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PubMed2004

Separation surgery for total vertical craniopagus twins.

OBJECTIVE: A pair of conjoined twins aged 11 months underwent investigations, followed by surgical separation in Singapore General Hospital in April 2001. They were joined at the skull vertex and facing in opposite directions. METHODS: Radiological investigations including cerebral angiography, magnetic resonance imaging and computerized tomographic scans were performed, leading to the diagnosis of total vertical craniopagus. There were two separate brains, with separate arterial circulations, but with a common superior sagittal sinus. Tissue expanders were inserted in the subgaleal space for 6 months of scalp expansion prior to surgery. Pre-operative planning involved the use of virtual reality equipment and life-sized polymer models of the conjoined skulls and brains. Surgical separation of the twins was achieved after approximately 100 h of operating time, using intraoperative image guidance, microsurgical techniques and intraoperative neurophysiologic monitoring. Reconstruction of the dura, calvarium and scalp was performed with artificial dura, absorbable plates and split skin grafts. Postoperative complications included focal cortical infarction, meningitis, and hydrocephalus. CONCLUSION AND OUTCOME: Despite these complications, the twins recovered satisfactorily and were discharged to their home country within 6 months. The 3-month outcome was minor disability in one twin and severe developmental delays in the other. Separation surgery is possible for complex cranially-conjoined twins but requires detailed planning and extensive surgical management.

Anesthesia, General↗
PubMed2004

Separation of craniopagus twins in the era of modern neuroimaging, interventional neuroradiology, and frameless stereotaxy.

INTRODUCTION: Craniopagus twinning is a rare embryological event occurring in 1 in 2.5 million births. CASE REPORT: We present our recent experience with the separation of total vertical craniopagus twins in the modern era of neuroimaging and interventional neuroradiology. Three-dimensional CT images revealed the twins' heads were axially rotated 30 degrees. MRI showed deficient dura between the brains of the twins, and some sharing of parietal brain tissue. Cerebral angiography showed a dominant arterial circulation of one twin with unilateral middle cerebral artery (MCA) branches feeding the other twin. The twins shared a common superior sagittal sinus in its middle segment where a circular sinus was formed. Prior to surgery, endovascular separation of the twins' arterial and venous circulations was achieved in part using tantalum coils for the MCA feeders in one twin, and balloon occlusion of the anterior superior sagittal sinus of the other. Using the ISG wand intraoperatively, surgery proceeded stepwise and included the circumferential removal of bone and opening of the dura, separation of the twins' brains along leptomeningeal planes, and identification of the major draining veins from the superficial cerebral cortex. DISCUSSION: The separation of craniopagus twins demands a multidisciplinary team approach. Utilizing preoperative neuroendovascular techniques to occlude shared vascular anastomotic channels, complex total vertical craniopagus twins can now be successfully separated in a one-stage procedure.

Child, Preschool↗
PubMed2002

The effect of sample treatment on separation profiles of tear fluid proteins: qualitative and semi-quantitative protein determination by an automated analysis system.

PURPOSE: Qualitative and quantitative determination of tear fluid components is of increasing interest in ophthalmology. Until now, for diagnosis and course control of some diseases of the anterior parts of the eye, different methods for tear fluid protein analysis are available. Results can be obtained by polyacrylamide gel electrophoresis (PAGE), immunochemistry, and high-performance liquid chromatography (HPLC). A new method for protein separation, identification and semi-quantitative determination on a chip-based micro-fluidic technique is used for the first time to investigate tear fluids. METHODS: Normal human reflex tears were obtained by stimulation with China mint oil and collected using glass capillary tubes. A lab-on-a-chip technology (developed by Agilent Technologies, Waldbronn, Germany, in co-operation with Caliper Technologies, Mountain View, California, USA) was used for separation and semi-quantitative determination of tear proteins. Tear fluid was separated on the Agilent 2100 Bioanalyzer in combination with the Protein 200 LabChip kit and the dedicated protein assay software. Time and temperature of the incubation with sample buffer were varied, and the influence of these parameters on protein separation profiles was studied. Tear proteins were also analysed by PAGE, and the results obtained by both methods were compared. RESULTS: The different proteins of tear fluid can be separated by the Agilent 2100 Bioanalyzer method in very short time. By this method, the molecular weight as well as the concentration of proteins can be determined. Data are automatically stored in digital format and can be retrieved and shared. Results of this technology were comparable with the protein pattern obtained by PAGE. It was confirmed by both methods that, depending on incubation time of tear fluid with sample buffer and on temperature, different protein pattern can be obtained from the tears of one specimen. CONCLUSION: Tear proteins - in contrast to serum or aqueous humour proteins - are very sensitive to changes in sample buffer temperature as well as incubation time with buffer. To obtain comparable results for tear fluid proteins, the sample buffer applied and the incubation time and temperature must be observed carefully. This can be demonstrated by both the new Agilent 2100 Bioanalyzer method and PAGE. These results are of importance when comparing tear fluid protein pattern for the diagnosis and course control of dry-eye syndrome and of other diseases of the anterior part of the eye.

