membrane skeleton probably consists in maintaining higher compressibility of the RBC membranes. This may partly provide conditions for conformational changes of RBC membrane proteins.

Variations of intracellular pH in human erythrocytes via K(+)(Na(+))/H(+) exchange under low ionic strength conditions.

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K(+) efflux and Cl(-) loss. When human erythrocytes were suspended in a physiological NaCl solution (pH(o) = 7.4), the measured pH(i) was 7.19 + or - 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH(i) to 7.70 + or - 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl(-) content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H(+) influx with the measured unidirectional K(+) efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H(+) influx and a K(+) efflux in LIS solution of 108.2 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 19.3 mmol (l(cells) hr)(-1), respectively. For bovine and porcine erythrocytes, in LIS media, H(+) influx and K(+) efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K(+)(Na(+))/H(+) exchanger, inhibited the K(+) efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K(+)(Na(+))/H(+) exchanger in the human erythrocyte membrane.

Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

Non-selective voltage-activated cation channel in the human red blood cell membrane.

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.

The influence of external surface potential and transmembrane potential on the passive transbilayer movement of phospholipids in the red blood cell membrane.

The passive transbilayer movement of spin-labelled analogues of phosphatidyl-choline (PC), phosphatidyl-ethanolamine (PE), and phosphatidyl-serine (PS) in red blood cell membranes was investigated at physiological and low ionic strength of the extracellular solution. Passive transbilayer movement of aminophospholipids PS and PE was measured in ATP-depleted cells. To discriminate between a possible surface potential and a transmembrane potential effect, NaCl in physiological ionic strength solution was replaced either by sucrose or by Na-tartrate (constant osmolarity). Neither in sucrose (low ionic strength) nor in Na-tartrate media a significant change of the translocation rate of the phospholipids was observed. From these results in can be concluded that changes of the external surface potential as well as of the transmembrane potential do not affect the passive transbilayer movement of phospholipids in human red blood cells.

Activation of a novel organic solute transporter in mammalian red blood cells.

1. Suspending human red blood cells in isotonic sucrose (low ionic strength, LIS) medium induces a significant increase in membrane transport of glutamine, glutamate, lactate, histidine, taurine, glycine, serine, choline and carnitine but not sorbitol or sucrose. 2. Progressive lowering of ionic strength by sucrose or NaCl replacement gave a similar activation profile for taurine influx as found earlier for residual K+(86Rb+) flux. 3. The induced taurine transport could be measured as enhanced influx and efflux. Influx was linear with external concentration up to 10 mM, largely insensitive to alteration in cell volume, and did not vary with red blood cell age. 4. Unlike previous results for residual K+ transport, altering transmembrane potential with gluconate or glucuronate media led to an increase in taurine influx similar to that observed in LIS media. Varying medium pH confirmed the effect was not due to alteration in pH. 5. The LIS-induced flux was sensitive to a variety of 'classical' anion transport inhibitors in the order of potency DNDS, DIDS, NPPB, DIOA, niflumic acid, furosemide (frusemide), glibenclamide, nitrendipine and bumetanide. 6. The taurine flux showed a temperature dependence similar to that of the LIS-induced residual K+ flux. High hydrostatic pressure (40 MPa), however, inhibited taurine flux but stimulated residual K+ influx in LIS media. 7. A significant enhanced taurine flux could be demonstrated in red blood cells of other species, including horse, cattle, pig and high and low potassium type sheep. 8. It is concluded that lowering ionic strength activates a transport pathway for organic molecules sharing some similarities with background Cl- channels and LIS-induced residual K+ fluxes. In the latter context, however, there are certain significant differences (effect of transmembrane potential; volume; pressure sensitivity; species distribution) which may be important, and the unequivocal identity of the two transport processes remains to be confirmed.

Investigation of low ionic strength effect on passive monovalent cation transport through erythrocyte membranes.

Effect of low ionic force on the passive transport of univalent cations through the erythrocyte membranes is considered. It is postulated that this effect is complex and cannot be explained on the basis of electrodiffusion. Data are presented on the already known transport pathways in the erythrocyte membranes for univalent cations. Characteristics of residual cation transport (the "leak" flux) through the erythrocyte membranes also affected by the low ionic force are presented.

Carrier-mediated residual K+ and Na+ transport of human red blood cells.

Residual, i.e., (ouabain, bumetanide, and EGTA)-insensitive K+ and Na+ influxes as well as effluxes of human red blood cells are enhanced in isotonic solutions of low (NaCl + KCl) concentration using sucrose to maintain constant osmolarity. Various carrier models were tested to fit the experimental data of these fluxes simultaneously. The residual K+ and Na+ fluxes can be described on the basis of a carrier mechanism of competing substrates with modifier sites.

Characterisation of the potassium influx in rat erythrocytes.

In the rat erythrocyte membrane five different transport pathways for K+ are present. In addition to the well characterised K+ transport via the Na+ pump, the Na,K,Cl cotransport and the Ca(2+)-activated K+ channel, there are a K,Cl cotransport and a residual (leak) K+ transport. The K,Cl cotransport is already present under physiological conditions, and can be stimulated by N-ethylmaleimide treatment but not by a cell volume increase. A low ionic strength stimulated increase of the residual K+ influx can be demonstrated in rat erythrocytes after suppressing the K,Cl cotransport pathway. Between 11 and 19 weeks of age, rats show significant differences in all transport pathways of the erythrocyte potassium influx. Using influx data from individual rats a significant correlation between the total K+ influx and the ouabain-sensitive K+ influx has been found. Maintaining the rats on a diet poor in essential fatty acids leads to a significant change of the linoleic acid content of the erythrocyte membrane phospholipids. However, no significant effect on the various K+ transport pathways has been found. An analysis of the fatty acid composition of the erythrocyte membrane phospholipids showed significant correlations between the content of oleic acid, and arachidonic acid, and the ouabain-sensitive K+ influx (as well as the total K+ influx).

Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition.

The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.

Investigation of monovalent cation influxes of diamide-treated human erythrocytes in solutions of different ionic strength.

Total and residual i.e. (ouabain + bumetanide + EGTA)-insensitive K+ as well as Na+ influxes were investigated in human erythrocytes before and after treatment with diamide (5 mM). In physiological and in low ionic strength solution these influxes were increased after diamide treatment. Diamide-treated cells do not exhibit significant differences between the total and residual influxes for both Na+ and K+. The diamide-induced cation fluxes in low ionic strength solution are significantly higher compared with the fluxes in physiological ionic strength solution. The diamide-induced K+ influx is not chloride-dependent, and replacement of NaCl by sodium methylsulfate does not significantly reduce this flux. A subsequent incubation of diamide-treated erythrocytes with dithioerythritol which restores the cellular glutathione level to its original value only partly decreases the enhanced K+ influx. From these results it can be concluded that electrodiffusion and K/Cl cotransport are not involved in the diamide-induced stimulation of the residual K+ influx of human erythrocytes.