Aqueous Humor↗
PubMed2003

Sulfur stable isotopes separate producers in marine food-web analysis.

Ecological applications of stable isotope analysis rely on different producers having distinct isotopic ratios to trace energy and nutrient transfer to consumers. Carbon (C) and nitrogen (N) are the usual elements analysed. We tested the hypothesis that producers unable to be separated using C and N would be separated by sulphur (S), by reviewing estuarine and marine food web studies using all three elements (total of 836 pairwise comparisons between producers). S had a wider range of values across all producers than C and N (S: 34.4, C: 23.3, N: 18.7 per thousand ), and a higher mean difference among producers (S: 9.3, C: 6.5, N: 3.3 per thousand ). We varied from 1 to 10 per thousand the distance producers must be apart to be considered separate. For each of these gap distances, S-separated producers tied on C and N in 40% or more of cases. Comparing the three elements individually, S had fewer tied pairs of producers for any gap distance than C or N. However, S also has higher within-producer variability. Statistical tests on simulated data showed that this higher variability caused S to be less effective than C for analysing differences among mean producer values, yet mixing models showed that S had the smallest confidence intervals around mean estimates of source contributions to consumers. We also examined the spatial and temporal scales over which S isotope signatures of the saltmarsh plant Spartina alterniflora varied. Differences between samples taken within tens of metres were smallest, but between samples hundreds of metres apart were as different as samples thousands of kilometres apart. The time between samples being taken did not influence S signatures. Overall, the use of S is recommended because it has a high probability of distinguishing the contribution of different producers to aquatic food webs. When two elements are employed, the combination of S and C separates more producers than any other combination.

Animals↗
PubMed2004

Modified extraperitoneal endoscopic separation of parts for abdominal compartment syndrome.

BACKGROUND: Standard therapy for abdominal compartment syndrome (ACS) is laparotomy and temporary abdominal wall closure with significant morbidity. The component separation technique allows for difficult abdominal closure. We studied a modified extraperitoneal endoscopic separation of parts technique on an animal model of ACS. METHODS: Twelve anesthetized pigs were instrumented for measurement of central venous pressure, arterial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, cardiac output, and intraabdominal pressure (IAP). ACS to 25 mmHg was created by infusing saline into an intraabdominally placed bag. Animals were divided in two equal groups. Pigs in group A underwent minimally invasive resection of the nerves supplying the rectus muscles bilaterally. Pigs in group B underwent minimally invasive modified component separation technique bilaterally. Change in IAP and other physiological parameters were recorded. RESULTS: (Group A) IAP increased significantly from 7.3 mmHg +/- 3.8 to 25.2 mmHg +/- 1.5 with infusion of saline. Following nerve transection on the right side there was a nonsignificant decrease in IAP from 25.2 mmHg +/- 1.5 to 22.3 mmHg +/- 1.4 and following nerve transection on the left side there was a further decrease in IAP to 20.3 mmHg +/- 1.9. (Group B) IAP increased significantly from 3.8 mmHg +/- 0.4 to 24.7 mmHg +/- 0.5 with infusion of saline. Following separation of parts on the right side there was a significant decrease in IAP from 24.7 mmHg +/- 0.5 to 15.0 mmHg +/- 1.7 and there was a further decrease in IAP to 11.3 mmHg +/- 1.4 following separation of parts on the left side. The only significant change in the physiological parameters measured was observed in CVP in both groups. CONCLUSION: We present a porcine model of extraperitoneal endoscopic release of abdominal wall components as a treatment option for ACS.

Abdomen↗
PubMed2005

Phase behavior of amorphous molecular dispersions II: Role of hydrogen bonding in solid solubility and phase separation kinetics.

PURPOSE: To determine the factors influencing "solid solubility" and phase separation kinetics of drugs from amorphous solid dispersions. METHODS: Solid dispersions of griseofulvin-poly(vinyl pyrrolidone) (PVP) and indoprofen-PVP were prepared using solvent evaporation technique. Dispersions demonstrating single Tg were exposed to 40 degrees C/69% RH for 90 days. Drug solid solubility in the polymer and phase separation rates were determined from changes in Tg of solid dispersions. FTIR spectroscopy and XRD were used to characterize drug-polymer interactions and drug crystallinity, respectively. RESULTS: Freshly prepared solid dispersion of up to 30% w/w griseofulvin and indoprofen were molecularly miscible with PVP. Hydrogen bonding was evident in indoprofen-PVP, but not in griseofulvin-PVP dispersions. When exposed to 40 degrees C/69% RH, griseofulvin phase separated completely, whereas the solid solubility of indoprofen was determined as 13% w/w. The first-order rate constants of phase separation for 10%. 20%, and 30% w/w griseofulvin dispersions were estimated as 4.66, 5.19, and 12.50 (x10(2)) [day(-1)], and those of 20% and 30% w/w indoprofen dispersions were 0.62 and 1.25 (x10(2)) [day(-1)], respectively. CONCLUSIONS: Solid solubility of griseofulvin and indoprofen in PVP is approximately 0% w/w and approximately 13% w/w, respectively. Drug-polymer hydrogen bonding in indoprofen-PVP dispersions favors solid solubility. Phase separation rate of drug from the solid dispersions depends on the initial drug content and the nature of drug-polymer interactions.

Hot Temperature↗
PubMed1987

Effect of spatial separation of stimulation sites on ventricular refractoriness during programmed electrical stimulation.

To determine if electrical stimulation during the basic ventricular drive train introduces local effects that influence ventricular refractoriness during endocardial stimulation in humans, 10 patients were studied. With a hexapolar catheter with 3 mm interelectrode distances positioned at the right ventricular apex, the ventricular functional and effective refractory periods were determined for unipolar cathodal stimuli of 2 and 5 times diastolic threshold. Stimulation was performed with both the scanning extrastimulus (S2) and basic ventricular drive stimuli (S1) delivered to the same site and to sites separated by 6 mm. The functional refractory period was not changed by delivering the basic drive and extrastimuli to the same as opposed to separate sites (273.5 +/- 16 vs 273.5 +/- 19 msec during stimulation at 2 times threshold and 270 +/- 16 vs 270 +/- 18 msec at 5 times threshold; p = NS). The longest S1-S2 interval at which S2 failed to capture (effective refractory period) was longer by 5.5 +/- 8.2 msec at twice threshold (0.05 less than p less than 0.10) and 5.5 +/- 3.5 msec at 5 times threshold stimulation (p less than 0.01) when the basic drive and extrastimuli were delivered to separate sites. This is consistent with later activation of the extrastimulus sites during the basic drive when the stimulation sites were separate. Thus, with this method of endocardial programmed electrical stimulation, separation of the stimulation sites for the basic drive and extrastimuli does not affect ventricular refractoriness determined by the extrastimulus technique.

Adult↗
PubMed1985

Separation and quantitation of molecular species from plant lipids by high-performance liquid chromatography.

Monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerol as well as phosphatidyl glycerol were isolated by conventional TLC and then separately subjected to HPLC for resolution of molecular species. Molecular species emerge in groups from reversed-phase columns during gradient elution. The groups are separated according to the sum of carbon and double bond numbers in fatty acyl pairs in linear relation to elution times. Therefore, it is possible to identify a species group with respect to carbon and double bond numbers by its retention time. The separation is monitored by recording the absorbance at 200 nm which depends on double bond combinations in acyl pairs. Diacylglycerols released from glyco- and phospholipids were separated as rho-anisoyl derivatives according to similar criteria. In this case separation was monitored at 250 nm, at which wavelength the absorbance is directly related to molar proportions. By calculating corrected 200-nm/250-nm absorbance ratios for different molecular species of rho-anisoyl diacylglycerols, relative response factors for different double bond combinations were obtained. The 200-nm absorbances of intact lipid species can be converted to molar proportions by division with these factors.

Chromatography, High Pressure Liquid↗
PubMed1986

High-performance liquid chromatographic separation of platinum complexes containing the cis-1,2-diaminocyclohexane carrier ligand.

Platinum drugs with the 1,2-diaminocyclohexane (dach) carrier ligand have shown great promise in cancer chemotherapy, but little is known about their metabolism in the body. Since it is possible to radiolabel the dach ligand, it should be possible to quantitate the biotransformation products of these drugs, provided a method were available to separate the biotransformation products. In this paper we describe a two-column high-performance liquid chromatography system which can be used to separate many likely dach-platinum biotransformation products from the parent compounds, and allow their identification. An initial separation on a reverse-phase Partisil ODS-3 column allowed resolution of the uncharged species. The peak fractions from this column were concentrated 10-fold and reinjected onto a cation exchange Partisil 10 SCX column to allow resolution of the positively charged species. This system allowed resolution of two prototype dach-platinum drugs, (cis-1,2-diaminocyclohexane)dichloroplatinum(II) and (cis-1,2-diaminocyclohexane)malonatoplatinum(II), the aquated species likely to form from these drugs, and the complexes formed when these compounds react with glutathione, metallothionein, and amino acids. By using cation exchange chromatography at pH 2.3 as well as pH 4 and by using 14C-labeled amino acids to determine stoichiometry, it was also possible to determine the most likely structures for some of the amino acid complexes. Most importantly, this system allowed clear separation of many of the likely biotransformation products tested from the biologically important aquated species. This system should prove useful for separating and identifying the biotransformation products of dach-platinum drugs in blood and urine, in tissue culture media, and inside the cell.

Antineoplastic Agents↗
PubMed1986

Sizing and separation of liposomes, biological vesicles, and viruses by high-performance liquid chromatography.

The ability of an HPLC gel exclusion column (TSK G6000PW) to separate lipid vesicles, viruses, and biological vesicles according to size was tested and compared with separations on Sephacryl S1000. The columns were calibrated using vesicular Stokes radii determined by quasielastic light scattering. The vesicles separated according to size on both types of column and remained intact during elution. Viruses of known diameters and clathrin-coated vesicles were also eluted as a function of size. The TSK G6000PW column was able to separate larger particles (greater than 500 nm) than the Sephacryl S1000, and, when used in combination with the TSK G5000PW column, gave more discrete separations of smaller particles (10 to 30 nm diameter). Moreover, the HPLC columns can be run significantly faster (10-20 min vs several hours) and give more precise results than Sephacryl S1000. Therefore, HPLC using a G6000PW column alone, or in combination with a G5000PW column, provides a rapid and accurate means of sizing and selecting specifically sized biological and artificial vesicles.

Acrylic Resins↗
PubMed1986

Separation of anionic oligosaccharides by high-performance liquid chromatography.

We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the amnionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (alpha 2.3 vs alpha 2.6) and/or location of alpha 2,3- and alpha 2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.

Anions↗
PubMed1988

Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis.

A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.

Amino Acids↗
PubMed1989

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments.

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.

Base Composition↗
PubMed1990

Separation of the DNA molecules beyond conventional size limits by gel electrophoresis with sodium dodecyl sulfate.

Electrophoretic mobility of DNA through polyacrylamide as well as agarose gels is greatly increased by sodium dodecyl sulfate (SDS). DNA molecules well beyond the conventionally separable size limits are separated readily and rapidly by gel electrophoresis with SDS in a conventional static electric field. Furthermore in optimal concentration gels DNA molecules of similar molecular sizes are separated better from one another in the presence of SDS than without it. Evidence is presented that SDS may act at least in part by altering conformation of DNA. This simple and readily available means for high resolution separation of hitherto impossible sizes of DNA molecules in polyacrylamide and agarose gels in an ordinary static electric field should find general use in molecular genetic analyses. Structural analyses of DNA-protein complexes are also facilitated by virtue of the simultaneous separation of the DNA and protein components on the same gel lane.

Chromatin↗
PubMed1990

Phase separation in phospholipid bilayers induced by biologically active polycations.

The interaction of various polyionenes with phospholipid bilayer membranes was explored by means of differential scanning calorimetry (DSC) with special reference to their antimicrobial activities. A strong interaction was observed between the polyionenes and acidic phospholipids, whereas zwitterionic phospholipid bilayers were not affected significantly by the polycations. Addition of the polyionenes was found to result in phase separation in mixed bilayer membranes composed of acidic and zwitterionic phospholipids. The ability to induce phase separation strongly depended on the structure of the polyionenes. Polyionenes with rigid spacers were found to be most effective to induce phase separation and to be most active in antimicrobial activity. Polyionenes with rigid and flexible spacers in the alternate fashion exhibited less activities which were similar to those of all flexible spacers. Furthermore, their mode of interaction with bilayers was again similar to those of all flexible spacers. Our results indicate that the rigid spacers are favorable for strong interaction with membranes which are assumed to be the target sites of the polycationic biocides, leading to the higher activity. Other factors affecting both the antimicrobial activity and the mode of interaction with membranes were molecular weight and hydrophobicity. With increasing molecular weight, both the activity and ability to induce phase separation increased. Introduction of hydrophilic groups into the spacers resulted in loss of activity and ability to induce phase separation. The antimicrobial activity and the mode of interaction with membranes were correlated and interpreted on the basis of conformational concept of the polyionenes in solution.

Bacteria↗
PubMed1983

Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients.

Quantitative plasma lipoprotein separation is usually performed by sequential ultracentrifugation which because of the necessity of repeated ultracentrifugation is extremely time consuming and results in changes in lipoprotein composition. We describe a simple, reliable, and rapid method for quantitative plasma lipoprotein separation which is based on ultracentrifugation of the plasma in a discontinuous density gradient media consisting of a saline solution of different densities placed over a plasma solution of 1.250 g/ml (with potassium bromide). After 48 hr of ultra-centrifugation in a swinging bucket rotor excellent lipoprotein separation was demonstrated. The lipoproteins were pure and there was significantly less loss of high density lipoprotein protein to the lipoprotein deficient plasma (d greater than 1.210 g/ml) when compared to the losses incurred during the sequential flotation method. The results of separating the plasma lipoproteins from subjects with dyslipoproteinemias by this method were demonstrated. We conclude that discontinuous density gradient ultra-centrifugation is the method of choice for quantitative plasma lipoprotein separation for both clinical and research use.

Adult↗
PubMed1982

Separation of acidic oligosaccharides by liquid chromatography: application to analysis of sugar chains of glycoproteins.

An ion-exchange, chromatographic system was developed to separate acidic oligosaccharides. Application of this system for the analysis of sialylated carbohydrate chains of glycoproteins was examined, and it was found that this system affords high resolution of acidic oligosaccharide isomers. (1) 3'-O-Neuraminyllactose, 6'-O-neuraminyllactose, and N-acetyl-6'-O-neuraminyllactosamine could be separated from bovine colostrum. (2) Subjection, to this system, of the oligosaccharide fraction released from bovine submaxillary mucin by treatment with alkaline borohydride resulted in good separation of the trisaccharide-alditol GlcNAc-(1 to 3)-[NeuAc-(2 to 6)]-GalNAcol and the disaccharide-alditol NeuAc- or NeuGc-(2 to 6)-GalNAcol. (3) When the serine- or threonine-linked oligosaccharide fraction obtained from human glycophorin A was subjected to this system, two monosialosylated, trisaccharide-alditol isomers, namely, NeuAc-(2 to 3)-Gal(1 to 3)-GalNAcol and Gal-(2 to 3)-[NeuAc-(2 to 6)]-GalNAcol, could be separated from each other. (4) The separation of so-called triantennary and biantennary, complex type of sugar chains of porcine thyroglobulin was achieved with this system.

Animals↗
PubMed1979

Dual-isotope separation technique for radioassay of cobalamin.

Cobalamin is assayed by a dual-isotope separation method using sodium [125I]iothalamate as a marker. Two systems are used: one in which the incompletely-separated bound fraction is counted and compared with the single-isotope method in which the bound fraction is separated by washing (Phadebas radiosorbent assay); and one in which an aliquot of the free fraction is counted. In the dual-isotope method counting bound fractions, about 97% of the supernatant is removed by pouring from silicone fluid separators. The results for serum samples obtained using dual- and single-isotope methods were similar (between run coefficients of variation 5--7%). Experimental errors were smaller in the dual-isotope method. A factor in the kit standards, presumably the absence of proteins, was found to affect the separation technique, resulting in relatively large experimental errors for standards in the single-isotope method. Washing the solid phase in the single-isotope method apparently resulted in a loss of bound isotope. In the dual-isotope method counting free fractions reasonable precision was obtained (coefficient of variation of serum samples 6.6%) even though only about 56% of the supernatant (free fraction) was counted.

Humans↗